CN116848141A

CN116848141A - Molecules that specifically act in tissues where cell death is observed

Info

Publication number: CN116848141A
Application number: CN202280012692.4A
Authority: CN
Inventors: 井川智之; 东正大
Original assignee: Chugai Pharmaceutical Co Ltd
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2023-10-03

Abstract

It is an object of the present invention to provide a molecule that specifically works in the tissue sought to be treated or targeted (e.g. affected tissue, abnormal tissue, etc.), but does not work or works less in healthy or normal tissue, which is capable of providing a molecule that can be used in a drug with reduced side effects while imparting a significant therapeutic or prophylactic effect. A molecule is provided that binds to a component of a cell or a portion thereof that is exposed to an extracellular environment due to cell death (e.g., a filament or histone that forms a cytoskeleton or a nuclear scaffold) and specifically functions in a tissue in which cell death is observed (e.g., affected tissue, abnormal tissue, etc.). More specifically, molecules are provided that confer signaling or blocking to cells in a tissue by binding one or more molecules, either directly or indirectly, to one or more membrane proteins in the cell to form a complex with a component or portion thereof.

Description

Molecules that specifically act in tissues where cell death is observed

Technical Field

The present invention relates to a molecule that specifically functions in a tissue in which cell death is observed and imparts signaling or blocking to cells in the tissue (e.g., immune cells or affected cells) by binding to components or portions thereof that make up the cells, wherein the components or portions thereof are exposed to the extracellular environment as a result of cell death; the molecules are useful as medicaments; use of said molecule in the manufacture of a medicament; pharmaceutical compositions comprising said molecules; a method for imparting signaling or blocking to cells in a tissue; a polynucleotide encoding said molecule; a vector comprising the polynucleotide; retaining cells of the vector; etc.

More particularly, the present invention relates to antigen binding molecules comprising a first moiety that binds to a first antigen that is a damage-associated molecular pattern (DAMP) and a second moiety that binds to an antigen different from the first antigen, and medical uses thereof.

Background

By taking advantage of the characteristics of cancer cells themselves and the Tumor Microenvironment (TME), cancer treatments have been developed that have localized (site-specific) activity primarily in cancer tissues. Some antibody therapies, such as Herceptin (Herceptin), cetuximab (Cetuximab), and the like, target cancer antigens that are mainly expressed in cancer cells, and exert cytotoxic activity mainly on tumor cells by ADCC, signal inhibition, and the like. Methods for delivering physiologically active ligands, such as cytokines, to solid cancers by immunocytokines comprising ligands fused to antibody molecules that bind to cancer antigens that are highly expressed on cancer cells are known.

It is well known that TMEs are often in a hypoxic and low nutritional state, resulting in cell death, particularly in solid cancers, and then found to be a dead cell-enriched environment. When cells die, there are many molecules released or exposed to the cell surface and are enriched in cancer, but not in normal tissues. Some molecules on dead cells are recognized by receptors expressed in phagocytes such as dendritic cells or macrophages, and then the dead cells are eliminated (cleared) by the phagocytes. Clec9A is known to be expressed in a subset of dendritic cells, for example, and binds to dead cells and ectopic F-actin (PTL 1 and PTL 2) exposed on the cell surface of dead cells.

There are many therapies that regulate cellular functions (e.g., T cell activation, B cell activation, induction of cell death, signal suppression, etc.). Many antibodies targeting co-stimulatory molecules are reported to be undergoing clinical research for cancer (NPL 1). For example, the anti-CD 137 agonist antibody Utomimrumab and the anti-CD 40 agonist antibody CDX-1140 are currently being developed clinically. These molecules are expected to exhibit antitumor effects by enhancing the activation of T cells directly or indirectly through dendritic cells, enhancing the tumor-inhibiting activity of macrophages, and the like. In early clinical trials, these antibodies showed some clinical response.

However, one of the current molecular concerns for regulating cellular function is systemic activity. These antibodies can bind to target molecules anywhere in the body, including normal tissues, and may cause toxicity. For example, anti-CD 137 antibodies (BMS) can cause hepatotoxicity.

Bispecific antibodies that target tumor antigens and antigens that modulate immune cell function have been evaluated as one of the possible strategies to overcome systemic toxicity. anti-CD 137 and tumor antigen binding bispecific antibodies show lower toxicity (NPL 2) compared to anti-CD 137 monospecific antibodies. However, in some cases, it is reported that the tumor antigen in the cancer tissue is lost, which may result in the loss of the anti-tumor effect of the tumor antigen specific antibody.

List of references

Patent literature

[PTL 1]WO 2013/053008 A2

[PTL 2]JP 2011-519959 A

Non-patent literature

[NPL1]Patrick et al.，Nature Reviews Drug Discovery，Vol.17，p.509-527，2018

[NPL2]Miguel Gaspar et al，Cancer Immunol Res.2020Jun；8(6)：781-793

Disclosure of Invention

Technical problem

It is an object of the present invention to provide strategies to overcome systemic toxicity caused by treatment that target one or more antigens expressed in affected tissues such as cancer and conditionally modulate cellular functions in the unique environment (e.g., TME) in the affected tissues. Furthermore, it is an object of the present invention to provide a molecule that specifically works in the tissue sought to be treated or targeted (e.g. affected tissue, abnormal tissue, etc.), but does not work or works less in healthy or normal tissue, which is capable of providing a molecule that can be used in a drug with reduced side effects while imparting a significant therapeutic or prophylactic effect.

In particular, it is an object of the present invention to provide antigen binding molecules that are capable of eliciting an immune response in diseased tissue, but not or less in healthy or normal tissue.

Solution to the problem

The present invention provides a molecule that binds to a component of a cell or a portion thereof (e.g., a filament or histone forming the cytoskeleton or nuclear framework) exposed to an extracellular environment as a result of cell death, the component of the cell or portion thereof being specifically observed in an affected tissue (e.g., TME), the molecule simultaneously binding to a target molecule (e.g., a membrane protein) on or within a cell (e.g., an immune cell, an affected cell such as a tumor cell, an autoreactive cell, and a virus-infected cell), the molecule specifically functioning in a tissue in which cell death is observed, such as an affected tissue, an abnormal tissue, and the like. More specifically, a molecule is provided that is capable of cross-linking between a component of a cell or portion thereof that is exposed to an extracellular environment as a result of cell death and a target molecule on or within a cell, such as an immune cell or an affected cell (e.g., cancer cells, autoreactive cells, and virus-infected cells), and imparting signaling or blocking to cells in a tissue. More specifically, by increasing cross-linking between a component of a cell or a portion thereof exposed to the extracellular environment due to cell death and a target molecule on or within a cell, such as an immune cell or an affected cell, by a plurality of molecules, it is achieved that increased signaling or increased signal blocking is conferred into the cell by the target molecule.

In particular, as outlined in the specification and below and in more detail in the examples, the present invention provides antigen binding molecules that bind to antigens found extracellularly, particularly in the extracellular space of diseased tissue such as cancer tissue and tissue affected by autoimmune disease. Without being bound by any theory, it is believed that these antigens are present extracellularly due to cellular damage and destruction that occurs in these diseased tissues. By being able to bind to these antigens, the antigen binding molecules of the invention elicit an immune response specific to the diseased tissue.

In particular, the following embodiments are part of the present invention, wherein the reader will understand that the specific embodiments included in the following are independently intended as specific embodiments for each general embodiment set forth below:

further, the present disclosure provides the following embodiments.

[A1] A molecule comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen,

wherein the first antigen is a component or portion thereof constituting a cytoskeleton, a cell membrane or an organelle, or a cytoplasmic protein or a metabolite, exposed to an extracellular environment due to cell death, and

Wherein the second antigen is a different antigen than the first antigen.

[A2] The molecule of [ A1], wherein the cell death is cell death in the affected tissue.

[A3] The molecule of [ A1] or [ A2], wherein the first antigen is a filament, a histone or a nucleosome comprising a histone, which forms a cytoskeleton or a nuclear scaffold.

[A4] The molecule of [ A3], wherein the first antigen is a mid-wire.

[A5] The molecule of [ A4], wherein the first antigen is F-actin.

[A6] The molecule of any one of [ A1] to [ A5], wherein the first antigen is an antigen formed by multimerization of a plurality of molecules.

[A7] The molecule of [ A1] or [ A2], wherein the first antigen is phosphatidylserine constituting a cell membrane.

[A8] The molecule of any one of [ A1] to [ A7], wherein the cell death is necrosis, apoptosis, autophagy cell death or unexpected cell death.

[A9] The molecule of any one of [ A1] to [ A8], wherein the second antigen is a membrane protein involved in signal transmission in a cell.

[A10] The molecule of [ A9], wherein the cell is an immune cell.

[A11] [ A10] wherein the immune cell is a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil, or a basophil.

[A12] The molecule of [ A9], wherein the cell is a tumor cell or a autoreactive cell.

[A13] The molecule of any one of [ A9] to [ a12], wherein a complex formed by binding one or more molecules to the first antigen is bound to one or more membrane proteins via the second moiety to confer signaling or blocking in the cell via the membrane proteins.

[A14] The molecule of [ A13], wherein the molecule imparts signal transduction through a membrane protein in a cell.

[A15] The molecule according to [ A13], wherein the molecule imparts signal blocking via a membrane protein in the cell.

[A16] The molecule of any one of [ A9] to [ a15], wherein the membrane protein is a membrane protein in a cell membrane or a membrane protein of an endosome.

[A17] The molecule of any one of [ a11] to [ a16], wherein the membrane protein is:

a membrane protein in the cell membrane of a T cell, said membrane protein selected from the group consisting of a T cell receptor, CD3, a co-stimulatory molecule and a co-inhibitory molecule; or (b)

A membrane protein in the cell membrane of a cell other than a T cell that binds to a costimulatory or cosuppression molecule in the T cell membrane.

[A18] [ A17] wherein the costimulatory molecule is CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR), tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1) or CD244 (2B 4).

[A19] [ A17] wherein the co-inhibitory molecule is CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R or CD200R.

[A20] The molecule of [ A17], wherein the membrane protein bound to the costimulatory molecule or the co-inhibitory molecule is CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN 1), VISTA, HHA 2, a member of the milk philin (butyl philin) family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX 40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL 5), CD112 (NECTIN-2), CD200 (MOX 1), CD48 (BCM-1) or Gal-9.

[A21] The molecule of any one of [ A1] to [ a20], wherein the first moiety is an antibody variable region or a single domain antibody (VHH).

[A22] The molecule of any one of [ A1] to [ a20], wherein the first antigen is F-actin and the first moiety is a molecule that binds to the F-actin or a portion thereof.

[A23] [ A22] wherein the molecule that binds to F-actin or a portion thereof is the extracellular domain of Clec9A or a portion thereof.

[A24] The molecule of any one of [ A9] to [ a23], wherein the second moiety is an antibody variable region or a single domain antibody (VHH), or a ligand.

[A25] The molecule of any one of [ A1] to [ A9], wherein:

the first antigen is F-actin;

the second antigen is a membrane protein involved in signal transmission in a cell;

the second moiety is one or two Fab regions comprising two Fab regions of an antibody variable region, the second moiety binding to a second antigen; and

the first moiety is a molecule that binds to F-actin or a portion thereof, linked by a linker or directly to the C-terminus of the Fc region linked to the variable region of the antibody.

[A26] The molecule of [ A25], wherein the cell is an immune cell.

[A27] The molecule of [ A26], wherein the immune cell is a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil, or a basophil.

[A28] The molecule of [ A25], wherein the cell is a tumor cell or a autoreactive cell.

[A29] The molecule of any one of [ a25] to [ a28], wherein a complex formed by binding one or more molecules to F-actin binds to one or more membrane proteins via the second moiety to confer signal transduction or blocking by the membrane proteins in the cell.

[A30] The molecule according to [ A29], wherein the molecule imparts a signal transmission through a membrane protein in a cell.

[A31] The molecule according to [ A29], wherein the molecule imparts signal blocking via a membrane protein in the cell.

[A32] The molecule of any one of [ a25] to [ a31], wherein the membrane protein is a membrane protein in a cell membrane or a membrane protein of an endosome.

[A33] The molecule of any one of [ a27] to [ a32], wherein the membrane protein is:

[A34] [ A33] wherein the costimulatory molecule is CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR). Tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1) or CD244 (2B 4).

[A35] [ A33] wherein the co-inhibitory molecule is CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R or CD200R.

[A36] [ A33] wherein the membrane protein bound to the costimulatory molecule or the co-inhibitory molecule is CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN 1), VISTA, HHA 2, a member of the milk philin (butyl philin) family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX 40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL 5), CD112 (NECTIN-2), CD200 (MOX 1), CD48 (BCM-1) or Gal-9.

[A37] The molecule of any one of [ a25] to [ a36], wherein the first moiety is an antibody variable region or a single domain antibody (VHH).

[A38] The molecule of any one of [ a25] to [ a37], wherein the molecule that binds to F-actin or a portion thereof is an extracellular region of Clec9A or a portion thereof.

[A39] The molecule of any one of [ a25] to [ a38], wherein the second moiety is an antibody variable region or a single domain antibody (VHH), or a ligand.

[A40] The molecule of any one of [ A1] to [ a20], wherein a compound comprising the first moiety and the second moiety.

[A41] The molecule of [ A40], wherein the compound is a compound that binds the first antigen and the second antigen when irradiated with light.

[B1] A pharmaceutical composition comprising a molecule of any one of [ A1] to [ a41 ].

[B2] The pharmaceutical composition of [ B1] for imparting signal transmission or blocking in a cell.

[B3] The pharmaceutical composition of [ B1] or [ B2] for imparting signaling or blocking in immune cells.

[B4] The pharmaceutical composition of any one of [ B1] to [ B3], for activating or preventing immunity.

[B5] The pharmaceutical composition of any one of [ B1] to [ B4], for use in the treatment or prevention of cancer or autoimmune disease.

[B6] The molecule of any one of [ A1] to [ A41], for use as a medicament.

[B7] The molecule of [ B6] for imparting signal transmission or blocking in a cell.

[B8] The molecule of [ B6] or [ B7] for conferring signal transmission or blocking in an immune cell.

[B9] The molecule of any one of [ B6] to [ B8], for activating or preventing immunity.

[B10] The molecule of any one of [ B5] to [ B9], for use in the treatment or prevention of cancer or autoimmune disease.

[B11] Use of a molecule according to any one of [ A1] to [ a41] in the manufacture of a medicament.

[B12] The use of [ B11], wherein the medicament is a medicament for imparting signal transmission or blocking in a cell.

[B13] The use of [ B11] or [ B12], wherein the medicament is a medicament for conferring signal transmission or blocking in an immune cell.

[B14] The use of any one of [ B11] to [ B13], wherein the medicament is a medicament for activating or preventing immunity.

[B15] The use of any one of [ B11] to [ B14], wherein the medicament is a medicament for treating or preventing cancer or autoimmune disease.

[B16] A method of conferring signaling or blocking in a cell comprising administering to a subject the molecule of any one of [ A1] to [ a41 ].

[B17] A method of activating or preventing immunity comprising administering to a subject the molecule of any one of [ A1] to [ a41 ].

[B18] The method of any one of [ B15] to [ B17], wherein the cell is an immune cell, a cancer cell, or an autoreactive cell.

[B19] A method of treating or preventing cancer or an autoimmune disease comprising administering to a subject the molecule of any one of [ A1] to [ a41 ].

[B20] A method for producing the molecule of any one of [ A1] to [ A39 ].

[B21] A polynucleotide encoding the molecule of any one of [ A1] to [ a39 ].

[B22] A polynucleotide, in particular DNA, encoding a molecule according to any one of [ A1] to [ A39 ].

[B23] A vector comprising the polynucleotide of [ B22 ].

[B24] A vector comprising a DNA sequence encoding an antigen binding molecule as defined in any one of [ A1] to [ a39], wherein the vector is particularly useful for recombinant protein expression.

[B25] A host cell retaining the vector of [ B23] or [ B24 ].

In addition, the present disclosure also provides, inter alia, the following embodiments [ C1] to [ C15]:

[C1] a molecule comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen,

Wherein the second antigen is a different antigen than the first antigen.

[C2] [ C1] wherein the cell death is cell death in the affected tissue.

[C3] The molecule of [ C1] or [ C2], wherein the first antigen is a filament, a histone or a nucleosome comprising a histone, which forms a cytoskeleton or a nuclear scaffold.

[C4] [ C3] wherein the first antigen is a mid-wire.

[C5] [ C4] wherein the first antigen is F-actin.

[C6] The molecule of any one of [ C1] to [ C5], wherein the first antigen is an antigen formed by multimerization of a plurality of molecules.

[C7] The molecule of any one of [ C1] to [ C6], wherein the cell death is necrosis, apoptosis, autophagy cell death, or unexpected cell death.

[C8] The molecule of any one of [ C1] to [ C7], wherein the second antigen is a membrane protein involved in signal transmission in a cell.

[C9] [ C8] wherein the cell is an immune cell.

[C10] The molecule of any one of [ C1] to [ C9], wherein the first moiety is an antibody variable region or a single domain antibody (VHH).

[C11] The molecule of any one of [ C1] to [ C9], wherein the first antigen is F-actin and the first moiety is a molecule that binds to the F-actin or a portion thereof.

[C12] The molecule of any one of [ C1] to [ C9], wherein:

the first antigen is F-actin;

the second moiety is one or two Fab regions comprising two Fab regions of an antibody variable region, the second moiety binding to the second antigen; and

[C13] A pharmaceutical composition comprising a molecule of any one of [ C1] to [ C12 ].

[C14] Use of a molecule according to any one of [ C1] to [ C12] in the manufacture of a medicament.

[C15] A method for producing the molecule of any one of [ C1] to [ C12 ].

In particular, the present disclosure still further provides the following embodiments [ D1] to [ D44]:

[D1] an antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a damage-associated molecular pattern (DAMP), and

wherein the second antigen is a different antigen than the first antigen.

[ D1.1] antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a tissue-derived antigen specific for a tissue state, wherein in particular the antigen is exposed to an extracellular environment, such as a damage-associated molecular pattern (DAMP), and

wherein the second antigen is different from the first antigen.

[D2] The antigen binding molecule of [ D1] or [ D1.1], wherein the second antigen is a membrane protein of an immune cell.

[D3] The antigen binding molecule of any one of [ D1] and [ D2], wherein the molecular pattern (DAMP) is exposed to an extracellular environment due to cell damage, in particular due to cell death.

[D4] The antigen binding molecule of any one of [ D1] to [ D3], wherein the molecule

A) Capable of providing a cell contact and/or signal due to said cell damage, wherein said contact and/or signal is related to said second antigen; and/or

B) The first portion comprising an Fc terminus of an antigen binding molecule; and/or

C) The antigen binding region comprised in the antigen binding molecule is preferably F (ab') ₂ Said second portion in the zone; and/or

D) Comprising at least one first moiety and at least one second moiety, in particular wherein the antigen binding molecule comprises one first moiety and one second moiety, more in particular wherein the antigen binding molecule comprises at least two, preferably two, first moieties and at least two, preferably two, second moieties; and/or

E) Wherein the compound is a compound capable of binding the first antigen and the second antigen upon light irradiation.

[D5] The antigen binding molecule of any one of [ D1] to [ D4], wherein the cell is damaged

i) Involving cell death, in particular cell death, and/or

ii) in affected tissues, in particular selected from tissues affected by a disorder or disease, in particular by cancer or autoimmune diseases, in particular in the Tumor Microenvironment (TME); and/or

iii) Caused by any one selected from the group consisting of necrosis, apoptosis, autophagy cell death, and unexpected cell death, and/or

iv) is cell death selected from the group consisting of necrosis, apoptosis, autophagic cell death, and unexpected cell death.

[D6] The antigen binding molecule of any one of [ D1] to [ D5], wherein the first antigen

i) Selected from the group consisting of a cytoskeletal component or part thereof, a cell membrane component or part thereof, an organelle component or part thereof, a cytoplasmic protein or part thereof, and a metabolite; and/or

ii) on any one selected from the group consisting of a filar (in particular an intermediate filar), a nucleosome, a cytoskeletal and/or a nuclear scaffold; and/or

iii) Capable of multimerizing a plurality of molecules; and/or

iv) in the presence of a multimeric molecule,

in particular wherein the first antigen is

A cytoskeletal component or part thereof, a cell membrane component or part thereof, an organelle component or part thereof, a cytoplasmic protein or part thereof, and a metabolite; and/or

ii) is located on any one selected from the group consisting of filaments (in particular intermediate filaments), nucleosomes, cytoskeleton and/or nuclear scaffold.

[D7] The antigen binding molecule of any one of [ D1] to [ D6], wherein the first antigen is selected from the group consisting of:

a) Actin, in particular F-actin; or (b)

B) Heat shock proteins, in particular selected from HSP90 and GRP78, in particular HSP90;

c) Phosphatidylserine (PS), particularly phosphatidylserine that is part of the cell membrane;

d) A histone or a component thereof,

e) Histone deacetylase complex subunits, in particular SAP130,

f) HMGN proteins, in particular selected from HMGB1 and HMGN1,

g) A growth factor derived from a cancer of the liver cells,

H)BCL-2；

i) Calreticulin, and

j) Cyclophilin a;

in particular wherein the first antigen is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90 and SAP130,

In particular wherein the first antigen is selected from the group consisting of F-actin, phosphatidylserine and HSP90,

in particular wherein the first antigen is selected from the group consisting of F-actin and HSP90,

preferably wherein the first antigen is F-actin.

[D8] The antigen binding molecule of any one of [ D1] to [ D7], wherein the first moiety is selected from the group consisting of:

a) An Ig-type binding moiety; or (b)

B) Binding polypeptides, particularly wherein the binding polypeptide is a non-Ig-type binding moiety.

[D9] The antigen binding molecule of any one of [ D1] to [ D8], wherein the first moiety is selected from the group consisting of:

a) A moiety capable of binding to actin, in particular F-actin;

b) A moiety capable of binding to a heat shock protein selected in particular from HSP90 and GRP 78;

c) A moiety capable of binding to Phosphatidylserine (PS), in particular phosphatidylserine as part of a cell membrane;

d) A moiety capable of binding to histone or a component thereof,

e) A moiety capable of binding to a histone deacetylase complex subunit, in particular SAP130,

f) A moiety capable of binding to an HMGN protein selected in particular from HMGB1 and HMGN1,

g) A moiety capable of binding to a hepatocellular carcinoma-derived growth factor,

H) A moiety capable of binding to BCL-2,

i) A moiety capable of binding to calreticulin, and

j) A moiety capable of binding to cyclophilin a;

in particular wherein said first part is selected from the group consisting of: a moiety capable of binding F-actin, a moiety capable of binding GRP78, a moiety capable of binding phosphatidylserine, a moiety capable of binding HSP90, and a moiety capable of binding SAP130,

in particular wherein said first part is selected from the group consisting of: a moiety capable of binding F-actin, a moiety capable of binding phosphatidylserine, and a moiety capable of binding HSP90,

in particular wherein the first moiety is selected from the group consisting of a moiety capable of binding F-actin and a moiety capable of binding HSP90,

preferably wherein the first moiety is a moiety capable of binding F-actin.

[D10] [ D9] the antigen binding molecule, wherein

A) The moiety capable of binding F-actin is a binding polypeptide, preferably a Clec9A polypeptide, and/or

B) The moiety capable of binding GRP78 is an Ig-type binding moiety, preferably from antibody GA20 or Mab159, and/or

The moiety C capable of binding phosphatidylserine is an Ig-type binding moiety, preferably from antibody 3G4, and/or

D) The moiety capable of binding HSP90 is an Ig-type binding moiety, preferably from antibody 1.5.1 or 6H8, and/or

E) The moiety capable of binding SAP130 is a binding polypeptide, preferably a mClec4e polypeptide;

in particular wherein the Ig type binding moiety is selected from the group consisting of: single domain antibodies (VHH), combinations of antibody heavy chain Variable (VH) and antibody light chain Variable (VL) regions, single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ ，

In particular wherein the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ A group of groups.

[D11] The antigen binding molecule of any one of [ D1] to [ D9], wherein

A) In the case where the first antigen is actin, preferably F-actin, the first moiety is the Clec9A polypeptide, or

B) In the case where the first antigen is GRP78, phosphatidylserine or HSP90, the first moiety is an Ig-type binding moiety, or

C) In the case where the first antigen is SAP130, the first moiety is a mClec4e polypeptide.

[D12] The antigen binding molecule of any one of [ D1] to [ D11], wherein in the case where the first antigen is actin or SAP130, the first binding moiety is an Ig-type binding moiety.

[D13] The antigen binding molecule of any one of [ D1] to [ D12], wherein the second antigen is a membrane protein of an immune cell, in particular a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a co-stimulatory molecule, or a co-inhibitory molecule;

in particular wherein the second antigen is a membrane protein involved in signal transmission in a cell, preferably a signaling receptor.

[D14] The antigen binding molecule of any one of [ D1] to [ D13], wherein the second moiety is an Ig-type binding moiety.

[D15] The antigen binding molecule of any one of [ D1] to [ D14], wherein the second antigen

A) Involved in signaling in cells;

Especially

a-1) wherein the second antigen is a membrane protein, and/or

A-2) wherein the second antigen is a signaling receptor, and/or

A-3) wherein the cells are i) immune cells, in particular wherein the immune cells are selected from the group consisting of T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophils and basophils, ii) tumor cells, or iii) autoreactive cells; and/or

B) Is an immune cell receptor antigen.

[D16] The antigen binding molecule of any one of [ D1] to [ D15], wherein the second antigen is selected from the group consisting of:

a) The membrane protein of the endosome,

b) Membrane proteins in the cell membrane of T cells,

in particular a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, costimulatory molecule or costibiter molecule,

in particular wherein the membrane protein capable of binding to a costimulatory or cosuppression molecule is selected from the group consisting of CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN 1), VISTA, HHA 2, a member of the milk-philin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX 40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL 5), CD112 (NECTIN-2), CD200 (MOX 1), CD48 (BCM-1) and Gal-9;

c) Membrane proteins in the cell membrane of cells other than T cells, in particular membrane proteins capable of binding to costimulatory molecules or costibiting molecules in the cell membrane of T cells,

in particular, wherein the co-stimulatory molecule is selected from the group consisting of CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR), tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1) and CD244 (2B 4), and/or wherein the co-stimulatory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R or CD200R, and/or

In particular, wherein the second antigen is selected from the group consisting of CD3, CD137, CD40 and CTLA4,

in particular, wherein the second antigen is selected from the group consisting of CD3 and CD137,

preferably, wherein the second antigen is CD3.

[D17] The antigen binding molecule of any one of [ D1] to [ D16], wherein the second moiety is selected from the group consisting of:

a) A moiety capable of binding to a membrane protein of an endosome,

b) A moiety capable of binding to a membrane protein in the cell membrane of a T cell,

in particular, a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, co-stimulatory molecule or co-inhibitory molecule,

in particular, wherein the membrane protein capable of binding to a costimulatory or co-inhibitory molecule is selected from the group consisting of CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN 1), VISTA, HHA 2, a member of the milk philin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX 40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL 5), CD112 (NECTIN-2), CD200 (MOX 1), CD48 (BCM-1) and Gal-9; and

c) A moiety capable of binding to a membrane protein in the cell membrane of a cell other than a T cell, in particular a membrane protein capable of binding to a costimulatory molecule or a costibiting molecule in the cell membrane of a T cell,

In particular, wherein the co-stimulatory molecule is selected from the group consisting of CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR), tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1) and CD244 (2B 4), and/or wherein the co-inhibitory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R or CD200R,

in particular, wherein the second part is selected from the group consisting of: a moiety capable of binding to CD3, a moiety capable of binding to CD137, a moiety capable of binding to CD40, and a moiety capable of binding to CTLA 4.

In particular, wherein the second moiety is selected from the group consisting of a moiety capable of binding CD3 and a moiety capable of binding CD 137.

Preferably, wherein the second moiety is a moiety capable of binding CD 3.

[D18] [ D17] an antigen binding molecule, wherein

A) The moiety capable of binding CD3 is an Ig-type binding moiety, and/or

B) The moiety capable of binding CD137 is an Ig-type binding moiety, and/or

C) The moiety capable of binding CD40 is an Ig-type binding moiety, and/or

D) The moiety capable of binding CTLA4 is an Ig-type binding moiety;

in particular, wherein the Ig-type binding moiety is selected from the group consisting of: single domain antibodies (VHH), combinations of antibody heavy chain Variable (VH) and antibody light chain Variable (VL) regions, single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ ，

In particular, wherein the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ A group of groups.

[D19] The antigen binding molecule of any one of [ D1] to [ D18], wherein the molecule is characterized in that a complex formed by binding one or more molecules to the first antigen is capable of binding to more than one membrane protein via the second moiety,

in particular, wherein

i) The complex formed by binding of one or more molecules to the first antigen can bind to one or more membrane proteins via the second moiety, thereby conferring signaling through the one or more membrane proteins in the cell, or

ii) a complex formed by binding one or more molecules to the first antigen is capable of binding to one or more membrane proteins via the second moiety, thereby conferring signal blocking in the cell via said one or more membrane proteins.

[D20] The antigen binding molecule of any one of [ D1] to [ D19], wherein the molecule is selected from the group consisting of:

a) A polypeptide complex, optionally a fusion protein,

b) A polynucleotide (preferably consisting of two entities, preferably linked by a linker), and

c) A medium-sized chemical compound (preferably consisting of two entities, preferably linked by a linker),

D) Chemical compounds of small size (preferably consisting of two entities, preferably linked by a linker), in particular wherein

A-1) the molecule is a polypeptide complex,

in particular, wherein the molecule is an antibody or antibody fragment thereof,

in particular, wherein the molecule is an antibody,

preferably, wherein the antibody is an IgG-type antibody and/or a bispecific antibody;

or (b)

A-2) the molecule is a polypeptide complex, which is a fusion protein,

in particular, wherein the molecule is an antibody, which is a fusion protein, or an antibody fragment thereof,

in particular, wherein the molecule is an antibody, which is a fusion protein,

preferably, wherein the antibody is a) an IgG-type antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain and/or b) a bispecific antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain.

[D21] The antigen binding molecule of any one of [ D1] to [ D20], wherein the molecule is an antibody.

[D22] The antigen binding molecule of [ D20] or [ D21], wherein the molecule is an antibody of the IgG type.

[D23] The antigen binding molecule of any one of [ D20] to [ D22], wherein the antibody is capable of binding to a membrane protein of an immune cell, and further comprising at least one binding polypeptide, preferably linked to an Fc region,

In particular, wherein the binding polypeptide is selected from the group consisting of Clec9A polypeptide and mClec4e polypeptide;

in particular, wherein the antibody is selected from the group consisting of:

an anti-CD 3 antibody comprising a Clec9A polypeptide,

an anti-CD 137 antibody comprising a Clec9A polypeptide,

an anti-CD 3 antibody comprising a mClec4e polypeptide, and

an anti-CD 137 antibody comprising a mClec4e polypeptide.

[D24] The antigen binding molecule of any one of [ D20] to [ D23], wherein the antibody is a bispecific antibody,

in particular, wherein the antigen binding molecules are membrane proteins directed against immune cells and bispecific antibodies directed against DAMP,

in particular, wherein the antibody is selected from the group consisting of:

bispecific anti-HSP 90 anti-CD 3 antibodies,

bispecific anti-HSP 90 anti-CD 3 antibodies;

bispecific anti-GRP 78 anti-CD 3 antibodies,

bispecific anti-GRP 78 anti-CD 137 antibody,

bispecific anti-phosphatidylserine anti-CD 3 antibodies, and

bispecific anti-phosphatidylserine anti-CD 137 antibodies.

[D25] The antigen binding molecule of any one of [ D1] to [ D24], wherein

i-1) said first moiety is an Ig-type binding moiety, or

i-2) wherein said first moiety is a binding polypeptide,

and/or

ii) the second moiety is an Ig-type binding moiety;

In particular, wherein

a) The first moiety is an Ig-type binding moiety selected from the group consisting of: single domain antibodies (VHH), combinations of antibody heavy chain Variable (VH) and antibody light chain Variable (VL) regions, single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ The method comprises the steps of carrying out a first treatment on the surface of the And/or

b) The second moiety is an Ig-type binding moiety selected from the group consisting of: single domain antibodies (VHH), combinations of antibody heavy chain Variable (VH) and antibody light chain Variable (VL) regions, single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ The method comprises the steps of carrying out a first treatment on the surface of the And/or

c) The binding polypeptide is a non-Ig-type binding moiety;

preferably, wherein

The first moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ Binding domains of the group of compositions, and/or

b) said second moiety is selected from VHH, scFv, scFv, fab and F (ab') ₂ Binding domains of the group consisting; and/or

Wherein the binding polypeptide is a non-Ig-type binding moiety, in particular a non-Ig-type binding moiety linked to the Fc domain of the antigen binding molecule.

[D26] The antigen binding molecule of any one of [ D20] to [ D25], wherein the molecule is selected from the group consisting of:

A) An antibody capable of binding to F-actin and an antigen involved in signal transmission in a cell;

b) An antibody capable of binding to F-actin and an immune cell receptor; or (b)

C) An antibody capable of binding HSP90 and an antigen involved in signal transmission in a cell;

d) Antibodies capable of binding HSP90 and immune cell receptors; or (b)

E) An antibody capable of binding GRP78 and an antigen involved in signal transmission in a cell;

f) An antibody capable of binding GRP78 and an immune cell receptor; or (b)

G) An antibody capable of binding to phosphatidylserine and an antigen involved in signal transmission in a cell;

h) An antibody capable of binding phosphatidylserine and an immune cell receptor;

i) An antibody capable of binding SAP130 and an antigen involved in signal transmission in a cell; and

j) An antibody capable of binding SAP130 and an immune cell receptor,

preferably, wherein the molecule is selected from the group consisting of:

a-1) an antibody capable of binding F-actin and CD 3;

a-2) an antibody capable of binding F-actin and CD 137;

c-1) an antibody capable of binding HSP90 and CD 3;

c-2) an antibody capable of binding HSP90 and CD 137;

e-1) an antibody capable of binding GRP78 and CD 3;

e-2) an antibody capable of binding GRP78 and CD 137;

G-1) an antibody capable of binding phosphatidylserine and CD 3;

g-2) an antibody capable of binding phosphatidylserine and CD 137;

i-1) an antibody capable of binding SAP130 and CD 3; and

i-2) antibodies capable of binding SAP130 and CD137,

[D27] the antigen binding molecule of any one of [ D20] to [ D26], wherein the molecule is selected from the group consisting of:

a) An antibody comprising two first moieties capable of binding F-actin and two second moieties involved in signal transmission in a cell, in particular wherein the first moieties are binding polypeptides, preferably Clec9A polypeptides, preferably linked to an Fc domain of an antibody, in particular wherein the second moieties are Ig-type binding moieties;

b) An antibody comprising two first portions capable of binding F-actin and two second portions that are immune cell receptor antigens, in particular wherein the first portions are binding polypeptides, preferably Clec9A polypeptides, preferably linked to an Fc domain of an antibody, in particular wherein the second portions are Ig-type binding portions;

c) An antibody comprising two first moieties capable of binding HSP90 and two second moieties involved in signal transmission in a cell, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

D) An antibody comprising two first moieties capable of binding HSP90 and two second moieties that are immune cell receptor antigens, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

e) An antibody comprising two first moieties capable of binding GRP78 and two second moieties involved in signal transmission in a cell, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

f) An antibody comprising two first moieties capable of binding GRP78 and two second moieties that are immune cell receptor antigens, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

g) An antibody comprising two first moieties capable of binding phosphatidylserine and two second moieties involved in signal transmission in a cell, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

h) An antibody comprising two first moieties capable of binding phosphatidylserine and two second moieties that are immune cell receptor antigens, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

I) An antibody comprising two first moieties capable of binding SAP130 and two second moieties involved in signal transmission in a cell, in particular wherein the first moieties are binding polypeptides, preferably mClec4e polypeptides, preferably linked to an Fc domain of an antibody, in particular wherein the second moieties are Ig-type binding moieties;

j) An antibody comprising two first parts capable of binding SAP130 and two second parts being immune cell receptor antigens, in particular wherein the first parts are binding polypeptides, preferably mClec4e polypeptides, preferably linked to the Fc domain of the antibody, in particular wherein the second parts are Ig-type binding parts,

preferably, wherein the molecule is selected from the group consisting of:

a1 An antibody comprising two first moieties capable of binding to F-actin and two second moieties capable of binding to CD3, in particular wherein the first moieties are binding polypeptides, preferably Clec9A polypeptides, preferably linked to the Fc domain of the antibody, in particular wherein the second moieties are Ig-type binding moieties;

a2 An antibody comprising two first moieties capable of binding to F-actin and two second moieties capable of binding to CD137, in particular wherein the first moieties are binding polypeptides, preferably Clec9A polypeptides, preferably linked to the Fc domain of an antibody, in particular wherein the second moieties are Ig-type binding moieties;

C1 An antibody comprising two first moieties capable of binding HSP90 and two second moieties capable of binding CD3, in particular wherein the first moieties are Ig-type binding moieties, in particular wherein the second moieties are Ig-type binding moieties;

c2 An antibody comprising two first moieties capable of binding HSP90 and two second moieties capable of binding CD137, in particular wherein the first moieties are Ig-type binding moieties, in particular wherein the second moieties are Ig-type binding moieties;

e1 An antibody comprising two first moieties capable of binding GRP78 and two second moieties capable of binding CD3, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

e2 An antibody comprising two first moieties capable of binding GRP78 and two second moieties capable of binding CD137, particularly wherein the first moieties are Ig-type binding moieties, particularly wherein the second moieties are Ig-type binding moieties;

g1 An antibody comprising two first moieties capable of binding phosphatidylserine and two second moieties capable of binding CD3, in particular wherein the first moieties are Ig-type binding moieties, in particular wherein the second moieties are Ig-type binding moieties;

G2 An antibody comprising two first moieties capable of binding phosphatidylserine and two second moieties capable of binding CD137, in particular wherein the first moieties are Ig-type binding moieties, in particular wherein the second moieties are Ig-type binding moieties;

i1 An antibody comprising two first moieties capable of binding SAP130 and two second moieties capable of binding CD3, in particular wherein the first moieties are binding polypeptides, preferably mClec4e polypeptides, preferably linked to the Fc domain of the antibody, in particular wherein the second moieties are Ig-type binding moieties;

i2 An antibody comprising two first moieties capable of binding SAP130 and two second moieties capable of binding CD137, in particular wherein said first moieties are binding polypeptides, preferably mClec4e polypeptides, preferably linked to the Fc domain of the antibody, in particular wherein said second moieties are Ig-type binding moieties.

[D28] A pharmaceutical composition comprising an antigen binding molecule of any one of [ D1] to [ D27 ].

[D29] An antigen binding molecule or pharmaceutical composition of any one of [ D1] to [ D28], for use

i) Medicine, and/or

ii) methods of treating medical conditions, particularly medical conditions involving cell death, and/or

iii) A method of treating a medical condition in a subject, the method comprising contacting the antigen binding molecule with a damaged cell,

iv) a method of treating a medical condition in a subject, the method comprising contacting the antigen binding molecule with a damage-associated molecular pattern (DAMP) in a patient, in particular further comprising contacting the antigen binding molecule with the second antigen,

v) a method of treating a medical condition in a subject, the method comprising administering the antigen binding molecule to a patient suffering from a condition involving cell damage, particularly cell death;

in particular wherein the method comprises bringing a plurality of antigen binding molecules into close proximity to the first and second antigens.

[D30] An antigen binding molecule or pharmaceutical composition of any one of [ D1] to [ D29], for use in medicine.

[D31] An antigen binding molecule or pharmaceutical composition of any one of [ D1] to [ D27], for use in a method of treating or preventing a medical disorder.

[D32] The antigen binding molecule or pharmaceutical composition for use as described in [ D31], wherein the medical condition is selected from the group consisting of cancer and an immune disease, preferably an autoimmune disease.

[D33] The antigen binding molecule or pharmaceutical composition for use according to any one of [ D29] - [ D32], wherein

i) The method comprises administering to the patient an antigen binding molecule according to any one of [ D1] to [ D27], and/or

ii) the method is a method for activating or preventing an immune response, and/or

iii) The method is used to confer signaling or blocking in a cell;

in particular, wherein

a) The method comprising contacting an antigen binding molecule with a damage-associated molecular pattern (DAMP) that is exposed to an extracellular environment due to cellular damage, particularly wherein the DAMP is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP130,

and/or

b) The method comprising contacting the antigen binding molecule with a cell selected in particular from the group consisting of a-i) an immune cell, a-ii) a tumor cell and a-iii) an autoreactive cell, in particular wherein the immune cell is selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil and a basophil,

and/or

c) The antigen binding molecule comprises at least one, preferably two Clec9A polypeptides, in particular wherein the antigen binding molecule further comprises c-1) at least two, preferably two, moieties involved in signal transmission in a cell, or c-2) at least two, preferably two, moieties that are immune cell receptor antigens.

[D34] An antigen binding molecule or pharmaceutical composition for use according to any one of [ D18] and [ D19], wherein the method is a method of treating or preventing cancer or an immune disease, preferably an autoimmune disease,

in particular wherein the method is a method of treating or preventing cancer,

in particular, wherein the method is a method of treating cancer.

[D35] A polynucleotide encoding an antigen binding molecule according to any one of [ D1] to [ D27 ].

[D36] A polynucleotide, in particular DNA, encoding a molecule according to any one of [ D1] to [ D27 ].

[D37] A vector comprising the polynucleotide of [ D35] or [ D36 ].

[D38] A vector comprising a DNA sequence encoding an antigen binding molecule as defined in any one of [ D1] to [ D27], wherein the vector is particularly useful for recombinant protein expression.

[D39] A host cell comprising the vector of [ D37] or [ D38 ].

[D40] A host cell comprising the vector of [ D37] or [ D38], wherein the host cell is particularly useful for recombinant protein expression of an antigen binding molecule encoded in the vector.

[D41] Use of at least a first part and at least a second part for the preparation of an antigen binding molecule comprising said at least first part and said at least second part, wherein

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

wherein the first antigen is a damage-associated molecular pattern (DAMP), and wherein the second antigen is different from the first antigen, preferably wherein the second antigen is a membrane protein of an immune cell.

[D42] Use of an antigen binding molecule for targeting cells in a tissue in which cell death is observed or to be detected, wherein

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

[D43] Use of an antigen binding molecule to induce or block signal transduction in cells in tissue in which cell death is observed

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

[D44] A kit for producing a molecule that specifically affects a tissue in which cell death is observed, the molecule comprising:

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

wherein the first antigen is a damage-associated molecular pattern (DAMP), and wherein the second antigen is different from the first antigen, preferably wherein the second antigen is a membrane protein of an immune cell, wherein

The kit comprises:

at least a first container comprising at least a first portion, and

at least a second container comprising at least a second portion.

In addition, the present disclosure also provides, inter alia, the following embodiments [ E1] to [ E32]:

[E1] an antigen binding molecule comprising:

(1) A first moiety that binds to a first antigen, wherein the first antigen is selected from the group consisting of actin, heat shock protein, phosphatidylserine, histone deacetylase complex subunit, HMGN protein, hepatocellular carcinoma-derived growth factor, BCL-2, calreticulin, and cyclophilin a, and

(2) A second moiety that binds to a second antigen, wherein the second antigen is different from the first antigen and is selected from a membrane protein on a T cell or an Antigen Presenting Cell (APC).

[E2] [ E1] an antigen binding molecule, wherein the actin is F-actin.

[E3] [ E1] wherein the heat shock protein is HSP90, GRP78, HSP70, HSP60, HSP72 or GP96.

[E4] [ E3] wherein the heat shock protein is HSP90 or GRP78.

[E5] [ E1] an antigen binding molecule, wherein the phosphatidylserine is part of a cell membrane.

[E6] [ E1] an antigen binding molecule, wherein the histone deacetylase complex subunit is SAP130.

[E7] [ E1] an antigen binding molecule, wherein the HMGN protein is HMGB1 or HMGN1.

[E8] [ E1] an antigen binding molecule, wherein the first antigen is selected from the group consisting of F actin, GRP78, phosphatidylserine, HSP90, and SAP130.

[E9][E1]To [ E8]]The antigen binding molecule of any one of claims, wherein the first moiety is selected from the group consisting of an IgG-type antibody, VHH, VH, VL, sdAb, scFv, single chain antibody, fab, and F (ab') ₂ Ig type binding moieties of the group.

[E10] The antigen binding molecule of any one of [ E1] to [ E8], wherein the first moiety is a non-Ig-type binding moiety.

[E11] [ E1] wherein the first antigen is F-actin and the first moiety is a Clec9A polypeptide.

[E12] [ E1] wherein the first antigen is selected from the group consisting of GRP78, phosphatidylserine, and HSP90, and the first moiety is an Ig-type binding moiety.

[E13] [ E1] an antigen binding molecule, wherein the first antigen is SAP130 and the first moiety is a mClec4E polypeptide.

[E14] The antigen binding molecule of any one of [ E1] to [ E13], wherein the second antigen is selected from the group consisting of T cell receptor, CD3, CD137, CD40, and CTLA 4.

[E15] The antigen binding molecule of any one of [ E1] to [ E14], wherein the second moiety is an Ig-type binding moiety.

[E16] The antigen binding molecule of any one of [ E1] to [ E15], wherein the molecule is an IgG-type antibody comprising both the first and second portions.

[E17] [ E16] wherein the IgG-type antibody is capable of binding to a membrane protein of an immune cell and comprises at least one binding polypeptide.

[E18] [ E17] wherein the binding polypeptide is linked to the Fc region of an IgG type antibody.

[E19] An antigen binding molecule of [ E17] or [ E18], wherein the binding polypeptide comprises a Clec9A polypeptide or a mClec4E polypeptide.

[E20] [ E17] wherein the antibody of the IgG type is selected from the group consisting of:

an anti-CD 3 antibody comprising a Clec9A polypeptide,

an anti-CD 137 antibody comprising a Clec9A polypeptide,

an anti-CD 3 antibody comprising a mClec4e polypeptide, and

an anti-CD 137 antibody comprising a mClec4e polypeptide.

[E21] [ E16] wherein the IgG-type antibody is a bispecific antibody.

[E22] [ E21] an antigen binding molecule, wherein the bispecific antibody is directed against a membrane protein of an immune cell and against a DAMP.

[E23] [ E21] an antigen binding molecule, wherein the bispecific antibody is selected from the group consisting of

Bispecific anti-HSP 90 anti-CD 3 antibodies,

bispecific anti-HSP 90 anti-CD 3 antibodies,

bispecific anti-GRP 78 anti-CD 3 antibodies,

bispecific anti-GRP 78 anti-CD 137 antibody,

bispecific anti-phosphatidylserine anti-CD 3 antibodies, and

bispecific anti-phosphatidylserine anti-CD 137 antibodies.

[E24] A pharmaceutical composition comprising an antigen binding molecule of any one of [ E1] to [ E23 ].

[E25] An isolated polynucleotide encoding the antigen binding molecule of any one of [ E1] to [ E23 ].

[E26] A vector comprising the polynucleotide of [ E25 ].

[E27] A host cell comprising the polynucleotide of [ E25 ].

[E28] A method of treating a medical condition involving cell damage or cell death in a subject suffering from a medical condition involving cell damage or cell death, comprising administering to the subject an antigen binding molecule of any one of [ E1] to [ E23 ].

[E29] A method of treating cancer in a subject in need thereof, comprising administering to the subject an antigen binding molecule of any one of [ E1] to [ E23 ].

[E30] A method of treating an immune disorder in a subject in need thereof, comprising administering to the subject the antigen binding molecule of any one of [ E1] to [ E23 ].

[E31] The method of [ E30], wherein the immune disease is an autoimmune disease.

[E32] A method comprising culturing the host cell of [ E27] under conditions suitable for expression of the antigen binding molecule and optionally recovering the antigen binding molecule.

Drawings

FIG. 1 schematically depicts a diagram of the technical concept encompassed and realized by the present invention in the case where the molecule of the present invention is an antibody-like molecule comprising a moiety that binds to F-actin that is exposed to the extracellular environment due to cell death, and also schematically depicts the formation of a complex of the molecule with F-actin, and the complex binding to one or more membrane proteins in a cell by the molecule to confer signaling or blocking to the cell.

FIG. 2 is a diagram schematically depicting the technical features of one embodiment of the molecule of the invention, which is an antibody-like molecule comprising a moiety that binds to a component of a cell or a portion thereof that is exposed to an extracellular environment due to cell death.

FIG. 3 is a diagram schematically illustrating the technical features of various embodiments of the molecules of the present invention, which are antibody-like molecules comprising a moiety that binds to a component of a cell or a portion thereof that is exposed to an extracellular environment due to cell death. For example, a circle (white) represents a "first portion" that binds to a "first antigen". In a further example, where two first moieties (white circles) are conjugated, at least one of the moieties binds to a "first antigen" and the other may bind to any antigen other than the "first antigen" and the "second antigen". In another example, at least one Fab arm binds to a "second antigen", while another Fab arm can bind to any antigen other than the "first antigen" and the "second antigen". In a further example, at least one Fab arm binds to a "second antigen" and another Fab arm can bind to a "first antigen". The conjugated first moiety (white circle) may bind to any antigen other than the "first antigen" and the "second antigen".

FIG. 4 is a graph showing measurement results of binding of Clec9A conjugated antibodies to living cells and necrotic cells or components thereof, respectively.

FIG. 5 shows a graph of the results of measuring T cell activation by aggregating Clec9A conjugated anti-CD 3 antibodies onto T cells in the presence of dead cells.

FIG. 6 is a graph showing the measurement results of binding of anti-phosphatidylserine or anti-HSP 90 to living or dead cells.

FIG. 7 shows a graph of the results of measuring CD3+ T cell activation by aggregating anti-phosphatidylserine// CD3 or anti-HSP 90// CD3 bispecific antibodies onto T cells in the presence of living or dead cells.

FIG. 8 is a graph showing the results of measuring CD137+ T cell activation by aggregating Clec9A conjugated anti-CD 137 antibodies onto T cells in the presence of living or dead cells.

FIG. 9 is a graph showing the results of measuring CD137+ T cell activation by aggregating anti-phosphatidylserine// CD137 antibodies onto T cells in the presence of living or dead cells.

FIG. 10 is a graph showing the results of CD3 activation by Clec9A conjugated antibodies in the presence or absence of F-actin.

Detailed Description

The following definitions and detailed description are provided to aid in the understanding of the invention shown herein. In particular, these definitions and detailed descriptions are used to explain the embodiments of groups A, B, C and D above.

Cell death

Several types of cell death are reported and fall into two types, programmed Cell Death (PCD) and non-PCD (Cell Research volume, pages347-364, 2019;Cell Biol.Int.,2019Jun;43 (6): 582-592;Nature Reviews Cancer,Vol.12,p.860-875, 2012). PCD is tightly regulated by signaling cascades and molecular definition mechanisms. Apoptosis is the most common PCD and many non-apoptotic PCD such as pyrosis (pyrosis), netois, necroptosis (Necroptosis), etc. have been reported in recent years. On the other hand, non-PCD is a biologically uncontrollable process and necrosis is classified as non-PCD.

In the present invention, "cell death" refers to apoptosis, which is one of the embodiments of Programmed Cell Death (PCD), of any kind or type of cell, including but not limited to; necrosis; accidental cell death; or regulated cell death (e.g., apoptosis, regulated necrosis, autophagic cell death, necrotic apoptosis, iron death, or cell coke death), including but not limited to somatic cells (e.g., epithelial cells, fibroblasts, mesenchymal cells, bone cells, muscle cells, neural cells, blood cells, etc.), germ cells, or affected or abnormal cells (e.g., tumor cells, autoreactive cells). Such cells may be located in any tissue including, but not limited to, epithelial tissue, muscle tissue, neural tissue, connective tissue. Herein, necrosis, a non-programmed cell death caused by injury or inflammation, also results in increased cell death. In particular, in the present invention, "cell death" may be cell death observed in affected tissues due to, for example, cancer or autoimmune diseases.

Cell damage

Also, in the present invention, "cell damage" is not particularly limited, and refers to any cell damage that results in exposure of DAMP. It includes in particular all forms of cell death described above.

First antigen/injury-related molecular pattern (DAMP)

In the present invention, the term "first antigen" is also referred to herein as "damage-related molecular pattern" or "DAMP", respectively, and is used interchangeably herein with "substance exposed to the extracellular environment due to cell damage", referring to a biomolecule associated with cell damage, which may also be referred to herein as "damaged cell-derived molecule", or "damaged cell-derived substance", "damaged cell-related molecule", "damaged cell-related substance", and "danger-related molecular pattern", respectively, which may be used interchangeably herein. These biomolecules and substances associated with cell damage may be up-regulated in expression at the time of cell damage, expressed on the cell surface, modified, passively diffused or actively secreted out of the cell. These changes are positively correlated with the presence or increase of damaged cells or with the decrease of intact cells. In other words, in cells that go to cell damage, the components constituting the intracellular cytoskeleton, cell membranes, organelles, cytoplasmic proteins, or metabolites, or portions thereof, are exposed to the extracellular environment, and any of these is included in the cell damage-related substance or biomolecule.

In any event, it is well known to those skilled in the art that "damage related molecular patterns" ("DAMP") are "substances that are exposed to the extracellular environment due to cell damage".

In the present invention, the terms "damage-related molecular pattern", "DAMP" and "substance exposed to the extracellular environment due to cell damage" may be referred to as "components or parts of cytoskeleton, cell membrane, organelle, cytoplasmic protein or metabolite, respectively, exposed to the extracellular environment due to cell damage", and may also be interchangeably referred to as "first antigen". That is, in the present invention, unless otherwise differently defined, the term "first antigen" refers to a composition or portion including, but not limited to, "cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite that is exposed to the extracellular environment due to cell damage, respectively.

In particular embodiments, the DAMP is selected from the group consisting of: a) Actin, in particular F-actin; b) Heat shock proteins, in particular selected from HSP90 and GRP78, in particular HSP90; c) Phosphatidylserine (PS), particularly phosphatidylserine that is part of the cell membrane; d) Histone or a component thereof, E) histone deacetylase complex subunits, in particular SAP130, F) HMGN protein, in particular selected from HMGB1 and HMGN1, G) hepatocellular carcinoma-derived growth factor, H) BCL-2, i) calreticulin, and J) cyclophilin a.

In the present invention, cell damage preferably involves cell death. In a preferred embodiment of the invention, the cell damage is cell death. Thus, in a further general aspect of the invention, the following applies:

first antigen and substance exposed to extracellular environment due to cell death

In the present invention, the term "first antigen" (also referred to as "substance exposed to the extracellular environment due to cell death") refers to a biomolecule associated with cell death, which in the present invention is also referred to as "dead cell derived molecule" or "dead cell derived substance", "dead cell related molecule", "dead cell related substance" and "danger related molecular pattern", which are used interchangeably. These biomolecules and substances associated with cell death may be up-regulated in expression, expressed on the cell surface, modified, passively diffused or actively secreted out of the cell upon cell death. These changes are positively correlated with the presence or increase of dead cells or with the decrease of living cells. In other words, in cells that undergo cell death, components constituting cytoskeleton, cell membrane, organelles, cytoplasmic proteins, or intracellular metabolites, or portions thereof are exposed to the extracellular environment, and any of these is included in substances or biomolecules associated with cell death.

That is, the term "a substance exposed to an extracellular environment due to cell death" (or a substance exposed to an extracellular environment due to cell damage "," damage-related molecular pattern ", or" DAMP ") may be referred to as" a component constituting a cytoskeleton, a cell membrane, an organelle, a cytoplasmic protein, or a metabolite, or a portion thereof exposed to an extracellular environment due to cell death ", or may be interchangeably used as" a first antigen "(or as" a component or portion of a cytoskeleton, a cell membrane, an organelle, a cytoplasmic protein, or a metabolite, respectively exposed to an extracellular environment due to cell damage "). That is, in the present invention, unless otherwise defined in a different sense, the term "first antigen" means a composition comprising a cytoskeleton, a cell membrane, an organelle, a cytoplasmic protein, or a metabolite, or a portion thereof, including but not limited to "exposure to an extracellular environment due to cell death" or "exposure to a cytoskeleton, a cell membrane, an organelle, a cytoplasmic protein, or a portion of a metabolite due to cell damage".

In any event, in the present invention, all examples and embodiments directed to "dead cell derived molecules", respectively, and the like, are specifically included in the DAMP of the present invention.

In the present invention, the term "cytoskeleton" refers to a structure of filaments in the cytoplasm that produces the intracellular and extracellular movement of cells as well as the physical energy required to maintain cell morphology. In the present invention, "filaments" include, but are not limited to, intermediate filaments, actin filaments, myosin filaments, and keratin filaments. Eukaryotic cells have actin filaments, intermediate filaments, myosin filaments, keratin filaments and microtubules as cytoskeleton. Actin filaments consist of actin; the intermediate silk is composed of vimentin, glial fibrillary protein, neurofilament protein, laminin and the like; microtubules consist of alpha tubulin and beta tube tubulin. In the present invention, the "cytoskeleton" that is exposed to the extracellular environment due to cell death is not limited to the protein components that form the cytoskeleton described above, and these protein components may be conjugated with one or more of any other proteins.

Biofilms, such as cell membranes, are composed of lipid bilayers in which proteins have been buried. In the present invention, components of biological membranes such as cell membranes include, but are not limited to, lipids and proteins. Lipids include, but are not limited to, phospholipids, glycolipids, and sterols. Proteins include, but are not limited to, transmembrane proteins such as ion channels, G Protein Coupled Receptors (GPCRs), proton pumps, and the like, lipid-anchored proteins such as G proteins, and the like, and surface membrane proteins such as enzymes, hormones, and the like.

In general, in the present invention, "membrane proteins" refer to membrane proteins generally known to those skilled in the art as part of or interacting with biological membranes and include several broad classes depending on their location, including in particular integral membrane proteins which are permanent parts of the cell membrane and which can penetrate the membrane (transmembrane) or bind to one side or the other of the membrane (integral single-site) and peripheral membrane proteins which bind transiently to the cell membrane (e.g. by so-called anchors or by some type of non-covalent interaction or combination of non-covalent interactions). The term specifically includes any membrane protein described herein.

In the present invention, "cell membrane or organelle exposed to extracellular environment due to cell death" includes, but is not limited to, the above-mentioned phospholipids or proteins, any biological membrane constituting organelles such as nucleus, ribosome, endoplasmic reticulum, golgi apparatus, mitochondria, etc., and proteins buried in the biological membrane.

In the present invention, "substances/molecules exposed to the extracellular environment due to cell death" ("dead cell-derived molecules") and DAMP include, but are not limited to, the above components or portions thereof, and any substances or molecules exposed to the extracellular environment due to cell death may be used in the present invention. Furthermore, the dead cell-derived molecule may be a complex of one or more of the above components or portions thereof. The dead cell-derived molecule may be an intact form or a truncated form of the protein. As a preferred embodiment, dead cell derived molecules include, but are not limited to, cytoskeleton, organelles, and cytoplasmic proteins in the cell. As a preferred embodiment, the cell membrane and any fragments of the cell membrane are also included in the dead cell-derived molecules. In the present invention, more preferred embodiments of dead cell-derived molecules include, but are not limited to, filaments that make up the cytoskeleton or the cytoskeleton, as well as nucleosomes or nucleosome-forming molecules.

In the present invention, as a non-limiting embodiment, dead cell-derived molecules include molecules that are released from cells into the extracellular environment as a result of immunogenic cell death, i.e., injury-related molecular patterns (DAMP) released as a result of immunogenic cell death. DAMPs include molecules reported by Dmitri et al, nature Reviews Cancer, vol.12, p.860-875, 2012. In other words, all DAMP described in Dmitri et al are specifically included as embodiments of DAMP according to the present invention.

Further and somewhat repeatedly, in the present invention, specific embodiments of the "substance exposed to the extracellular environment due to cell death" or "component or portion thereof that constitutes the cytoskeleton, cell membrane, organelle, cytoplasmic protein, or metabolite that is exposed to the extracellular environment due to cell death" and "DAMP" include, but are not limited to, filaments or histones that form the cytoskeleton or nuclear skeleton (e.g., intermediate filaments such as F-actin), phosphatidylserine, heat shock proteins, and proteins, RNAs, DNAs, or metabolites that further produce the modified molecule or complex, or combinations thereof. Herein, the protein may be a full-length protein or a fragment thereof.

In addition, molecules such as glypican and cohesin can be expressed on the surface of living cells, but modified by the microenvironment enriched in dead cells, and these molecules are also included in dead cell derived molecules. Furthermore, under normal physiological conditions, biglycan, decorin and photoprotein glycans (1 umicans) can also be sequestered in the extracellular matrix, but they are released proteolytically as dead cells increase. These molecules are also included in the present invention as dead cell related molecules in the present invention.

The dead cell-derived molecule may also be a molecule that is released, exposed or abundantly expressed on the cell surface when cell death is induced. All cells, such as somatic and germ cells, can produce dead cell-derived molecules. A common feature of solid tumors is the presence of hypoxia and hypotrophic conditions derived from reduced blood flow. These hypoxic conditions and low nutritional status stress tumor cells and lead to massive cell death in solid tumors. In addition to tumors, cell death is commonly found in many types of autoimmune diseases, such as vitiligo, type I diabetes, and the like, as cell death is involved in the pathogenesis of autoimmune diseases. Vitiligo, for example, is an autoimmune disease caused by melanocyte destruction. It has been reported that, among vitiligo, there are various types of autoantibodies which recognize laminin A, tyrosinase-related protein 1, HSP90, HSP70, etc. (Hamid et al, pigmentary Disorders 2015, 2:6). These molecules are usually located in the cytosol or nucleus in a steady state. Because antibodies are raised against molecules that are located extracellular, these molecules must be released or exposed to the plasma membrane upon cell death.

In the present invention, the source of the dead cell-derived molecules is not limited to the sources described above. Any molecule or substance in or on the cell exposed to the extracellular environment in which cell death is observed can be a source of dead cell-derived molecules. Whether a molecule or substance is a dead cell-derived molecule can be confirmed by examining whether the molecule or substance is one that is exposed to the extracellular environment when it induces cell death.

Methods including, but not limited to, checking and confirming whether induction of cell death can be employed.

Methods for detecting dead cells or dead cell-derived molecules or substances may involve detecting one or more characteristics associated with dead cells, an increase in the process, or a decrease in the proportion of living cells. Such methods include, but are not particularly limited to, detecting cells that are positive for staining by a DNA binding dye that is impermeable to living cells, an amine reactive dye that is impermeable to living cells, measuring a decrease in enzymatic activity, such as lactate dehydrogenase, in the supernatant of dead cells, or intracellular ATP levels associated with metabolically active living cells. In addition, methods for detecting dead cells as reported by Riss can be used (Riss, cytotoxicity Assays: in Vitro Methods to Measure Dead Cells; may 1, 2019).

In the present invention, it is possible to determine or confirm whether a certain molecule is a dead cell-derived molecule by comparing a cell culture supernatant to which a cytotoxic drug such as mitoxantrone or mitomycin C is added with a cell culture supernatant that has not been treated with the drug. By performing proteomic and/or metabolomic analysis of the proteins and/or metabolites contained in each of the supernatant of the cell culture cultured in the presence of the cytotoxic drug and the supernatant of the cell culture without the drug added, the relatively large number of molecules detected in the supernatant of the cell culture with the cytotoxic drug added can be determined to be dead cell-derived molecules (i.e., substances exposed to the extracellular environment due to cell death). Here, the drug added to the cell culture is not limited to mitoxantrone or mitomycin C, and any drug may be used as long as it has cytotoxicity.

In the present invention, the method of inducing cell death is not limited to the above-described method using a cytotoxic drug. For example, cell death may be physically induced by freeze-thawing treatment of the cells.

Furthermore, in the present invention, it is possible to determine or confirm whether certain molecules or substances are dead cell-derived molecules or dead cell-derived substances, for example, by preparing cells treated or untreated with the above-described drugs, wherein the treatment with the drugs induces cell death, and measuring and comparing the degree of expression of one or more membrane proteins in each group of cells using flow cytometry. The membrane proteins found in drug-treated cells with increased expression relative to untreated cells were identified as molecules that could be used as dead cell-derived molecules.

In the present invention, dead cell-derived molecules, such as but not limited to RNA, DNA, filiform action, histones, phosphatidylserine or heat shock proteins, as described above, may be quantified using any quantification means and/or method known to those skilled in the art, e.g., column chromatography, mass spectrometry (the term being used interchangeably herein with mass spectrometry), ELISA, etc. Any other means and/or method of quantification may be used as long as the dead cell-derived molecules of the present invention can be quantified.

Can be combined with

In the context of the present invention, the terms "capable of binding" and "binding" are used interchangeably and denote the ability of a moiety to bind an antigen, in particular under physiological conditions, i.e. conditions found in animals, in particular humans. Specific exemplary methods for determining the capabilities are described below. As used herein, terms such as "ability to bind to an antigen" may also be designated as terms such as "binding affinity to an antigen". Furthermore, in the case of antibodies, the ability may also be described by using the term "antibodies against.

A first moiety that binds to a first antigen

In the present invention, a "first part" of a "molecule comprising a first part that binds to a first antigen and a second part that binds to a second antigen" is bound to a "first antigen" (as described above, which is also interchangeably referred to as "a substance exposed to the extracellular environment due to cell death", and "a component constituting a cytoskeleton, a cell membrane, an organelle, a cytoplasmic protein, or a metabolite, or a part thereof exposed to the extracellular environment due to cell death").

The "first portion" of a molecule of the invention may have any structure as long as it binds to the "first antigen" as described above. The structure of the "first portion" may include, but is not limited to, a polypeptide or portion thereof, or a small or medium chemical compound or portion thereof, or a polynucleotide or portion thereof. The polypeptide or portion thereof includes, but is not limited to, a cell membrane protein expressed on a cell (e.g., an immune cell such as a dendritic cell) or portion thereof (e.g., an extracellular domain, any unique domain thereof; antibodies (including, but not limited to, human antibodies, chimeric antibody antibodies, humanized antibodies, and VHH antibodies) or antigen binding domains (also known as portions, or fragments of antibodies). Antigen binding domains of antibodies include, but are not limited to, antibody heavy chain Variable (VH) regions, antibody light chain Variable (VL) regions (preferably a combination of antibody heavy chain Variable (VH) regions and antibody light chain Variable (VL) regions), single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab, and F (ab') ₂ 。

The polypeptide or part thereof may also be an antigen binding polypeptide, e.g. a module of the a domain called Avimer, having about 35 amino acids comprised in vivo cell membrane proteins (WO 2004/044011 and WO 2005/040229), an adnectin with a 10Fn3 domain derived as a protein binding domain from the glycoprotein fibronectin expressed on the cell membrane (WO 2002/032925); affibody with an IgG binding domain scaffold constituting a triple helix bundle consisting of 58 amino acids of protein A (WO 1995/001937); DARPins (designed ankyrin repeat protein), which is a molecular surface exposed region of Ankyrin Repeat (AR), each region having subunits of 33 amino acid residue structure folded into turns, two antiparallel helices and loops (WO 2002/020565); an anti-cargo protein (anticholin) having four loop regions linked to 8 antiparallel chains bent toward the central axis at one end of the barrel structure, a structure highly conserved in lipocalin molecules such as neutrophil gelatinase-associated cargo protein (NGAL) (WO 2003/029462); and recessed regions in the inner parallel sheet structure of the horseshoe fold consisting of repeated Leucine Rich Repeat (LRR) modules of Variable Lymphocyte Receptors (VLRs) of immunoglobulin-free structure seen in the acquired immune system of invertebrates such as lampreys or braille (WO 2008/016854).

One embodiment of the invention that belongs to the polypeptide or part thereof that "binds to the first part of the first antigen" as described above includes, but is not limited to, a cell membrane protein (e.g. receptor) or part thereof (e.g. extracellular domain, any unique domain thereof) expressed on a cell. One representative of the receptors or portions thereof that fall within the scope of the present invention that "bind to the first portion of the first antigen" includes, but is not limited to, scavenger receptors, toll-like receptors (TLRs), C-type lectins (CLECs) expressed on dendritic cells, such as Clec9A, NOD-like receptors (NLR) (J Biol chem.2014Dec 19;289 (51): 35237-45), and the like, or portions thereof. One of the preferred embodiments of the "first part binding to the first antigen" in the present invention is Clec9A, which is a C-type lectin expressed on Dendritic Cells (DCs) and which binds to the cytoskeletal forming the filar complex F-actin when the F-actin complex is exposed to the extracellular environment due to cell death. In the present invention, the "first portion that binds to the first antigen" in the present invention includes, but is not limited to, the whole polypeptide, an extracellular domain (ECD) having any amino acid length, a C-type lectin domain (CTLD), or any portion of Clec9A (GenBank: NP-001192292.1).

A second moiety that binds to a second antigen

In the present invention, a "second moiety" of a "molecule comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen" binds a "second antigen" different from the "first antigen" as described above. The "second antigen" may be any antigen as long as it is different from the first antigen. In the present invention, a "second antigen" is also referred to as a "target molecule" that is targeted by a "second moiety" comprising a first moiety that binds to a first antigen and a second moiety that binds to a second antigen. After targeting the "second antigen" by the "second portion" of the molecules of the invention, the target molecule, i.e. "second antigen", may lead to the desired outcome of the indicated disease, e.g. immune activation for combating infection or cancer, immune suppression for autoimmune diseases and/or Cytokine Release Syndrome (CRS), direct inhibition of cell growth of cancer, or promotion of cell regeneration after tissue injury.

In the present invention, one common embodiment of an immune activation target includes, but is not limited to, CD3 ("CD epsilon"), CD4, CD8, CD28, OX40, 4-1BB, and T Cell Receptor (TCR) expressed on T cells, which results in activation of cytotoxic or T helper functions to eliminate pathogens, infected or transformed cells. Target molecules (i.e., "secondary antigens") may also be expressed on Antigen Presenting Cells (APCs), including XCR1, clec9A, DEC-205, DCIR2, TLR3, TLR5, flt3 on Dendritic Cells (DCs). Activation of DCs may indirectly result in clearance of pathogens and cancer cells by initiating and activating adaptive immune cell types, including cytotoxic and helper T cells. One suitable example is the targeting of CD40 agonists to CD40 (ESMO Open.2019;4 (Suppl 3): e 000510) to more effectively activate cytotoxic T cells by enhancing antigen cross presentation and co-stimulatory capacity, thereby achieving an anti-tumor effect. Furthermore, a synergistic effect leading to a stronger priming capacity of antigen-treating immune cells can be achieved by parallel activation of polyinosinic acid: polycytidylic acid (poly-IC) sensor receptor TLR3 also expressed by DC (J Clin invest.2018;128 (10): 4387-4396). In other cases, inhibition of the target molecule may be desirable, e.g., antagonizing CTLA-4, PD-1, LAG-3, TIM-3, VISTA on T cells, to prevent inhibition of immune responses against tumor cells. The molecule to be targeted (i.e. "second antigen") may also be any molecule expressed on or in other immune cell types (e.g. natural killer cells, macrophages, neutrophils), and their corresponding activating or inhibiting receptors. The oncogenic receptor on the cancer cell or the immune escape molecule on the transformed or infected cell may also be targeted (i.e. "second antigen").

In the present invention, the "second antigen" as briefly described above includes, but is not limited to, membrane proteins expressed on cell membranes and/or endosomal membranes. One embodiment of a "second moiety" bound membrane protein (i.e., a second antigen) includes, but is not limited to, a membrane protein involved in signal transmission in a cell, expressed on the cell membrane and/or endosome of an immune cell, such as a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil, and a basophil. A further embodiment of the membrane protein (i.e., the second antigen) that binds to the "second moiety" includes, but is not limited to, a membrane protein expressed on tumor cells or autoreactive cells.

In the present invention, membrane proteins belonging to the above "second antigen" include, but are not limited to, co-stimulatory molecules such as CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR). Tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1) or CD244 (2B 4); or a co-inhibitory molecule such as CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R or CD200R; or a costimulatory or cosuppression molecule, such as CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN 1), VISTA, HHA 2, a member of the milk-philin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX 40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL 5), CD112 (NECTIN-2), CD200 (BCMX 1), CD48 (BCMX-1) or Gal-9.

As further embodiments of membrane proteins belonging to the above "second antigen", it includes, but is not limited to CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3 (e.g., CD3 epsilon), CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD29, CD30L, CD32, CD33 (p 67 protein), CD34, CD38, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD 62, CXCR3, CXCR 4; fibroblast Activation Protein (FAP), fas, cytokine receptors such as IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-10R, IL-12R, IL-15R, IL-17R, IL-18R, IL-20R or IL-31R; integrin families such as α1β1 (VLA-1), α2β1 (VLA-2), α3β1 (VLA-3), α4β1 (VLA-4), α5β1 (VLA-5), α6β1 (VLA-6), α7β1, α8β1, α9β1, α11bβ3 (GPIIb/IIIa), αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, αLβ2 (LFA-1), αMβ2 (Mac-1), αxβ2, αdβ2, α4β7; TNF-RI, TNF-RII, TNFRSF 10A (TRAIL R1Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID), TNFRSF10D (TRAIL R4DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPGOCIF, TR 1), TNFRSF12 (TWEAK FN 14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, hveA, LIGHT R, TR 2), TNFRSF16 (NGFR p75 NTR), TNFRSF17 (BCMA), TNFRSF18 (TNGITRAITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RETRICEL), TNSF 1A (TNRI) and TNFRRI 120, p 55-60), TNFRSF1B (TNF RIICD120B, p 75-80), TNFRSF26 (TNFRH 3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX 40ACT35, TXGP 1R), TNFRSF6 (Fas Apalpha-1, APT1, CD 95), TNFRSF6B (DcR 3M68, TR 6), TNFRSF7 (CD 27), TNFRSF8 (CD 30), TNFRSF9 (4-1 BBCD137, ILA), TNFRSF21 (DR 6), TNFRSF22 (DcTRAIL R2TNFRH 2), TNFRST23 (DcTRAIL R1TNFRH 1), TNFRSF25 (DR 3Apo-3, LARD, TR-3, TRAMP, WSL-1), TLR (Toll-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10.

In the present invention, "second antigen" also includes, but is not limited to, soluble proteins that can bind to the membrane proteins described above. In the present invention, soluble proteins belonging to the "second antigen" include, but are not limited to, cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IFN- β, IFN- γ, IL-18, IL21, IL-23 and IL-27.

In the present invention, soluble proteins belonging to the "second antigen" further include, but are not limited to, the following molecules or any soluble form of the molecule (e.g., truncated forms of membrane proteins): 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF 2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE-2, activin A, activin AB, activin B, activin C, activin RIA ALK-2, activin RIBALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressees, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha 1-antitrypsin, alpha V/beta 1 antagonists, ANG, ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av/B3 integrin, axl, B2M, B7-1, B7-2, B7-H, B lymphocyte stimulating factor (BlyS), BACE-1, bad, BAFF, BAFF-R, bag-1, BAK, bax, BCA-1, BCAM, bcl, BCMA, BDNF, B-ECGF, bFGF, BID, bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, osteogenic protein, BMP-4, BMP-2B, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8 a, OP-2), BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, B-NGF, BMP-4, BMP-2B, BMP-5, BMP-6Vgr-1, BMPR-8 (BRPR-6), BOK, bombesin, and a peptide, bone derived neurotrophic factor, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin A, cathepsin E, and cathepsin E cathepsin X/Z/P, CBL, CCI, CCK2, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2 cathepsins X/Z/P, CBL, CCI, CCK2, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26 CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CCR5, CCR1, CCR2, CCR10, CCR2, CCL, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, dcR3, DC-SIGN, decay accelerating factor, des (1-3) -IGF-I (brain IGF-1), dhh, digoxin, DNAM-1, dnase, dpp, DPPIV/CD26, dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOSEot, eosinophil chemokine 1 (eotaxin 1), ep, ephrin (ephrin) B2/Ephb4, ePO, ERCC, E-selectin, ET-1, factor IIa factor VII, factor VIIIc, factor IX, fibroblast, activated protein (FAP), fas, fcR1, FEN-1, ferritin, FGF-19, FGF-2, FGF3, FGF-8, FGFR-3, fibrin, FL, FLIP, flt-3, flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, gas6, GCP-2, GCSF, GD2, GD3, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (tubocurarine), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR- α1, GFR- α2, GFR- α3, gITR, glucagon, glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic Growth Factor (HGF), hep B gp120, heparanase, her2/neu (ErbB-2), her3 (ErbB-3), her4 (ErbB-4), herpes Simplex Virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA high molecular weight melanoma associated antigen (HMW-MAA), HIV gp120, HIVIIIB gp 120V3 loop, HLA-DR, HM1.24, HMFG PEM, HRG, hrk, human cardiac myosin, human Cytomegalovirus (HCMV), human Growth Hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, ig, igA receptor, igE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18R, IL-21, IL-23, IL-27, IL-12, IL-18, interferon-alpha, interferon-beta, interferon-gamma, inhibin, iNOS, insulin chain a, insulin chain B, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4 beta 1, integrin alpha 4 beta 7, integrin alpha 5 (alpha V), integrin alpha 5 beta 1, integrin alpha 5 beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1bp1, LBP, LDGF, LECT, and lefty, lewis-Y antigen, lewis-Y associated antigen, LFA-1, LFA-3, lfo, LIF, LIGHT, lipoprotein, LIX, LKN, lptn, L-selectin, LT-a, LT-B, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP-1, MMP-10, MMP-11, MMPP-12, MMP-2, MMPP-12, MMP-2, and the like, MMPP-13, MMPP-14, MMP-15, MMPP-2, MMPP-24, MMPP-3, MMP-7, MMP-8, MMP-9, MPIF, mpo, MSK, MSP, mucin (Muc 1), MUC18, a spurious inhibitory substance, mug, muSK, NAIP, NAP, NCAD, N-C adhesin, NCA 90, NCAM, enkephalinase (neprilysin), neurotrophin-3, -4, or-6, neurturin, nerve Growth Factor (NGF), NGFR, NGFbeta, nNOS, NO, NOS, npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p, p95, PADPR, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF, PGE, PGF, PGI2, PGJ2 PIN, PLA2, placental alkaline phosphatase (PLAP), plGF, PLP, PP, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate Specific Membrane Antigen (PSMA), PTEN, PTHrp, ptk, PTN, R, RANK, RANKL, RANTES, RANTE, relaxin A chain, relaxin B chain, renin, respiratory Syncytial Virus (RSV) F, RSV Fgp, ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor alpha/beta), tdT, TECK, TEM, TEM5, TEM7, TEM8, TERT, testicle PLAP-like alkaline phosphatase, tfR, TGF, TGF- α, TGF- β -generic specificity, TGF- βri (ALK-5), TGF- βrii, TGF- βriib, TGF- βriii, TGF- β1, TGF- β2, TGF- β3, TGF- β4, TGF- β5, thrombin, thymus Ck-1, thyroid stimulating hormone, tie, TIMP, TIQ, tissue factor, TMEFF2, tmpo, TMPRSS2, TNF- α/β, TNF- β, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR 4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, tri), TNFRSF10D (TRAIL R4DcR2, trdd), frodsf 11 (rand) TRANCE R), TNFRSF11B (OPG OCIF, TR 1), TNFRSF12 (TWEAK RFN 14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, hveA, LIGHT R, TR 2), TNFRSF16 (NGFR p75 NTR), TNFRSF17 (BCMA), TNFRSF18 (GITRAITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNRICD 120A, p 55-60), TNFRSF1B (TNFRRICD 120B, p 75-80), TNFRSF26 (TNFRH 3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX 40 35, TX1R), TNFRSF5 (CD 40p 50), TNFRSF6 (Fas-1, APT1, CD 95), TNFRSF6 (DK 6, CD 68, TNFRSF6 (CD 8), TNFRSF 30 (TNFRSF 6, CD 8) TNFRSF9 (4-1 BB CD137, ILA), TNFRSF21 (DR 6), TNFRSF22 (DcTRAIL R2 tnfrsh 2), TNFRSF 23 (DcTRAIL R1 tnfrsh 1), TNFRSF25 (DR 3Apo-3, LARD, TR-3, TRAIL, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL 2), TNFSF11 (TRAIL/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL 2), TNFSF13B (BAFF BLYS, TALL1, smank, TNFSF 20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL 1A/VEGI), TNFSF18 (TR ligand AITR, TL 6), tnff 1A (TNF-a Conectin, DIF), TNFSF 2), TNFSF1B (TNF-B ITa, TNFSF 1), TNFSF3 (LTb TNFC, p 33), TNFSF4 (OX 40 ligand gp34, TXGP 1), TNFSF5 (CD 40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD 27 ligand CD 70), TNFSF8 (CD 30 ligand CD 153), TNFSF9 (4-1 BB ligand CD137 ligand), TP-1, t-PA, tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, trk, TROP-2, TXX (toll-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, 8, TLR9, TLR10, TSG, TSLP, TSCA 125, tumor-associated antigen-expressed by TLR-Y-associated antigen-expressed by TLR-2, VCAM-23, VCVE-2, and the like receptor, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA-1, VLA-4, VNR integrin, vascular blood friend factor, WIF-1, WNT2B/13, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT, WNT16, XCL1, XCL2, XCR1, XEDAR, XIAP, XPD, HMGB1, igA, CD81, CD97, CD98, DDR1, DKK1, EREG, hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, kallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31R, nav1.1, nav1.2, nav1.3, nav1.4, nav1.5, nav1.6, nav1.7, nav1.8, navl.9, EPCR, C1q, C1R, C1s, C2a, C2B, C3a, C3B, C4a, C4B, C5a, C5B, C6, C7 C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, adhesive proteoglycan-1, adhesive proteoglycan-2, adhesive proteoglycan-3, adhesive proteoglycan-4, LPA, S1P and hormone or growth factor receptors.

Although examples of the molecules listed above also include receptors (membrane proteins), these receptors (membrane proteins) that are even in soluble form in body fluids (e.g., truncated forms) can be used as "second antigens" (target molecules) to which the "second moiety" of the present invention binds. One non-limiting example of a soluble form of such a receptor (membrane protein) may include a protein represented by soluble IL-6R as described by Mullberg et al (J.Immunol. (1994) 152 (10), 4958-4968).

In the present invention, where the "second antigen" is a soluble protein, such as IL-6, the molecule comprising the first and second portions of the invention binds to a dead cell-derived molecule (i.e., the first antigen) via the first portion of the molecule, such as the F-actin complex, and binds to IL-6 (i.e., the second antigen) via the second portion of the molecule to neutralize IL-6 in the affected tissue, more particularly wherein dead cell-derived molecules occur as a result of cell death in the affected tissue as compared to in unaffected tissue. When IL-6 is neutralized, the immune response elicited by immune cells is indirectly regulated.

In the present invention, the "second moiety" which binds to the "second antigen" as described above may have any structure as long as it binds to the "second antigen" as described above. The structure of the "second portion" may include, but is not limited to, a polypeptide or portion thereof, or a small or medium chemical compound or portion thereof, or a polynucleotide or portion thereof. The polypeptide or portion thereof includes, but is not limited to, an antibody (including, but not limited to, a human antibody, a chimeric antibody, a humanized antibody, and a VHH antibody) or an antigen binding domain (also referred to as a portion, or a fragment of an antibody). The antigen binding domain of an antibody includes but is not limited to Not limited to antibody heavy chain Variable (VH) regions, antibody light chain Variable (VL) regions (preferably a combination of antibody heavy chain Variable (VH) regions and antibody light chain Variable (VL) regions), single domain antibodies (sdabs), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ 。

A molecule comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen

In the present invention, one embodiment of the "molecule comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen" includes, but is not limited to, a molecule having the following characteristics.

i. An antibody, wherein "the first moiety that binds to the first antigen" has been conjugated to the Fc region and at least one of the two fabs binds to the "second antigen". A schematic example of this embodiment is shown in fig. 2. Alternatively, the "first portion that binds to the first antigen" may bind to one of the Fab arms instead of or in addition to the Fc region. Furthermore, more than two "first moieties that bind to a first antigen" may be conjugated to an Fc region. Fig. 3 schematically shows other various examples of embodiments of the molecules of the invention. As shown in fig. 3, two Fab arms can be conjugated to the Fc region in various forms. In this embodiment, one example of a "first moiety that binds to a first antigen" is all or part of Clec9A or VHH.

ii antibodies, wherein one Fab arm binds to a "first antigen" and the other Fab arm binds to a "second antigen". An example of this embodiment may be a bispecific antibody or bispecific antigen binding domain, wherein one Fab arm binds to a dead cell-derived molecule (e.g. F-actin complex) as described above, and the other Fab arm binds to a membrane protein expressed on the cell membrane of a cell (e.g. immune cell, cancer cell, autoreactive cell). The antibodies or antibody-like molecules in this example may take various forms, as shown in fig. 3.

A polypeptide comprising a "first moiety that binds to a first antigen" and a "second moiety that binds to a second antigen", wherein the first moiety and the second moiety are fused with or without a linker. An example of this embodiment is a fusion protein comprising the extracellular domain of Clec9A and a ligand for any membrane protein expressed on the cell membrane of a cell (e.g., immune cell, cancer cell, autoreactive cell).

iv a chemical compound comprising a "first moiety that binds to a first antigen" and a "second moiety that binds to a second antigen". An example of this embodiment may be a chemical compound comprising at least two moieties, one of which binds to a dead cell-derived molecule as described above (e.g., F-actin complex, nucleosomes as a complex of histone and nucleic acid), and the other of which binds to a membrane protein expressed on the cell membrane of a cell (e.g., immune cell, cancer cell, autoreactive cell). Such compounds may be carried out by using bioconjugate techniques (Bioconjugate Techniques,3rd Edition,2013,Greg T.Hermanson). For example, where the "first antigen" is the nucleosome of a histone and nucleic acid complex and the "second antigen" is mTOR (the mechanical target of rapamycin), the compound may be one that comprises distamycin as the "first moiety" that binds to nucleic acid and rapamycin as the "second moiety" that binds to mTOR, and further comprises photoactive groups (e.g., aryl azide, biaziridine, psoralen). Upon receipt of light (photo) energy by the compound, the first antigen and the second antigen are cross-linked by the compound, and binding of the second moiety to the second antigen on the cell membrane imparts signal transmission or signal blocking into the cell.

Imparting signal transmission or signal blocking

In the present invention, the "imparting of signalling or signal blocking" into a cell by means of a "molecule comprising a first part binding a first antigen and a second part binding a second antigen" may be achieved by providing a modulation in the cell, such as agonistic activity, antagonistic activity, allosteric modulation, conformational change, internalization, stabilization or any modulation of the target molecule (i.e. the second antigen) by the molecule of the present invention.

Affected tissue

In the present invention, the term "affected tissue" refers to tissue that finds any condition or feature different from normal tissue and is unique to the disease. One example of affected tissue includes, but is not limited to, tumor tissue, inflammatory tissue, tissue associated with autoimmune disease, and the like. In such affected tissues, cell death as described above occurs more frequently than in normal tissues to produce dead cell-derived molecules as described above.

Tumor tissue

In the present invention, the term "tumor tissue" refers to a tissue comprising at least one tumor cell. Typically, tumor tissue consists of a population of tumor cells that constitute the tumor bulk (parenchyma), and connective tissue and blood vessels that exist between the tumor cells and support the tumor (stroma). In some cases, these are clearly distinguishable, but in some cases they are intermixed. In some cases, there are cells, such as immune cells, that have penetrated into the tumor tissue. In contrast, "non-tumor tissue" refers to living tissue other than tumor tissue. Healthy tissue/normal tissue that is not diseased is representative of such non-tumor tissue.

Inflammatory tissue

In the present invention, "inflamed tissue" includes but is not limited to the following.

i. Joint tissue associated with rheumatoid arthritis or osteoarthritis.

ii lung tissue associated with bronchial asthma.

Digestive organ tissue associated with inflammatory bowel disease, crohn's disease or ulcerative colitis.

iv fibrotic tissue associated with liver, kidney or lung fibrosis.

Tissue associated with immune rejection by organ transplantation.

vascular tissue or cardiac tissue associated with arteriosclerosis or heart failure.

visceral adipose tissue associated with metabolic syndrome.

Skin tissue associated with atopic dermatitis or any other dermatitis.

Spinal nerve tissue associated with herniated disk or chronic back pain.

Bone tissue associated with a fracture.

Tissue associated with burn.

Tissue of organ tissue injured by mechanical, physical or chemical forces.

Antigens formed by multimerization of multiple molecules

In the present invention, one embodiment of the "first antigen" includes, but is not limited to, "antigen formed by multimerization of a plurality of molecules". In the present invention, "antigen formed by multimerization of a plurality of molecules" includes, but is not limited to, an antigen formed by multimerization of a plurality of proteins or portions thereof and/or lipids constituting a cytoskeleton, a biological membrane (e.g., a cell membrane), a nucleosome, a chaperone, or the like.

Proteins or portions thereof include, but are not limited to, proteins or portions thereof that constitute the cytoskeleton consisting of actin filaments, intermediate filaments, myosin filaments, keratin filaments, microtubules, and the like. Biofilms, such as cell membranes, are composed of various complexes formed by different proteins, different lipids and/or one or more proteins and one or more lipids. Such proteins include, but are not limited to, transmembrane proteins such as ion channels, G Protein Coupled Receptors (GPCRs), proton pumps, lipid-anchored proteins such as G proteins and the like, and surface membrane proteins such as enzymes, hormones and the like. Lipids include, but are not limited to, phospholipids, glycolipids, sterols, and the like. As described above, in affected tissues, such as those associated with cancer, inflammation, autoimmune diseases, and the like, protein and/or lipid complexes in biological membranes (e.g., cell membranes) or portions thereof are exposed to the extracellular environment as a result of cell death. Such complexes or portions thereof are also included as "first antigens" (also referred to as dead cell-derived molecules) as described above.

Nucleosomes are complexes of histones and nucleic acids, chaperones consisting mainly of Heat Shock Proteins (HSPs). These are also included as "first antigens" (also known as dead cell derived molecules).

Membrane proteins as "second antigen" expressing cells

As described above, in the present invention, the "second antigen" includes, but is not limited to, a membrane protein expressed on a cell membrane and/or an endosomal membrane involved in signal transmission in a cell. In the present invention, such cells expressing a membrane protein as a "second antigen" may be any kind or type of cells, including but not limited to immune cells (e.g., T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophils, and basophils), somatic cells (e.g., epithelial cells, fibroblasts, mesenchymal cells, bone cells, muscle cells, neural cells, blood cells, etc.), germ cells, or affected or abnormal cells (e.g., tumor cells, autoreactive cells). Such cells may be located in any tissue, including but not limited to epithelial tissue, muscle tissue, nerve tissue, connective tissue, or affected tissue associated with cancer, inflammatory and autoimmune diseases, and the like. Preferred embodiments of cells expressing a "second antigen" (also referred to as a target molecule) may be immune cells, such as T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophils, and basophils. Other preferred embodiments of cells expressing a "second antigen" (also referred to as a target molecule) may be cells in affected tissues associated with cancer, inflammatory and autoimmune diseases, and the like.

In the present invention, the binding of the "molecule comprising a first moiety binding to a first antigen and a second moiety binding to a second antigen" of the present invention as described above to a substance (molecule) exposed to the extracellular environment due to cell death as described above ("dead cell derived molecule") can be examined, for example, by preparing cells treated or untreated with a cytotoxic drug such as mitoxantrone or mitomycin (treatment of the drug induces cell death), and measuring and comparing the extent of binding of the molecule to each drug treated or untreated cell using flow cytometry. The increased degree of binding of the molecules of the invention as described above to the drug-treated cells suggests that the molecules of the invention specifically bind to dead cell-derived molecules as described above.

The present invention provides a molecule that binds to a component of a cell or a portion thereof that is exposed to an extracellular environment as described above due to cell death ("first antigen", also referred to as dead cell-derived molecule) and simultaneously binds to a target molecule ("second antigen"; e.g., a membrane protein as described above) on or within a cell (e.g., an immune cell, an affected cell such as a tumor cell, an autoreactive cell, and a virus-infected cell). The molecule is capable of cross-linking between a component of a cell or portion thereof exposed to the extracellular environment as a result of cell death and a target molecule on or within a cell, such as an immune cell or an affected cell (e.g., cancer cells, autoreactive cells, and virus-infected cells), and imparting signaling or blocking to cells in a tissue.

In the present invention, the above-described signal transmission or blocking into cells by the molecules of the present invention can be examined, for example, as follows.

i. Cells are provided that are treated or untreated with a cytotoxic drug, the treatment of which induces cell death.

ii provides the following molecules: molecules (e.g., antibodies) that bind to, e.g., CD3 or a co-stimulatory or co-inhibitory molecule expressed in an immune cell ("second antigen") [ control molecules ], as well as molecules having a moiety that binds thereto ("second antigen") and a moiety that binds to, e.g., F-actin ("first antigen"; one of the embodiments of dead cell-derived molecules) [ molecules of the invention ].

Providing a reporter cell that has been modified to give a detectable signal when signalling occurs through the molecule bound to (ii).

iv culturing the reporter cells with each molecule of (ii) in the presence of the drug-treated cells of (i) or the supernatant of a drug-treated cell culture, or the supernatant of untreated cells or untreated cells.

Measuring the reporter signal in each cell culture of (iv) and comparing the reporter signal for each cell culture so measured.

The detection of a higher reporter signal in the cell culture of the molecule of the invention of (ii) in the presence of the drug-treated cells or the supernatant of the drug-treated cell culture than in the cell culture of the molecule of the invention of (ii) in the presence of untreated cells or the supernatant of the untreated cell culture, indicates that the molecule of the invention of (ii) confers signaling into the cells by binding to an antigen such as CD3 or a co-stimulatory or co-inhibitory molecule expressed in immune cells ("second antigen").

The detection of a higher reporter signal in the cell culture of the molecule of the invention of (ii) in the presence of the supernatant of the drug-treated cell or drug-treated cell culture than in the control cell culture of (ii) in the supernatant of the drug-treated cell or drug-treated cell culture, indicates that the molecule of the invention of (ii) confers a stronger signaling to the cell than the control molecule, wherein the control molecule does not bind to a dead cell-derived molecule such as F-actin ("first antigen").

In the present invention, any kind and/or any type of reporter, and any kind and/or any type of reporter cell may be used according to the "second antigen", as long as it is possible to detect the induction of signal transmission into the cell by binding of the molecule of the present invention (ii) above to the "second antigen" (e.g. CD 3) or co-stimulatory or co-inhibitory molecules expressed in immune cells.

In the present invention, the "stronger" signal in the above (vii) means that the difference in maximum activity is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold or 1000-fold. The "stronger" signal in (vii) above may also be at least one of an EC50 value or an IC50 difference of 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold or 1000-fold.

When terms described or referenced below are used in the present invention, these terms have meanings (including but not limited to) as described or referenced below.

Furthermore, the techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are generally employed using conventional methodologies, such as the widely used methodologies described in: sambrook et al, molecular Cloning: a Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y.; current protocols in molecular biology (F.M.Ausubel et al, editions, (2003)); enzymatic methods series (Academic Press, inc.): PCR 2: practical methods (M.J.MacPherson, B.D.Hames and G.R.Taylor edit (1995)), harlow and Lane edit (1988) antibodies, laboratory manuals and animal cell cultures (R.I.Freshney edit (1987)); oligonucleotide synthesis (m.j. Gait, edit, 1984); molecular biology methods, humana Press; cell biology: laboratory Manual (J.E.Cellis editions, 1998) academic Press; animal cell culture (r.i. freshney) edit, 1987); cell and tissue culture profiles (J.P.Mather and P.E.Roberts, 1998) Plenum Press; cell and tissue culture: laboratory procedures (A.Doyle, J.B.Griffiths and D.G.Newell et al, 1993-8) J.Wiley and Sons; experimental immunology handbook (d.m. weir and c.c. blackwell editions); mammalian cell gene transfer vectors (J.M.Miller and M.P.Calos. Eds., 1987); and (2) PCR: polymerase chain reaction (Mullis et al, 1994); current protocols for immunology (j.e. coligan et al, editions, 1991); short protocols in molecular biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.transitions, 1997); antibodies (P.Finch, 1997); antibody: one practical method (D.Catty editions, IRL Press, 1988-1989); monoclonal antibodies: one practical method (p.shepherd and c.dean editions, oxford university press, 2000); antibody was used: laboratory Manual (E.Harlow and D.Lane (Cold spring harbor laboratory Press 1999), antibodies (M.Zanetti and J.D.Capra editions Harwood Academic Publishers, 1995), and cancer: oncology principles and practices (V.T.DeVita et al editions J.B.Lippincott Company, 1993).

Amino acids

Herein, amino acids are described by single letter codes or three letter codes or both, such as Ala/A, leu/L, arg/R, lys/K, asn/N, met/M, asp/D, phe/F, cys/C, pro/P, gln/Q, ser/S, glu/E, thr/T, gly/G, trp/W, his/H, tyr/Y, ile/I or Val/V.

Amino acid changes

For amino acid changes in the amino acid sequence of the antigen binding molecule (also referred to herein as "amino acid substitutions" or "amino acid mutations"), known methods such as site-directed mutagenesis (Kunkel et al (proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be suitably used. In addition, several known methods can also be used as amino acid changing methods to replace unnatural amino acids (Annu Rev. Biophys. Biomol. Structure. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Protein Express) that includes a tRNA that has an unnatural amino acid that binds to a complementary amber suppressor tRNA, which is a codon UAG (amber codon) that is one of the stop codons, is suitable for use.

In the present invention, when describing the site of amino acid change, the meaning of the term "and/or" includes each combination of "and" or "as appropriate. Specifically, for example, "amino acid substitutions at positions 33, 55, and/or 96" include variants with the following amino acid changes: (a) position 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) amino acids at positions 33, 55, and 96.

Further, as the expression indicating the amino acid change herein, it is possible to appropriately use the expression of a code showing 1 letter or 3 letters of the amino acid before and after the change, respectively, before and after the number indicating the specific position. For example, the change N100bL or Asn100bLeu used when substituting amino acids contained in the antibody variable region means substitution of Asn with Leu at position 100b (numbering according to Kabat). That is, the number indicates the amino acid position according to Kabat numbering, the 1-letter or 3-letter amino acid code written before the number indicates the amino acid before substitution, and the 1-letter or 3-letter amino acid code written after the number indicates the amino acid after substitution. Similarly, the change P238D or Pro238Asp used when substituting the amino acid of the Fc region contained in the antibody constant region indicates substitution of Pro at position 238 (numbering according to EU) with Asp. That is, the numbers indicate amino acid positions according to EU numbering, 1 letter or 3 letter amino acid codes written before the numbers indicate amino acids before substitution, and 1 letter or 3 letter amino acid codes written after the numbers indicate amino acids after substitution.

Polypeptides

As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) that are linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and not to a product of a particular length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other term used to refer to a chain of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms.

The term "polypeptide" also means the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptides may be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from the specified nucleic acid sequences. It can be produced in any manner, including by chemical synthesis. The size of a polypeptide as described herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, polypeptides that do not have a defined three-dimensional structure but can take a number of different conformations are referred to as unfolded.

Percent (%) amino acid sequence identity

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not taking any conservative substitutions into account as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 was used to generate% amino acid sequence identity values. ALIGN-2 sequence comparison computer program was written by Genntech, inc., source code was submitted with the user document to the United states copyright office, washington, inc., 20559, U.S. copyright accession number TXU510087. The ALIGN-2 program is publicly available from Genntech, inc. of san Francisco, calif., or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including the digital UNIX v4.0d. All sequence comparison parameters were set by the ALIGN-2 program and did not change. In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence A to, and or against, a given amino acid sequence B (which may alternatively be expressed as a given amino acid A having or comprising a particular% amino acid sequence identity to, and against, a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program ALIGN-2, wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. All% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless specifically indicated otherwise.

Recombinant methods and compositions

Antibodies, antigen binding molecules, antigen binding domains, including those belonging to the invention, can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. Where the molecule of the invention is an antibody, in one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acids may encode amino acid sequences comprising the VL of the antibody and/or amino acid sequences comprising the VH (e.g., the light chain and/or heavy chain of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp2/0 cell). In one embodiment, a method of making a molecule of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a molecule (e.g., an antibody) as described above under conditions suitable for expression of the molecule (e.g., an antibody) as described above, and optionally recovering the molecule (e.g., an antibody) as described above from the host cell (or host cell culture medium).

For recombinant production of a molecule (e.g., an antibody) as described above, nucleic acids encoding the molecule (e.g., an antibody) as described above are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., in the case where the molecule is an antibody, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing a vector encoding a molecule of the invention include prokaryotic or eukaryotic cells as described herein. For example, the molecules of the invention may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of the molecules of the invention in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199 and 5,840,523 (see also Charlton, methods in Molecular Biology, vol.248 (b.k.c. lo, ed., humana Press, totowa, NJ, 2003), pages 245-254, describing expression of antibody fragments in e.coli), the molecules of the invention can be isolated from bacterial cell pastes in the soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the molecules of the invention, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce molecules of the invention with a partially or fully human glycosylation pattern. See gemmgross, nat. Biotech.22:1409-1414 (2004) and Li et al, nat. Biotech.24:210-215 (2006).

Suitable host cells for expressing the glycosylated molecules of the invention are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing PLANTIBODIES for antibody production in transgenic plants) ^TM Technology).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth may be useful. Examples of other useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293 cells as described in Graham et al, J.Gen. Virol.36:59 (1977); baby hamster kidney cells (BHK); mouse support cells (TM 4 cells, e.g., as described in Mather, biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as Mather et al, annals N.Y. Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, proc.Natl. Acad. Sci.usa 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see Yazaki and Wu, methods in Molecular Biology, vol.248 (b.k.c.lo, ed., humana Press, totowa, n.j.), pp.255-268 (2003).

Recombinant production of the molecules of the invention may be performed in a similar manner as described above, by using a host cell comprising one or more vectors (e.g., a host cell that has been transformed with one or more vectors) comprising a nucleic acid encoding an amino acid sequence comprising the entire molecule or a portion thereof.

In the present invention, as described above, one embodiment of the molecule of the invention, i.e. "comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen" as described above, is an antibody or antigen binding domain/molecule thereof, which may comprise a further moiety conjugated to e.g. an Fc region. In the present invention, such molecules may also be referred to as antigen binding molecules. Where the molecule is an antigen binding molecule, for example, having multiple specificities for two or more different antigens, it may be referred to as a multi-specific antigen binding molecule.

Antigen binding molecules and multispecific antigen binding molecules

As used herein, the term "antigen binding molecule" refers to any molecule comprising an antigen binding site or any molecule having binding activity to an antigen, and may further refer to such a molecule, e.g., a peptide or protein of about five amino acids or more in length. Peptides and proteins are not limited to those derived from organisms, for example, they may be polypeptides produced from artificially designed sequences. They may also be any naturally occurring polypeptide, synthetic polypeptide, recombinant polypeptide, etc. Scaffold molecules comprising known stable conformational structures such as the alpha/beta barrel as scaffold and wherein a portion of the molecule is made into an antigen binding site are also one embodiment of the antigen binding molecules described herein.

By "multispecific antigen-binding molecule" is meant an antigen-binding molecule that specifically binds more than one antigen. The term "bispecific" refers to an antigen binding molecule that is capable of specifically binding to at least two different antigenic determinants. The term "trispecific" refers to antigen binding molecules capable of specifically binding to at least three different antigenic determinants.

Antigen binding domains

As described above, in the case where the molecule of the invention, i.e. "comprising a first part that binds a first antigen and a second part that binds a second antigen", is for example an antigen binding molecule such as an antibody (in some embodiments, it is also referred to as antigen binding molecule as described above), "second part that binds a second antigen" and (if appropriate) further "first part that binds a first antigen" may be an antigen binding domain of an antibody or derived from an antibody.

The term "antigen binding domain" refers to a portion of an antibody that comprises a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen binding domains include, for example, "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv 2)", "Fab" and "F (ab') ₂ ”。

Ig-type binding moieties

The terms "Ig-type binding moiety", "Ig-type binding moiety" and the like as used herein are well known to those skilled in the art and generally refer to an antigen binding portion of an Ig-type (immunoglobulin-type) which is well known to the skilled person. In certain embodiments herein, it includes all "antigen binding domains" described herein (provided that these domains include at least one immunoglobulin structure), and it specifically includes all examples of the latter term described herein. In this context, preferably, the Ig-type binding moiety is selected from the group consisting of single domain antibodies (VHH), antibody heavy chain variable regions (VH), antibody light chain variable regions (VL), single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ A group of groups. More preferably, the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ A group of groups. It includes, in particular, the polypeptides consisting of "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv 2)", "Fab" and "F (ab') ₂ "any one of the group consisting, in particular any one of the specific embodiments described elsewhere herein.

Variable region

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVR). (see, e.g., kit et al, kuby Immunology, 6 th edition, w.h. freeman and co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to antigens may be used to screen libraries of complementary VL or VH domains, respectively, to isolate antibodies that bind to a particular antigen. See, e.g., portland et al, j.immunol.150:880-887 (1993); clarkson et al, nature 352:624-628 (1991).

HVR or CDR

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and/or forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ("antigen contact"). Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), which terms are used interchangeably herein to refer to the portion of the variable region that forms the antigen binding region. Typically, an antibody comprises six HVRs: three of VH (H1, H2, H3) and three of VL (L1, L2, L3).

Exemplary HVRs herein include:

(a) Hypervariable loops (Chothia and Lesk, J.Mol. Biol.196:901-917 (1987)) present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3);

(b) CDRs at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD (1991));

(c) Antigen contacts at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al, J.mol. Biol.262:732-745 (1996)); and

(d) Combinations of (a), (b) and/or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.

HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 are also referred to as "H-CDR1", "H-CDR2", "H-CDR3", "L-CDR1", "L-CDR2" and "L-CDR3", respectively.

Capable of binding to antigen

Whether an antibody variable region is "capable of binding to an antigen" can be determined by methods known in the art. This can be determined, for example, by electrochemiluminescence (ECL method) (BMC Research Notes 2011, 4:281).

Specifically, for example, a low-molecular antibody consisting of a region capable of binding to an antigen (e.g., fab region) of an antigen binding molecule to be detected biotin-labeled or a monovalent antibody thereof (an antibody lacking one of two Fab regions carried by a usual antibody) is mixed with an antigen (Ru complex) labeled with a sulfo label, and the mixture is added to a streptavidin-immobilized plate. In this procedure, the biotin-labeled antigen binding molecules to be tested bind to streptavidin on the plate. The sulfo tag was allowed to emit light, and a luminescent signal was detected by using a Sector Imager 600 or 2400 (MSD k.k.), etc., to confirm the binding of the antigen to the above region of the antigen binding molecule to be measured. Alternatively, the measurement may be performed by ELISA or FACS (fluorescence activated cell sorting ), ALPHASCREN (Amplified Luminescent Proximity Homogeneous Assay, enhanced luminescence near homogeneity measurement), or BIACORE method based on the phenomenon of Surface Plasmon Resonance (SPR), etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

Specifically, for example, the measurement can be performed using an interaction analyzer Biacore (GE Healthcare Japan corp.) based on the Surface Plasmon Resonance (SPR) phenomenon. Biacore analyzers include any model, such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any sensor chip of Biacore, such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip, may be used as the sensor chip. Proteins capturing the antigen binding molecules of the invention (e.g., protein a, protein G, protein L, anti-human IgG antibodies, anti-human IgG-Fab, anti-human L chain antibodies, anti-human Fc antibodies, antigen proteins, or antigen peptides) are immobilized on the sensor chip by, for example, amine-conjugated, disulfide-conjugated (disulfide coupling), aldehyde-conjugated methods. Antigens are injected as analytes on the chip and interactions are measured to obtain a sensorgram. In this procedure, the concentration of the antigen may be selected in the range of several μ to several pM depending on the strength of interaction (e.g., KD, etc.) of the assay sample.

Alternatively, the antigen, rather than the antigen binding molecule, may be immobilized on a sensor chip, such that the antibody sample to be evaluated interacts with the antigen. From the dissociation constant (KD) value calculated from the sensorgram of the interaction, or from the degree of increase in sensorgram of the antigen binding molecule sample after the action relative to the pre-action level, it can be confirmed whether the antibody variable region of the antigen binding molecule of the present invention has binding activity to an antigen.

In some embodiments, the binding activity or affinity of an antibody variable region of the invention to an antigen of interest (i.e., antigen) is assessed using, for example, a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare) at 37 ℃ or 25 ℃ (for antigen). Anti-human Fc (e.g., GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen binding molecules or antibody variable regions are captured on the anti-Fc sensor surface and then the antigen is injected onto the flow cell. The capture level of the antigen binding molecule or antibody variable region is targeted at 200 Resonance Units (RU). Recombinant human antigen can be injected at a dose of 400 to 25nM, which is prepared by serial dilution of two-fold followed by dissociation. In the presence of 20mM ACES,150mM NaCl,0.05% Tween 20, 0.005% NaN ₃ All antigen binding molecules or antibody variable regions and analytes were prepared in ACES pH 7.4. With 3M MgCl per cycle ₂ Regenerating the sensor surface. Binding affinities were determined by processing and fitting the data to a 1:1 binding model using, for example, biacore T200 evaluation software version 2.0 (GEHealthcare) or Biacore 8K evaluation software (GE Healthcare). KD values were calculated to assess the specific binding activity or affinity of antigen binding domains.

ALPHASScreen is implemented by the ALPHA technique using two types of beads (donor and acceptor) based on the following principle: by biological interactions between the molecules bound to the donor beads and the molecules bound to the acceptor beads, a luminescent signal is only detected when the two beads are in close proximity. The laser-excited photosensitizer within the donor bead converts ambient oxygen to singlet oxygen having an excited state. Singlet oxygen diffuses around the donor beads to the acceptor beads near the donor beads, thereby causing chemiluminescent reactions within the beads, ultimately emitting light. When the molecules bound to the donor beads and the molecules bound to the acceptor beads do not interact, the singlet oxygen generated by the donor beads does not reach the acceptor beads. Therefore, no chemiluminescent reaction occurs.

One (ligand) of the substances observing the interaction is immobilized on a gold film of the sensor chip. Light is irradiated from the back surface of the sensor chip such that total reflection occurs at the interface between the gold thin film and the glass. As a result, sites where the reflection intensity (SPR signal) decreases are formed in a part of the reflected light. The other (analyte) of the substance observing the interaction is injected to the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, thereby changing the refractive index of the solvent at the surface of the sensor chip. The change in refractive index shifts the position of the SPR signal (conversely, dissociation of the binding molecule returns the signal to its original position). The Biacore system plots displacement amounts, i.e., mass changes on the surface of the sensor chip, on the ordinate, and displays the mass changes as measured data (sensor map) in relation to time. The amount of analyte bound to the ligand captured on the surface of the sensor chip (the amount of change in response on the sensor pattern before and after analyte interaction) can be determined from the sensor pattern. However, since the amount of binding also depends on the amount of ligand, comparison must be made under conditions where substantially the same amount of ligand is used. Dynamics, i.e., association rate constant (ka) and dissociation rate constant (KD), can be determined from the curves of the sensorgram, and affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also preferred for the BIACORE method. Examples of inhibition assays are described in Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010.

For example, a biotin-labeled antigen binding molecule to be tested is allowed to bind to streptavidin on a donor bead, while a Glutathione S Transferase (GST) -labeled antigen is allowed to bind to an acceptor bead. In the absence of the competing second antigen, the antigen binding molecule to be tested interacts with the antigen to produce a signal of 520 to 620 nm. Unlabeled second antigen competes with the antigen for interaction with the antigen binding molecule to be tested. The decrease in fluorescence due to competition can be quantified to determine the relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin and the like is known in the art. The antigen may be tagged with GST by methods suitably employed, including, for example, fusing a polynucleotide encoding the antigen in a flame (in flag) to a polynucleotide encoding GST; the resulting fusion gene is expressed by a cell or the like having a vector capable of expressing it, and then purified using a glutathione column. Preferably, the obtained signals are analyzed using, for example, software GRAPHPAD PRISM (GraphPad Software, inc., san Diego) adapted to a one-site competition model based on non-linear regression analysis.

The tag is not limited to a GST tag and may be performed using any tag, such as, but not limited to, a histidine tag, MBP, CBP, flag tag, HA tag, V5 tag, or c-myc tag. Binding of the antigen binding molecule to be tested to the donor beads is not limited to binding using the biotin-streptavidin reaction. In particular, when the antigen binding molecule to be tested comprises Fc, a possible method involves binding the antigen binding molecule to be tested to the donor beads via an Fc recognition protein, such as protein a or protein G.

Fab molecule/Fab region/Fab arm

In the present invention, the terms "Fab molecule", "Fab region" or "Fab arm" are used interchangeably to refer to a protein consisting of VH and CH1 domains of the heavy chain ("Fab heavy chain") and VL and CL domains of the light chain ("Fab light chain") of an immunoglobulin.

₂ Fab, F (ab ') and Fab'

"Fab" consists of a single light chain and CH1 domains and variable domains from a single heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

“F(ab′) ₂ "or" Fab "is produced by treating an immunoglobulin (monoclonal antibody) with proteases such as pepsin and papain, by which is meant attachment by disulfide bonds present between the hinge regions of the two H chains, respectivelyAn antibody fragment produced by the near digestion of an immunoglobulin (monoclonal antibody). For example, papain cleaves IgG upstream of disulfide bonds present between the hinge regions of the respective two H chains to generate two homologous antibody fragments, wherein the L chain comprising VL (L chain variable region) and CL (L chain constant region) is linked to the H chain fragment comprising VH (H chain variable region) and chγ1 (γ1 region of H chain constant region) by disulfide bonds at its C-terminal region. These two homologous antibody fragments are each referred to as Fab'.

“F(ab′) ₂ "consists of two light chains and two heavy chains comprising a constant region of a CH 1 domain and a portion of a CH2 domain, thereby forming a disulfide bond between the two heavy chains. F (ab') ₂ Can be preferably prepared as follows. Partially digesting the whole monoclonal antibody or the monoclonal antibody comprising the desired antigen binding site with a protease such as pepsin; the Fc fragment was removed by adsorption onto a protein a chromatography column. The protease is not particularly limited as long as it can cleave the whole antibody in a selective manner under the appropriately set enzymatic reaction conditions such as pH to produce F (ab') ₂ And (3) obtaining the product. Such proteases include, for example, pepsin and ficin.

Fused/fused

The terms "fused" and "fusion" refer to two or more different polypeptides (e.g., a first polypeptide that binds a first antigen and a second polypeptide that binds a second antigen; a Fab arm and an Fc domain) linked directly by a peptide bond or by one or more peptide linkers.

Thus, as used herein, the term "fusion protein" (which may be used interchangeably with the term "fusion polypeptide") preferably means that two or more different polypeptides are linked covalently directly or via one or more peptide linkers. A specific example of a fusion protein herein is an antigen binding molecule of the invention, wherein an antibody (or at least an Ig-type binding moiety) is linked to a binding polypeptide that is not an Ig-type binding moiety.

Exchange Fab

"swapped" Fab molecules (also referred to as "swapped Fab") refer to Fab molecules in which the variable or constant regions of the Fab heavy and light chains are swapped, i.e., the swapped Fab molecules comprise a peptide chain consisting of a light chain variable region and a heavy chain constant region, and a peptide chain consisting of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which Fab light and Fab heavy chain variable regions are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. In contrast, in an exchange Fab molecule in which the constant regions of the Fab light and Fab heavy chains are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the exchange Fab molecule.

Conventional Fab

In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native form, i.e., comprising a heavy chain consisting of a heavy chain variable region and a constant region (VH-CH 1) and a light chain consisting of a light chain variable region and a constant region (VL-CL). The term "immunoglobulin molecule" refers to a protein having a naturally occurring antibody structure. For example, igG class immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains disulfide-bonded. From the N-terminal to the C-terminal, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH 1, CH2 and CH 3), also known as heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain, also known as a light chain constant region. The heavy chain of an immunoglobulin may be designated as one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM), some of which may be further divided into subtypes such as gamma 1 (IgG 1), gamma 2 (IgG 2), gamma 3 (IgG 3), gamma 4 (IgG 4), alpha 1 (IgA 1) and alpha 2 (IgA 2). Based on the amino acid sequence of its constant domain, the light chain of an immunoglobulin can be assigned to one of two types called kappa and lambda. Immunoglobulins consist essentially of two Fab molecules and an Fc domain, which are linked by an immunoglobulin hinge region.

Affinity for

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a first or second moiety; an antibody or antigen binding domain) and its binding partner (e.g., a first or second antigen; or antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a first moiety and a first antigen; a second moiety and a second antigen; an antigen binding molecule and an antigen, or an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may include different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by accepted methods known in the art, including those described herein. One particular method of measuring affinity is Surface Plasmon Resonance (SPR).

Method for determining affinity

In certain embodiments, each of the first or second portions of the invention, comprising a molecule that binds to the first portion of the first antigen and to the second portion of the second antigen, and the dissociation constant (KD) of the antigen-binding molecule or antibody to its antigen (i.e., first antigen, second antigen) as described above, is 1. Mu.M or less, 120nM or less, 100nM or less, 80nM or less, 70nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 2nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10nM or less ^-8 M or less, 10 ^-8 M to 10 ^-13 M，10 ^-9 M to 10 ^-13 M). In certain embodiments, the KD of the molecules of the invention for the first antigen and the second antigen falls within the range of 1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60-80nM, respectively.

In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). For example, the molecules of the invention are directed against antigens (i.e., first antigen, second antigen)Solution binding affinity was measured by the following steps in the presence of a titration series of unlabeled antigen: using the minimum concentration% ¹²⁵ I) The labeled antigen balances the molecules and then captures the bound antigen with a plate coated with antibodies that bind the molecules (see, e.g., chen et al, j.mol. Biol.293:865-881 (1999)). To establish assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml of molecular-conjugated capture antibody (Cappel Labs) in 50mM sodium carbonate (pH 9.6), followed by blocking with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23 ℃) for 2 to 5 hours. In the non-adsorbed plate (Nunc# 269620), 100pM or 26pM [ ¹²⁵ I]Antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al, cancer res.57:4593-4599 (1997)). Then incubating the target molecule overnight; however, incubation may last for a longer period of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture was transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. After the plate was dried, 150. Mu.l/well scintillator (MICROSICINT-20) was added ^TM The method comprises the steps of carrying out a first treatment on the surface of the Packard) and at TOPCount ^TM Plates were counted on a gamma counter (Packard) for 10 minutes. The concentration of each Fab that produced less than or equal to 20% of maximum binding was selected for competitive binding assays.

According to another embodiment, kd is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, assays using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000 (BIACORE, inc., piscataway, NJ) were performed with immobilized antigen CM5 chips at 25 ℃ in about 10 Response Units (RU). In one embodiment, the carboxymethylated dextran biosensor chip (CM 5, BIACORE, inc.) is activated with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the supplier. The antigen was diluted to 5. Mu.g/ml (. About.0.2. Mu.g) with 10mM sodium acetate, pH4.8, and then injected at a flow rate of 5. Mu.l/min to achieve about 10Conjugated proteins of Response Units (RU). After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, double serial dilutions of Fab (0.78 nM to 500 nM) were made in a solution containing 0.05% polysorbate 20 (TWEEN-20 ^TM ) Surfactant in PBS (PBST) at 25 degrees C at a flow rate of about 25 microliters/min. Association rate (k) was calculated by fitting the association and dissociation sensorgrams simultaneously using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) evaluation software version 3.2) _on ) Dissociation rate (k) _off ). The equilibrium dissociation constant (Kd) is calculated as the ratio k _off /k _on . See, for example, chen et al, j.mol. Biol.293:865-881 (1999). If the association rate of the above surface plasmon resonance measurement exceeds 10 ⁶ M ^-1 s ^-1 The association rate can be determined by: by using fluorescence quenching techniques to measure the increase or decrease in fluorescence emission intensity (excitation = 295nM; emission = 340nM,16nM bandpass), 20nM of the molecule-bound antibody in PBS, ph7.2 at 25 ℃, in the presence of increased antigen concentrations, as in a spectrometer such as a spectrophotometer (Aviv Instruments) equipped with stop flow or 8000 series of SLM-AMINCO with stirred cuvettes ^TM Measured in a spectrophotometer (thermo spectronic).

According to the above-described method of measuring the affinity of the molecules of the invention for antigens, the person skilled in the art can perform affinity measurements for various antigens for any other kind or type of molecules of the invention.

Antibodies to

The term "antibody" is used herein in its broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein.

Antibody fragments

An "antibody fragment" (which may also be referred to as a portion of an antibody) refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., pluckaphun, in The Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore eds, springer-Verlag, new York, pp.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. Fab and F (ab') which contain salvage receptor binding epitope residues and have increased in vivo half-life ₂ See U.S. Pat. No. 5,869,046 for discussion of fragments. Diabodies are antibody fragments having two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat Med 9, 129-134 (2003); and Hollinger et al Proc Natl Acad Sci USA, 6444-6448 (1993). Three-chain antibodies (triabody) and four-chain antibodies (tetrabody) are also described in Hudson et al, nat Med 9, 129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (domatis, inc., waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibody tablets, as described herein Segments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E.coli or phage).

In certain embodiments herein, the antibody fragment is an Ig-type binding moiety.

Variable fragment (Fv)

Herein, the term "variable fragment (Fv)" refers to the smallest unit of an antigen-binding site derived from an antibody consisting of a pair of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Skerra and Pluckaphun found in 1988 that uniform and active antibodies could be prepared from E.coli periplasmic fractions by inserting an antibody gene downstream of the bacterial signal sequence and inducing expression of the gene in E.coli (Science (1988) 240 (4855), 1038-1041). In Fv prepared from Zhou Zhiji minutes, VH associates with VL in a manner that binds to antigen.

₂ scFv, single chain antibody and sc (Fv)

Herein, the terms "scFv", "single chain antibody" and "sc (Fv) ₂ "refers to antibody fragments comprising a single polypeptide chain derived from the variable regions of the heavy and light chains, but not the constant regions. In general, single chain antibodies also comprise polypeptide linkers between the VH and VL domains, which enable the formation of the desired structure believed to allow antigen binding. Single chain antibodies are discussed in detail in Pluckaphun, the Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore, eds., springer-Verlag, new York,269-315 (1994) ". See also International patent publication WO 1988/001649; U.S. Pat. nos. 4,946,778 and 5,260,203. In particular embodiments, single chain antibodies may be bispecific and/or humanized.

scFv is a single chain low molecular weight antibody in which the VH and VL that form the Fv are linked together by a peptide linker (proc.Natl. Acad.Sci.U.S.A. (1988) 85 (16), 5879-5883). VH and VL may be held in close proximity by peptide linkers.

sc(Fv) ₂ Is a single chain antibody in which four variable regions of two VL and two VH are linked by a linker such as a peptideThe adaptors are ligated to form single strands (J immunol. Methods (1999) 231 (1-2), 177-189). The two VH and the two VL may be derived from different monoclonal antibodies. Such sc (Fv) ₂ Preferably comprising, for example, bispecific sc (Fv) recognizing two epitopes present in a single antigen ₂ As disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc (Fv) ₂ Can be produced by methods known to those skilled in the art. For example sc (Fv) ₂ May be prepared by linking the scFv via a linker such as a peptide linker.

Herein, sc (Fv) ₂ Comprises two VH units and two VL units, starting from the N-terminus of a single chain polypeptide, in VH, VL, VH and VL ([ VH)]-linker- [ VL]-linker- [ VH]-linker- [ VL]) Is a sequential arrangement of (a). The order of the two VH units and the two VL units is not limited to the above-described form, and they may be arranged in any order. Exemplary forms are listed below.

[ VL ] -linker- [ VH ] - [ VL ].

[ VH ] -linker- [ VL ],

[ VH ] -linker- [ VL ].

[ VL ] -linker- [ VH ],

[ VL ] -linker- [ VH ] -linker- [ VL ] -linker- [ VH ].

sc(Fv) ₂ The molecular form of (C) is also described in detail in WO 2006/132352. From these descriptions, one skilled in the art can appropriately prepare the desired sc (Fv) ₂ To produce the polypeptide complexes disclosed herein.

Antibody class

"class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: igA, igD, igE, igG and IgU, some of which may be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

Unless otherwise indicated, light chain constant region amino acid residues are numbered herein by Kabat et al, and heavy chain constant region amino acid residue numbers are numbered by the EU numbering system, also known as EU index numbering, as set forth in Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991.

Frame

"framework" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Accordingly, HVR and FR sequences generally occur in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

Human shared frame

A "human consensus framework" is a framework representing the most common amino acid residues in a human immunoglobulin VL or VH framework sequence selection. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subgroup of sequences is the subgroup in Kabat et al Sequences of Proteins of Immunological Interest, fifth Edition, NIH Publication 91-3242, bethesda MD (1991), vols.1-3. In one embodiment, for VL, the subgroup is subgroup κI of Kabat et al, supra. In one embodiment, for VH, the subgroup is subgroup III of Kabat et al, above.

Chimeric antibodies

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy and/or light chain variable region is derived from a particular source or species, while the remainder of the heavy and/or light chain variable region is derived from a different source or species.

Humanized antibodies

"humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all of at least one and typically two variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally comprises at least a portion of an antibody constant region derived from a human antibody. An antibody, such as a "humanized version" of a non-human antibody, refers to an antibody that has been humanized. "humanized antibody variable region" refers to the variable region of a humanized antibody.

Human antibodies

A "human antibody" is an antibody having an amino acid sequence that corresponds to an amino acid sequence produced by a human or human cell or derived from an antibody of non-human origin that utilizes a human antibody repertoire or other human antibody coding sequences. This definition of human antibodies explicitly excludes humanized antibodies that comprise non-human antigen binding residues (e.g., CDRs). "human antibody variable region" refers to the variable region of a human antibody.

Polynucleotide (nucleic acid)

"Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and/or analogue thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may comprise modifications that are performed post-synthetically, for example, conjugated to a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as those having uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those containing pendent (pendant) moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), bands Those with intercalators (e.g., acridine, psoralen, etc.), those with chelators (e.g., metals, radiometals, boron, oxidized metals, etc.), those with alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and polynucleotides in unmodified form. Furthermore, any hydroxyl groups typically present in the sugar may be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages of additional nucleotides, or may be conjugated to a solid or semi-solid support. The 5 'and 3' terminal OH groups may be phosphorylated or partially substituted with amines or organic end capping groups of 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to standard protecting groups. Polynucleotides may also comprise similar forms of ribose or deoxyribose known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl-, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl nucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, those wherein the phosphate is replaced by P (O) S ("thioester"), P (S) S ("dithioester"), (O) NR ₂ ("amic esters"), P (O) R, P (O) OR ', CO, OR CH2 ("methylal"), wherein each R OR R' is independently H OR a substituted OR unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, OR aralkyl (araldyl). Not all bonds in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

Isolated (nucleic acid)

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been isolated from a component of its natural environment. Isolated nucleic acid molecules also include nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but which are present extrachromosomally or at a chromosomal location different from their natural chromosomal location.

Carrier body

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors". The vector may be introduced into the host cell using virus or electroporation. However, the introduction of the vector is not limited to in vitro methods. For example, the vector may also be introduced into the subject directly using in vivo methods.

A subject

In the present invention, there is no particular limitation on the subject. According to a preferred embodiment herein, the subject is an animal, preferably a mammal, more preferably a human.

In a generally preferred embodiment herein, the subject (including but not limited to any medical use) relates to an animal subject, preferably a mammalian subject, more preferably a human subject.

Thus, in a preferred embodiment herein, the first antigen is an antigen of animal, preferably mammalian, more preferably human origin. Thus, in a preferred embodiment herein, the second antigen is an antigen of animal, preferably mammalian, more preferably human origin. Thus, in a preferred embodiment herein, the antibodies herein are antibodies of animal, preferably mammalian, more preferably human origin. In alternative embodiments, the antibodies herein are humanized antibodies.

Host cells

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, irrespective of the number of passages. The offspring may not be exactly identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny selected or selected for the same function or biological activity in the primary transformed cells are included herein.

Specificity (specificity)

By "specific" is meant that a molecule that specifically binds to one or more binding partners does not exhibit any significant binding to molecules other than the partners. In addition, "specificity" is also used when an antigen binding site has specificity for a particular epitope of a plurality of epitopes contained in an antigen. An antigen binding molecule is also described as "antigen binding molecule has/exhibits specificity for/against an antigen" if it specifically binds to an antigen. When an epitope that binds to an antigen binding site is contained in a plurality of different antigens, an antigen binding molecule that contains the antigen binding site can bind to various antigens having the epitope.

In addition, the molecules of the invention (i.e., "molecules comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen") can be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, it is preferable to insert a sugar chain addition sequence into the molecule so that the sugar chain produces a desired effect.

Joint

Linkers for linking two or more different components (e.g., polypeptides, peptides) include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers such as those disclosed in Protein Engineering,9 (3), 299-305, 1996. However, peptide linkers are preferred in the present disclosure. The length of the peptide linker is not particularly limited and may be appropriately selected by one skilled in the art according to purpose. The length is preferably 5 amino acids or more (the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), particularly preferably 15 amino acids. For example, when sc (Fv) ₂ When three peptide linkers are included, they may all be the same or different in length.

For example, such peptide linkers include:

Ser，

Gly-Ser，

Gly-Gly-Ser，

Ser-Gly-Gly，

Gly-Gly-Gly-Ser(SEQ ID NO：11)，

Ser-Gly-Gly-Gly(SEQ ID NO：12)，

Gly-Gly-Gly-Gly-Ser(SEQ ID NO：13)，

Ser-Gly-Gly-Gly-Gly(SEQ ID NO：14)，

Gly-Gly-Gly-Gly-Gly-Ser(SEQ ID NO：15)，

Ser-Gly-Gly-Gly-Gly-Gly(SEQ ID NO：16)，

Gly-Gly-Gly-Gly-Gly-Gly-Ser(SEQ ID NO：17)，

Ser-Gly-Gly-Gly-Gly-Gly-Gly(SEQ ID NO：18)，

(Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 13)) n, and

(Ser-Gly-Gly-Gly-Gly(SEQ ID NO：14))n，

wherein n is an integer of 1 or more. The length or sequence of the peptide linker can be selected accordingly by those skilled in the art depending on the purpose.

Synthetic linkers (chemical cross-linkers) are commonly used to cross-link peptides, examples include:

n-hydroxysuccinimide (NHS),

disuccinimidyl suberate (DSS),

bis (sulfosuccinimide) suberate (BS 3),

dithiobis (succinimidyl propionate) (DSP),

dithiobis (sulfosuccinimidyl propionate) (DTSSP),

ethylene glycol bis (succinimidyl succinate) (EGS),

ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS),

disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfo-DST),

bis [2- (succinimidyloxycarbonyloxy) ethyl ] sulfone (BSOCOES), and

bis [2- (sulfosuccinimidyloxycarbonyloxy) ethyl ] sulfone (sulfo-BSOCOES). These crosslinking agents are commercially available.

For example, typically, three linkers are required to link together the four antibody variable regions. The joints to be used may be of the same type or of different types.

Fc region

The term "Fc region" or "Fc domain" refers to a region comprising a fragment consisting of the hinge or portion thereof and the CH2 and CH3 domains in an antibody molecule. The Fc region of an IgG class refers to, but is not limited to, the region from, for example, cysteine 226 (EU numbering (also referred to herein as EU numbering)) to the C-terminus or from proline 230 (EU numbering) to the C-terminus. The Fc region may preferably be obtained by: for example, monoclonal antibodies such as IgG1, igG2, igG3 or IgG4 are partially digested with proteolytic enzymes such as pepsin, and then the fractions adsorbed on the protein A column or protein G column are re-eluted. The proteolytic enzyme is not particularly limited as long as the enzyme is capable of digesting a full-length antibody under reaction conditions (e.g., pH) appropriately set by the enzyme to form Fab or F (ab') ₂ And (3) obtaining the product. Examples thereof may include pepsin and papain.

An Fc region derived from, for example, naturally occurring IgG may be used as the "Fc region" of the present invention. Naturally occurring IgG herein refers to a polypeptide containing the same amino acid sequence as naturally occurring IgG and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. Naturally occurring human IgG refers to, for example, naturally occurring human IgG1, naturally occurring human IgG2, naturally occurring human IgG3 or naturally occurring human IgG4. Naturally occurring IgG also includes variants derived spontaneously therefrom, and the like. In Sequences of proteins of immunological interest of NIH publication No. 91-3242, a plurality of allotype sequences based on gene polymorphism are described as constant regions of human IgG1, human IgG2, human IgG3 and human IgG4 antibodies, any of which can be used in the present invention. In particular, the sequence of human IgG1 can have DEL or EEM as the amino acid sequence of EU numbering positions 356-358.

In some embodiments, the Fc domain of an antibody belonging to a molecule of the invention consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises CH2 and CH3IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stably associating with each other. In one embodiment, the molecules of the invention may comprise no more than one Fc domain.

In one embodiment, the Fc domain that a molecule may comprise is an IgG Fc structure. In a particular embodiment, the Fc domain is an IgG1Fc domain. In another embodiment, the Fc domain is an IgG1Fc domain. In a further specific embodiment, the Fc domain is a human IgG1Fc region.

Production and purification of multispecific antibodies

As described above, in the case where the molecule of the invention, i.e. "comprising a first part that binds a first antigen and a second part that binds a second antigen", is for example an antigen binding molecule such as an antibody (which is also referred to as an antigen binding molecule as described above), in some embodiments the "second part that binds a second antigen" and (if appropriate) further "first part that binds a first antigen" may be an antigen binding domain of an antibody or derived from an antibody. In one embodiment, the molecule, i.e., an antigen binding molecule such as an antibody, may be a multispecific antigen binding molecule such as bispecific, trispecific, and tetraspecific antibodies or antigen binding molecules.

In one example, the multispecific antigen-binding molecules described herein comprise two different antigen-binding portions (e.g., a "first antigen-binding portion" (i.e., a first portion) and a "second antigen-binding portion" (i.e., a second portion) fused to one or the other of the two subunits of an Fc domain, so that the two subunits of an Fc domain are typically contained in two different polypeptide chains.

Thus, in certain embodiments, the Fc domain of the multispecific antigen-binding molecules described herein comprises modifications that promote the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the Fc domain of human IgG is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

In particular embodiments, the modification is a so-called "mortar" modification, including a "mortar" modification in one of the two subunits of the Fc domain and a "mortar" modification in the other of the two subunits of the Fc domain.

For example in US 5,731,168; US7,695,936; pestle and mortar techniques are described in Ridgway et al, prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method includes introducing a protrusion ("slug") at the interface of the first polypeptide and a corresponding cavity ("socket") in the interface of the second polypeptide, such that the protrusion may be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the first polypeptide interface with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide.

Thus, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the multispecific antigen-binding molecule, the amino acid residues are substituted with amino acid residues having a greater side-chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residues are substituted with amino acid residues having a smaller side-chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

Protrusions and cavities may be formed by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or peptide synthesis.

In a specific embodiment, the threonine residue at position 366 is replaced with a tryptophan residue in the CH3 domain of the first subunit of the Fc domain (T366W), and the tyrosine residue at position 407 is replaced with a valine residue in the CH3 domain of the second subunit of the Fc domain (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A).

In yet another embodiment, the serine residue at position 354 is additionally substituted with a cysteine residue in the first subunit of the Fc domain (S354C), and the tyrosine residue at position 349 is additionally substituted with a cysteine residue in the second subunit of the Fc domain (Y349C). The introduction of these two cysteine residues results in the formation of disulfide bonds between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248,7-15 (2001)).

In other embodiments, other techniques for promoting association between H chains and between L and H chains with a desired combination may be applied to the multispecific antigen-binding molecules of the present invention.

For example, techniques for inhibiting undesired H chain associations by introducing electrostatic repulsion at the interface of the second constant region or the third constant region (CH 2 or CH 3) of an antibody H chain can be applied to multispecific antibody associations (WO 2006/106905).

In a technique for inhibiting undesired H chain association by introducing electrostatic repulsion at the interface of CH2 or CH3, examples of amino acid residues contacted at the interface of another constant region of the H chain include regions corresponding to residues at positions 356, 439, 357, 370, 399 and 409 of EU numbering in the CH3 region.

More specifically, examples include antibodies comprising two types of H chain CH3 regions, wherein 1 to 3 pairs of amino acid residues in the first H chain CH3 region selected from the amino acid residue pairs shown in (1) to (3) below carry the same type of charge: (1) Amino acid residues comprised at EU numbering positions 356 and 439 in the CH3 region of the H chain; (2) Amino acid residues comprised at EU numbering positions 357 and 370 in the H chain CH3 region; and (3) comprises amino acid residues at EU numbering positions 399 and 409 in the CH3 region of the H chain.

Furthermore, the antibody may be an antibody in which the pair of amino acid residues in the second H chain CH3 region different from the above-mentioned first H chain CH3 region is selected from the pair of amino acid residues of (1) to (3) above, wherein 1 to 3 pairs of amino acid residues corresponding to the pair of amino acid residues of (1) to (3) above carrying the same type of charge in the above-mentioned first H chain CH3 region have opposite charges to the corresponding amino acid residues of the above-mentioned first H chain CH3 region.

Each of the amino acid residues shown in (1) to (3) above approaches each other during association. The person skilled in the art can find out positions corresponding to the amino acid residues of the above-mentioned (1) to (3) in the desired H chain CH3 region or H chain constant region by homology modeling or the like using commercially available software, and the amino acid residues at these positions can be appropriately modified.

In the above antibodies, the "charged amino acid residues" are preferably selected from, for example, amino acid residues comprised in any one of the following groups:

(a) Glutamic acid (E) and aspartic acid (D); and

(b) Lysine (K), arginine (R), and histidine (H).

In the above antibody, the phrase "carrying the same charge" means, for example, that 2 or more amino acid residues are all selected from the amino acid residues contained in any one of the above groups (a) and (b). The phrase "carrying opposite charges" means, for example, when at least one amino acid residue of two or more amino acid residues is selected from the amino acid residues contained in any one of the above groups (a) and (b), the remaining amino acid residues are selected from the amino acid residues contained in the other groups.

In a preferred embodiment, the above antibodies may have their first H chain CH3 region and second H chain CH3 region crosslinked by disulfide bonds.

In the present invention, the amino acid residues to be modified are not limited to the amino acid residues of the antibody variable region or antibody constant region described above. Those skilled in the art can identify interfacial amino acid residues in mutant polypeptides or heteromultimers by methods such as homology modeling using commercially available software; amino acid residues at these positions can then be modified to modulate association.

In addition, other known techniques may be used to form multispecific antibodies. Association of polypeptides having different sequences can be efficiently induced by CH3 complementary binding using a strand-exchange engineering domain CH3 generated by changing a portion of an antibody's H chain CH3 to a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of another H chain CH3 (Protein Engineering Design & Selection,23;195-202, 2010). This known technique can also be used to efficiently form target multispecific antibodies.

Furthermore, as in WO 2011/028952, WO2014/018572 and Nat Biotechnol.2014Feb;32 (2): 191-8 using an association of antibodies CH1 and CL, and an association of VH and VL; techniques for producing bispecific antibodies using separately prepared monoclonal antibody combinations (Fab arm exchange) as described in WO2008/119353 and WO 2011/131746; techniques for modulating the association between antibody heavy chains CH3 as described in WO2012/058768 and WO 2013/067302; as described in WO 2012/023553, techniques for producing a multispecific antibody consisting of two types of light chains and one type of heavy chain; techniques for producing multispecific antibodies, and the like, using two bacterial cell lines that individually express one of the chains of an antibody comprising a single H chain and a single L chain, as described by Christoph et al (Nature Biotechnology Vol.31, p 753-758 (2013); can be used for the formation of multispecific antibodies.

Alternatively, even in the case where the target multispecific antibody cannot be efficiently formed, the multispecific antibody can be obtained by separating and purifying the target multispecific antibody from the produced antibody. For example, a method of imparting isoelectric point differences by introducing amino acid substitutions into the variable regions of two types of H chains, thereby enabling purification of two types of homo-and hetero-poly (heteromeric) antibodies of interest by ion exchange chromatography has been reported (WO 2007114325). Heretofore, as a method of purifying a hetero-antibody, a method of using protein a in order to purify a hetero-dimeric antibody comprising a mouse IgG2a H chain which binds to protein a and a rat IgG2b H chain which does not bind to protein a has been reported (WO 98050431 and WO 95033844). Furthermore, heterodimeric antibodies can be efficiently purified by: an H chain comprising amino acid residues at EU numbering positions 435 and 436, which are IgG protein a binding sites, replaced with Tyr, his, or an amino acid that produces a different protein a affinity, or an H chain with a different protein a affinity is used to alter the interaction of each H chain with protein a, then purified using a protein a column.

Furthermore, an Fc region having improved C-terminal heterogeneity of the Fc region can be suitably used as the Fc region of the present invention. More specifically, the present invention provides an Fc region produced by deleting glycine at position 446 and lysine at position 447 as specified by EU numbering from the amino acid sequences of two polypeptides constituting the Fc region derived from IgG1, igG2, igG3 or IgG 4.

The multispecific antigen-binding molecules prepared as described herein may be purified by techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens that bind to the multispecific antigen-binding molecules may be used. For example, for affinity chromatography purification of a multispecific antigen-binding molecule, a matrix with protein a or protein G may be used. Sequential protein a or G affinity chromatography and size exclusion chromatography can be used to isolate multi-specific antigen binding molecules. The purity of the multispecific antigen-binding molecule can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high performance liquid chromatography, and the like.

Antibody dependent cell-mediated cytotoxicity

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells bearing an antigen, followed by killing of the target cells with cytotoxins. Primary cells mediating ADCC NK cells express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. Ravetch and Kinet, annu.rev.immunol.9: table 3 on page 464 of 457-492 (1991) summarizes FcR expression on hematopoietic cells. In order to assess ADCC activity of a target molecule, an in vitro ADCC assay may be performed, such as those described in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta). Useful effector cells for such assays include PBMCs and NK cells. Alternatively or additionally, for example in a process such as Clynes et al pnas (USA) 95:652-656 (1998) in vivo, the ADCC activity of a target molecule is assessed in an animal model.

Complement dependent cytotoxicity

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1 q) to antibodies (appropriate subclasses) that bind to their cognate antigens. To assess complement activation, CDC assays can be performed, for example, as in Gazzano-Santoro et al, j.immunol.methods 202:163 (1996). For example, polypeptide variants having altered amino acid sequences of the Fc region (polypeptides having variant Fc regions) and increased or decreased C1q binding capacity are described in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642. See, for example, idusogie et al j. Immunol.164:4178-4184 (2000).

Pharmaceutical composition

In one aspect, the present disclosure provides pharmaceutical compositions comprising a molecule of the invention as described above, e.g. "comprising a first moiety that binds a first antigen and a second moiety that binds a second antigen".

In certain embodiments, the pharmaceutical compositions of the invention are used to confer signaling or signal blocking to cells (e.g., immune cells, cells in affected tissue, as described above), in other words, directly or indirectly activate, modulate, suppress or inhibit immune cells, or suppress or inhibit cells (e.g., tumor cells) in affected tissue. In one embodiment, the pharmaceutical composition of the invention is a therapeutic agent for inducing cytotoxicity. In certain embodiments, the pharmaceutical compositions of the present disclosure are pharmaceutical compositions for the treatment and/or prevention of cancer, autoimmune diseases, inflammation, viral infection, and the like.

In the present invention, the pharmaceutical composition for treatment may also be referred to as a therapeutic agent (including but not limited to cytostatic agents, anticancer agents, etc.).

In the present invention, the terms "cancer", "cancerous" and "cancerous" refer to or describe the physiological condition of a mammal, which is typically characterized by unregulated cell growth/proliferation. In the present invention, the term "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, as well as all pre-cancerous and cancerous cells and tissues. The terms "cancer", "canceration", "cell proliferative disorder", "proliferative disorder" and "tumor" as described herein are not mutually exclusive. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

In the present invention, cancers include, but are not limited to, for example, stomach cancer, head and neck cancer, esophageal cancer, colorectal cancer, lung cancer, mesothelioma, liver cancer, ovarian cancer, breast cancer, colon cancer, kidney cancer, skin cancer, muscle tumor, pancreatic cancer, prostate cancer, testicular cancer, uterine cancer, bile duct cancer, meckel cell cancer, bladder cancer, thyroid cancer, schwannoma, adrenal cancer, anal cancer, central nervous system tumor, neuroendocrine tissue tumor, penile cancer, pleural tumor, salivary gland tumor, vulval cancer, thymoma, lymphoma, myeloid leukemia, childhood cancer (wilms tumor, neuroblastoma, sarcoma, hepatoblastoma, and germ cell tumor). More preferred cancer types include gastric, colorectal, lung, mesothelioma, liver, breast, skin, lymphoma and myeloid leukemia, but are not limited thereto (Tumori. (2012) 98, 478-484; tumor Biol. (2015) 36, 4671-4679;Am J Clin Pathol (2008) 130, 224-230;Adv Anat Pathol (2014) 21, 450-460; med Oncol (2012) 29, 663-669;Clinical Cancer Research (2004) 10, 6612-6621;Appl Immunohistochem Mol Morphol (2009) 17, 40-46;Eur J Pediatr Surg (2015) 25, 138-144;J Clin Pathol (2011) 64, 587-591;Am J Surg Pathol (2006) 30, 1570-1575; oncology (73, 389-394;Diagnostic Pathology (2010) 64,1-6;Diagnostic Pathology (2015) 34,1-6;Am J Clin Pathol (2008) 129, 899-906;Virchows Arch (2015) 466, 67-76). More preferred types of cancer include gastric cancer, lung cancer, mesothelioma, liver cancer, breast cancer, skin cancer, lymphoma and myeloid leukemia.

In the present invention, "autoimmune diseases" include, but are not limited to, vitiligo, type I diabetes, and the like.

In the present invention, "inflammation" includes, but is not limited to, inflammation in the following tissues.

i. Joint tissue associated with rheumatoid arthritis or osteoarthritis.

ii lung tissue associated with bronchial asthma.

iv fibrotic tissue associated with liver, kidney or lung fibrosis.

Tissue associated with immune rejection by organ transplantation.

vii visceral adipose tissue associated with metabolic syndrome.

Skin tissue associated with atopic dermatitis or any other dermatitis.

Spinal nerve tissue associated with herniated disk or chronic back pain.

Bone tissue associated with a fracture.

Tissue associated with burn.

xii is a tissue of an organ tissue injured by mechanical, physical or chemical external forces.

If desired, the pharmaceutical compositions of the invention may be formulated with one or more molecules of the invention. For example, activation, modulation, suppression or inhibition of the immune response of immune cells, or the cytotoxic effect of cells expressing an antigen, may be enhanced by a mixture of two or more molecules of the invention.

Pharmaceutical compositions comprising a molecule of the invention are prepared by mixing a molecule of the invention having the desired purity as described above with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, including, but not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (HYLENEX (registered trademark), baxter International, inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/026086 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient, preferably those active ingredients having complementary activities that do not adversely affect each other, as desired for the particular indication being treated. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

If necessary, the molecules of the present invention as described above may be encapsulated in microcapsules (microcapsules made of hydroxymethyl cellulose, gelatin, polymethyl methacrylate, etc.) and made into components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Science th edition", oslo Ed. (1980)), furthermore, methods of preparing agents as sustained release agents are known, and these methods may be applied to antigen binding molecules of the present disclosure (j.biomed.mater.res. (1981) 15, 267-277; chemtech. (1982) 12, 98-105; U.S. Pat. No. 3773719; european patent application nos. EP58481 and EP133988; biopolymers (1983) 22, 547-556).

Pharmaceutical compositions comprising a molecule of the invention as described above may be administered to a patient orally or parenterally. Parenteral administration is preferred. In particular, such methods of administration include injection, nasal administration, pulmonary administration, and transdermal administration. Injections include, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, pharmaceutical compositions comprising a molecule of the invention as described above may be administered topically or systemically by injection. Furthermore, an appropriate administration method may be selected according to the age and symptoms of the patient. For each administration, the dose administered may be selected from the range of, for example, 0.0001mg to 1,000mg/kg body weight. Alternatively, the dosage may be selected from the range of, for example, 0.001 mg/body to 100,000 mg/body per patient. However, the dosage of the pharmaceutical composition of the present invention is not limited to these dosages.

The present invention provides a method of conferring signal transmission or signal blocking in a cell (e.g. an immune cell, a cell in an affected tissue, as described above), in other words, directly or indirectly activating, modulating, suppressing or inhibiting an immune cell, or suppressing or inhibiting a cell in an affected tissue (e.g. a tumor cell), comprising administering a molecule of the invention as described above to a subject in need thereof. In one embodiment, the method of the invention is used to induce cytotoxicity in the affected tissue as described above. The invention also provides a method of treating and/or preventing cancer, autoimmune diseases, inflammation, viral infection, etc., comprising administering a molecule of the invention as described above to a subject in need thereof.

The present disclosure also provides a kit for use in the method of the invention comprising the molecule of the invention as described above. The kit may be packaged with additional pharmaceutically acceptable carriers or media, or instructions describing how to use the kit, and the like.

In another aspect of the invention, articles of manufacture are provided that contain materials for the treatment, prevention and/or diagnosis of the above-described conditions. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be made of a variety of materials such as glass or plastic. The container contains the composition, either by itself or in combination with another composition effective to treat, prevent and/or diagnose the condition, and may have a sterile access (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the invention.

The label or package insert indicates that the composition is to be used to treat the selected condition. Furthermore, the article of manufacture may comprise (a) a first container having a composition therein, wherein the composition comprises a molecule of the invention as described above; and (b) a second container having a composition contained therein, wherein the composition contains a further cytotoxic or other therapeutic agent.

The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

Package insert

The term "package insert" is used to refer to instructions that are typically contained in commercial packages of therapeutic products, including information about the indication, usage, dosage, administration, combination therapy, contraindications and/or warnings of use of such therapeutic products.

Pharmaceutical preparation

The term "pharmaceutical formulation" or "pharmaceutical composition" is used interchangeably to refer to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation is to be administered.

Pharmaceutically acceptable carrier

By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

Treatment of

As used herein, "treatment" (and grammatical variations thereof, such as "treatment") or "treatment" used interchangeably herein refers to clinical intervention to attempt to alter the natural course of a treated individual, and may be used to prevent or progress during a clinical pathology process. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a disease state, alleviating or improving prognosis. In some embodiments, the antigen binding molecules or antibodies of the present disclosure are used to delay the onset of a disease or to slow the progression of a disease.

Other Agents and treatments

The molecules of the invention as described above may be administered in combination with one or more other therapeutic agents. For example, a molecule of the invention as described above may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise any active ingredient suitable for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, cytostatic agent, cytotoxic agent, apoptosis activator, or agent that increases sensitivity of the cell to an apoptosis inducer. In particular embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

Such other agents are suitably present in combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of the molecules of the invention used as described above, the type of disorder or treatment, and other factors discussed above. The molecules of the invention as described above are generally used at the same dosages and routes of administration as described herein, or about 1 to 99% of the dosages described herein, or any dosages and any routes of administration as empirically/clinically determined to be appropriate.

Such combination therapies as described above include the combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and the separate administration, in which case the administration of the molecules of the invention as described above may occur before, simultaneously with and/or after the administration of additional therapeutic agents and/or adjuvants. The molecules of the invention as described above may also be used in combination with radiation therapy.

Hereinafter, specific embodiments of the present invention are set forth in more detail, which are directed in part to, but not limited to, the embodiments outlined above for D1 to D37. All definitions and further detailed description given above relate in particular also to the following embodiments.

That is, the present invention is particularly directed to antigen binding molecules comprising (1) at least one first moiety that binds a first antigen, and (2) at least one second moiety that binds a second antigen; wherein the first antigen is a damage-associated molecular pattern (DAMP), and wherein the second antigen is a different antigen than the first antigen.

Furthermore, the present invention relates in particular to an antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the second antigen is different from the first antigen.

According to the present invention, the term "tissue-state specific tissue-derived antigens" relates to antigens that are exposed to the extracellular environment due to various cell death programs that are characterized in organs and tissues as a result of microbial infection, cellular stress, injury and chemotherapy exposure. Apoptosis, necrotic apoptosis and pyro-apoptosis are well known as various cell death programs. Dying and dead cells release various self-proteins and bioactive chemicals that originate from the cytoplasm, nucleus, endoplasmic reticulum and mitochondria. These endogenous factors are named pathogen-associated molecular patterns (PAMPs), lesion-associated molecular patterns, cell death-associated molecular patterns (CDAMPs) and sirens (alarmins) (Beatriz Sangiuliano et al, mediators Inflammation (2014) 2014: article ID 821043, pages 1-14).

In general, herein, the damage-associated molecular pattern (DAMP) is preferably exposed to the extracellular environment due to cell damage, in particular due to cell death. Preferably, due to the cell damage, the molecule is capable of providing a cell contact and/or signal, wherein the contact and/or signal is related to the second antigen.

Generally, herein, the cell damage preferably i) involves cell death, in particular cell death, and/or ii) occurs in affected tissues, in particular selected from tissues affected by a condition or disease, in particular from tissues affected by cancer or autoimmune diseases, in particular in the Tumor Microenvironment (TME); and/or iii) caused by any one selected from necrosis, apoptosis, autophagic cell death and unexpected cell death, and/or iv) is cell death selected from the group consisting of necrosis, apoptosis, autophagic cell death and unexpected cell death.

Thus, in certain embodiments herein, the cell damage preferably involves cell death, particularly cell death. Thus, in certain embodiments herein, the cell damage preferably occurs in affected tissues, particularly selected from tissues affected by a condition or disease, particularly tissues affected by cancer or autoimmune disease, particularly in the Tumor Microenvironment (TME). Thus, in certain embodiments herein, the cell damage is preferably caused by any one selected from the group consisting of necrosis, apoptosis, autophagy cell death, and unexpected cell death. Thus, in certain embodiments herein, the cell damage is preferably cell death selected from the group consisting of necrosis, apoptosis, autophagy cell death, and unexpected cell death.

The first antigen as defined herein is not particularly limited, and certain preferred embodiments correspond to those described above.

In fact, the first antigen may be any DAMP. DAMPs are generally well known and include in particular all DAMPs described elsewhere herein, including those molecules described elsewhere herein that are exposed to the extracellular environment due to cell death.

In certain preferred embodiments, the first antigen is a cytoskeletal component or portion thereof, a cell membrane component or portion thereof, an organelle component or portion thereof, a cytoplasmic protein or portion thereof, and a metabolite.

In certain preferred embodiments, the first antigen is located on any one selected from the group consisting of a filar (particularly an intermediate filament), a nucleosome, a cytoskeleton and/or a nuclear scaffold.

In certain preferred embodiments, the first antigen is capable of multimerizing a plurality of molecules. In certain additional/alternative preferred embodiments, the first antigen is present in a multimeric molecule.

In a specific embodiment, the first antigen is selected from the group consisting of a cytoskeletal component or part thereof, a cell membrane component or part thereof, an organelle component or part thereof, a cytoplasmic protein or part thereof, and a metabolite, and is located on any one selected from the group consisting of a filament (particularly an intermediate filament), a nucleosome, a cytoskeletal, and a nuclear scaffold.

In certain preferred embodiments, the first antigen is selected from the group consisting of: a) Actin, in particular F-actin; b) Heat shock proteins, in particular selected from HSP90 and GRP78, in particular HSP90; c) Phosphatidylserine (PS), particularly phosphatidylserine that is part of the cell membrane; d) Histone or a component thereof, E) histone deacetylase complex subunits, in particular SAP130, F) HMGN protein, in particular selected from HMGB1 and HMGN1, G) hepatocellular carcinoma-derived growth factor, H) BCL-2, i) calreticulin, and J) cyclophilin a.

In certain preferred embodiments, the first antigen is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP 130; more preferably selected from the group consisting of F-actin, phosphatidylserine and HSP90; even more preferably selected from the group consisting of F-actin and HSP90, and in particular, the first antigen is F-actin.

The first part defined herein is not particularly limited, and certain preferred embodiments correspond to those described above.

The present embodiment relating to the primary antigen may additionally or alternatively be further defined by reference to the first portion.

In certain preferred embodiments, the first moiety is an Ig-type binding moiety.

In other preferred embodiments, the first moiety is a binding polypeptide. Preferably, the binding polypeptide is a non-Ig-type binding moiety.

According to certain preferred embodiments, the first moiety is selected from the group consisting of: a) A moiety capable of binding actin, in particular F-actin; b) A moiety capable of binding to a heat shock protein, in particular a heat shock protein selected from the group consisting of HSP90 and GRP 78; c) A moiety capable of binding Phosphatidylserine (PS), in particular phosphatidylserine as part of a cell membrane; d) A moiety capable of binding to histone or a component thereof, E) a moiety capable of binding to a histone deacetylase complex subunit, in particular SAP130, F) a moiety capable of binding to HMGN protein (in particular selected from HMGB1 and HMGN 1), G) a moiety capable of binding to hepatocellular carcinoma-derived growth factor, H) a moiety capable of binding to BCL-2, I) a moiety capable of binding to calreticulin and J) a moiety capable of binding to cyclophilin a.

In a particularly preferred embodiment, the first moiety is selected from the group consisting of: a moiety capable of binding to F-actin, a moiety capable of binding to GRP78, a moiety capable of binding to phosphatidylserine, a moiety capable of binding to HSP90, and a moiety capable of binding to SAP 130. More preferably, wherein the first portion is selected from the group consisting of: a moiety capable of binding to F-actin, a moiety capable of binding to phosphatidylserine, and a moiety capable of binding to HSP 90. In particular, the first moiety is selected from the group consisting of a moiety capable of binding to F-actin and a moiety capable of binding to HSP90, and in particular the first moiety is a moiety capable of binding to actin, preferably F-actin.

In certain embodiments, wherein the first antigen is actin, preferably F-actin, the first moiety is an Ig-type binding moiety. In other preferred embodiments, wherein the first antigen is actin, preferably F-actin, the first moiety is a binding polypeptide, preferably Clec9A polypeptide.

In certain embodiments, wherein the first antigen is GRP78, the first moiety is an Ig-type binding moiety, preferably an Ig-type binding moiety from any of the corresponding antibodies described herein, more preferably an Ig-type binding moiety from antibody GA20 or Mab 159.

In certain embodiments, wherein the first antigen is phosphatidylserine, the first moiety is an Ig-type binding moiety, preferably an Ig-type binding moiety from any of the corresponding antibodies described herein, more preferably an Ig-type binding moiety from antibody 3G 4.

In certain embodiments, wherein the first antigen is HSP90, the first moiety is an Ig-type binding moiety, preferably an Ig-type binding moiety from any of the corresponding antibodies described herein, more preferably an Ig-type binding moiety from antibody 1.5.1 or 6H 8.

In certain embodiments, wherein the first antigen is SAP130, the first moiety is an Ig-type binding moiety. In other preferred embodiments, wherein the first antigen is SAP130 and the first moiety is a binding polypeptide, preferably an mClec4e polypeptide.

Preferably, also in the above embodiments, the Ig-type binding moiety is selected from the group consisting of a single domain antibody (VHH), an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ A group of groups.

More preferably, the Ig-type binding moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ A group of groups.

In a preferred general embodiment, the molecule comprises said first moiety at the Fc terminus of the antigen binding molecule.

The second antigen as defined herein is not particularly limited, and certain preferred embodiments correspond to those described above.

In a particularly preferred embodiment, the second antigen is a membrane protein of an immune cell. Preferably, the second antigen is a membrane protein in the cell membrane of a T cell selected from the group consisting of T cell receptor, CD3, CD137, CD40, CTLA4, costimulatory molecule, and costibiting molecule.

In a preferred embodiment, the second antigen herein is a membrane protein involved in signal transmission in a cell, preferably wherein the cell is i) an immune cell, in particular wherein the immune cell is selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, an NK cell, an NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil and a basophil, ii) a tumor cell, or iii) an autoreactive cell.

In certain preferred embodiments, the second antigen is a signaling receptor. In certain preferred embodiments, the second antigen is an immune cell receptor antigen. In certain preferred embodiments, the second antigen is a signaling receptor and immune cell receptor antigen.

In certain embodiments, the second antigen is an endosomal membrane protein. In a specific embodiment, the second antigen is a membrane protein in the cell membrane of a T cell, in particular a membrane protein of a T cell membrane selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a co-stimulatory molecule or a co-inhibitory molecule. In a particular embodiment, the second antigen is a membrane protein in the cell membrane of a cell other than a T cell, in particular it is capable of binding to a co-stimulatory or co-inhibitory molecule in the cell membrane of a T cell.

In certain preferred embodiments, the costimulatory molecule is selected from the group consisting of CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR). Tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B 4).

In certain preferred embodiments, the co-inhibitory molecule is selected from the group consisting of CD279 (PD-1), CD152 (CTLA-4), TIGIT, BTLA, CD366 (Tim-3), CD96, CD112R or CD 200R.

In certain preferred embodiments, the membrane protein capable of binding to a costimulatory or co-inhibitory molecule is selected from the group consisting of CD274 (PD-L1), CD273 (PD-L2), CD80, CD86, CD276 (ICOSL), CD276 (VICN 1), VISTA, HHA 2, a member of the milk philin family, CD270 (HVEM), CD137L (TNFSF-9), CD252 (OX 40L), CD70 (TNFSF-7), CD40 (TNFRSF-7), GIRL (TNFSF-18), CD155 (PVR, NECL 5), CD112 (NECTIN-2), CD200 (MOX 1), CD48 (BCM-1) and Gal-9.

In a particularly preferred embodiment, the second antigen is selected from the group consisting of CD3, CD137, CD40 and CTLA4, preferably from the group consisting of CD3 and CD137, more preferably the second antigen is CD3.

The second part as defined herein is not particularly limited and certain preferred embodiments correspond to those described above.

The present embodiment relating to the second antibody may additionally or alternatively be further defined by reference to the second part.

In a preferred embodiment, the second moiety is an Ig type binding moiety. Thus, in a particularly preferred embodiment, the invention relates to an antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

Wherein the first antigen is a Damage Associated Molecular Pattern (DAMP),

wherein the second antigen is different from the first antigen, and

wherein the second antigen is a membrane protein of an immune cell, and wherein the second moiety is an Ig-type binding moiety.

In a particularly preferred embodiment, the invention relates to an antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a Damage Associated Molecular Pattern (DAMP),

the first moiety is an Ig-type binding moiety,

wherein the second antigen is different from the first antigen,

wherein the second antigen is a membrane protein of an immune cell, and

wherein the second moiety is an Ig type binding moiety.

In a further particularly preferred embodiment, the present invention relates to an antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a Damage Associated Molecular Pattern (DAMP),

wherein the first part is Clec9a or mClec4e,

wherein the second antigen is different from the first antigen,

Wherein the second antigen is a membrane protein of an immune cell, and

wherein the second moiety is an Ig type binding moiety.

In certain embodiments, the second moiety is a moiety that is capable of binding to a membrane protein of an endosome.

In a particular embodiment, the second moiety is a moiety capable of binding to a membrane protein in the cell membrane of a T cell (preferably a membrane protein of a T cell membrane selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, co-stimulatory molecule, or co-inhibitory molecule).

In a particular embodiment, the second moiety is a moiety capable of binding to a membrane protein of a cell membrane of a cell other than a T cell (in particular, which is capable of binding to a co-stimulatory molecule or co-inhibitory molecule in the cell membrane of a T cell).

In certain preferred embodiments, the costimulatory molecule is selected from the group consisting of CD28 (TP 44), CD278 (ICOS), CD27 (TNFRSF-7), CD30, CD134 (OX 40), CD357 (GITR), tim-2, CD28H (TMIGD 2), CD137 (4-1 BB, TNFRSF-9), CD226 (DNAM-1), and CD244 (2B 4).

In particularly preferred embodiments, the second moiety is selected from the group consisting of a moiety capable of binding CD3, a moiety capable of binding CD137, a moiety capable of binding CD40, and a moiety capable of binding CTLA4, more preferably from the group consisting of a moiety capable of binding CD3 and a moiety capable of binding CD137, even more preferably the second moiety is a moiety capable of binding CD 3.

In the above embodiments, preferably, the moiety capable of binding CD3 is an Ig-type binding moiety. In the above embodiments, preferably, the moiety capable of binding CD137 is an Ig-type binding moiety. In the above embodiments, preferably, the moiety capable of binding CD40 is an Ig-type binding moiety. In the above embodiments, preferably, the moiety capable of binding CTLA4 is an Ig-type binding moiety.

Preferably, the Ig-type binding moiety is selected from the group consisting of: single domain antibodies (VHH), combinations of antibody heavy chain Variable (VH) and antibody light chain Variable (VL) regions, single domain antibodies (sdAb), single chain Fv (scFv), single chain antibodies, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ 。

In a preferred general embodiment, the molecule is preferably F (ab') in the antigen binding region of the antigen binding molecule ₂ The second portion is contained in the zone.

In a preferred general embodiment, the molecule comprises at least one first moiety and at least one second moiety, in particular wherein the antigen binding molecule comprises one first moiety and one second moiety, more in particular wherein the antigen binding molecule comprises at least two, preferably two, first moieties and at least two, preferably two, second moieties.

In certain embodiments herein, the antigen binding molecule is capable of binding to a first antigen and a second antigen upon light irradiation.

In a preferred general embodiment, the antigen binding molecule is characterized in that the complex formed by binding one or more molecules to the first antigen is capable of binding more than one membrane protein via the second moiety. In certain embodiments, a complex formed by binding one or more molecules to a first antigen is capable of binding to one or more membrane proteins via a second moiety to confer signaling in a cell via the one or more membrane proteins. In other particular embodiments, the complex formed by binding of one or more molecules to the first antigen is capable of binding to one or more membrane proteins via the second moiety, thereby conferring signal blocking in the cell via the one or more membrane proteins.

In general, the antigen binding molecules herein may be selected from the group consisting of: a) a polypeptide complex, which is optionally a fusion protein, B) a polynucleotide (preferably consisting of two entities, preferably connected by a linker), C) a medium-sized chemical compound (preferably consisting of two entities, preferably connected by a linker), and D) a small chemical compound (preferably consisting of two entities, preferably connected by a linker).

In a preferred general embodiment herein, the antigen binding molecule is a polypeptide complex.

In a particularly preferred embodiment herein, the antigen binding molecule is an antibody or a fragment of an antibody, in particular an antibody. In certain embodiments, the antibodies are further characterized as described elsewhere herein.

In certain preferred embodiments, the antibody, antigen binding molecule is an IgG type antibody. In certain preferred embodiments, the antibody, antigen binding molecule is a bispecific IgG type antibody.

In other preferred general embodiments herein, the antigen binding molecule is a polypeptide complex, which is a fusion protein. Preferably, the molecule is an antibody or fragment thereof as a fusion protein, in particular an antibody as a fusion protein.

Preferably, the antibody is an IgG type antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain, in particular an antibody having at least one binding polypeptide, preferably two binding polypeptides, fused to its Fc domain.

The binding polypeptide may be fused to the antigen binding molecule using one or more linkers, preferably any of the linkers described elsewhere herein.

Generally, in certain preferred embodiments, wherein the antigen binding molecule is an antibody, i-1) the first moiety is an Ig-type binding moiety, or i-2) the first moiety is a binding polypeptide, and/or ii) the second moiety is an Ig-type binding moiety.

In certain preferred embodiments, wherein the antigen binding molecule is an antibody (particularly a fusion protein), the antibody is preferably capable of binding to a membrane protein of an immune cell, and further comprises at least one binding polypeptide, preferably linked to an Fc region.

Preferably, the binding polypeptide is selected from the group consisting of Clec9A polypeptide and mClec4e polypeptide, more preferably the antibody is selected from the group consisting of an anti-CD 3 antibody comprising a Clec9A polypeptide, an anti-CD 137 antibody comprising a Clec9A polypeptide, an anti-CD 3 antibody comprising a mClec4e polypeptide, and an anti-CD 137 antibody comprising a mClec4e polypeptide.

In a particular embodiment, the antigen binding molecule comprises at least one, preferably two Clec9A polypeptides, in particular wherein the antigen binding molecule further comprises i) at least two, preferably two, moieties involved in signal transmission in a cell, or ii) at least two, preferably two, moieties that are immune cell receptor antigens.

Similarly, in a particular embodiment, the antigen binding molecule comprises at least one, preferably two mClec4e polypeptides, in particular wherein the antigen binding molecule further comprises i) at least two, preferably two, moieties involved in signal transmission in a cell, or ii) at least two, preferably two, moieties that are immune cell receptor antigens.

In a generally preferred embodiment, wherein the antigen binding molecule is an antibody, the antibody is a membrane protein directed against immune cells and a bispecific antibody directed against DAMP, more preferably the antibody is selected from the group consisting of a bispecific anti-HSP 90 anti-CD 3 antibody, a bispecific anti-HSP 90 anti-CD 3 antibody; bispecific anti-GRP 78 anti-CD 3 antibodies, bispecific anti-GRP 78 anti-CD 137 antibodies, bispecific anti-phosphatidylserine anti-CD 3 antibodies, and bispecific anti-phosphatidylserine anti-CD 137 antibodies.

Generally, in certain preferred embodiments, wherein the antigen binding molecule is an antibody, i-1) the first moiety is an Ig-type binding moiety, or i-2) the first moiety is a binding polypeptide, and ii) the second moiety is an Ig-type binding moiety.

In particular embodiments herein, a) the first moiety is an Ig-type binding moiety selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain Variable (VH) region and an antibody light chain Variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ A group of; and/or b) the second moiety is an Ig-type binding moiety selected from the group consisting of a single domain antibody (VHH), a combination of an antibody heavy chain Variable (VH) region and an antibody light chain Variable (VL) region, a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, fv, single chain Fv2 (scFv 2), fab and F (ab') ₂ A group of; and/or c) the binding polypeptide is a non-Ig type binding moiety.

In a further preferred specific embodiment herein, a) the first moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ The binding domain of the group consisting, and/or b) the second moiety is selected from the group consisting of VHH, scFv, scFv, fab and F (ab') ₂ Binding domains of the group consisting; and/or c), wherein the binding polypeptide is a non-Ig-type binding moiety, in particular a non-Ig-type binding moiety linked to the Fc domain of the antigen binding molecule.

In a preferred specific embodiment herein, the antigen binding molecule is an antibody selected from the group consisting of: a) An antibody capable of binding to F-actin and an antigen involved in signal transmission in a cell; b) An antibody capable of binding to F-actin and an immune cell receptor; c) An antibody capable of binding HSP90 and an antigen involved in signal transmission in a cell; d) Antibodies capable of binding HSP90 and immune cell receptors; e) An antibody capable of binding GRP78 and an antigen involved in signal transmission in a cell; f) An antibody capable of binding GRP78 and an immune cell receptor; g) An antibody capable of binding to phosphatidylserine and an antigen involved in signal transmission in a cell; h) An antibody capable of binding phosphatidylserine and an immune cell receptor; i) An antibody capable of binding SAP130 and an antigen involved in signal transmission in a cell; and J) an antibody capable of binding SAP130 and an immune cell receptor.

More preferably, the antibody is selected from the group consisting of: a-1) an antibody capable of binding F-actin and CD 3; a-2) an antibody capable of binding F-actin and CD 137; c-1) an antibody capable of binding HSP90 and CD 3; c-2) an antibody capable of binding HSP90 and CD 137; e-1) an antibody capable of binding GRP78 and CD 3; e-2) an antibody capable of binding GRP78 and CD 137; g-1) an antibody capable of binding phosphatidylserine and CD 3; g-2) an antibody capable of binding phosphatidylserine and CD 137; i-1) an antibody capable of binding SAP130 and CD 3; i-2) antibodies capable of binding SAP130 and CD 137.

In a preferred specific embodiment herein, the antibody is selected from the group consisting of:

more preferably, the antibody is selected from the group consisting of:

A further general aspect of the invention relates to pharmaceutical compositions comprising the antigen binding molecules described herein.

In a further general aspect, the invention relates to an antigen binding molecule of the invention for use in medicine, and/or ii) in a method for treating a medical condition, in particular a medical condition involving cell death, and/or iii) in a method for treating a medical condition in a subject, the method comprising contacting the antigen binding molecule with damaged cells, and/or iv) in a method for treating a medical condition in a subject, the method comprising contacting the antigen binding molecule with a damage related molecular pattern (DAMP) in a patient, in particular further comprising contacting the antigen binding molecule with the second antigen, and/or v) in a method for treating a medical condition in a subject, the method comprising administering the antigen binding molecule to a patient suffering from a condition involving cell damage, in particular cell death.

Thus, in certain preferred embodiments, there is provided an antigen binding molecule of the invention for use in medicine. Thus, in certain preferred embodiments, there is provided an antigen binding molecule of the invention for use in a method of treating a medical condition, in particular a medical condition involving cell damage, in particular cell death. Thus, in certain preferred embodiments, there is provided an antigen binding molecule of the invention for use in a method of treating a medical condition in a subject, the method comprising contacting the antigen binding molecule with a damaged cell. Thus, in certain preferred embodiments, there is provided an antigen binding molecule of the invention for use in a method of treating a medical condition in a subject, the method comprising contacting the antigen binding molecule with a damage-associated molecular pattern (DAMP) in a patient, in particular further comprising contacting the antigen binding molecule with the second antigen. Thus, in certain preferred embodiments, there is provided an antigen binding molecule of the invention for use in a method of treating a medical condition in a subject, the method comprising administering the antigen binding molecule to a patient suffering from a condition involving cell damage, in particular cell death.

In a related further general aspect, the present invention relates to a pharmaceutical composition for any of the above uses of the molecules of the invention.

In certain preferred embodiments, the methods of treatment of the invention comprise bringing a plurality of antigen binding molecules into close proximity with a first and second antigen.

In certain preferred embodiments, the methods of treatment of the present invention are methods of activating or preventing an immune response. Preferably, the therapeutic methods of the invention are methods for activating an immune response.

In certain preferred embodiments, the therapeutic methods of the invention are for conferring or blocking signal transmission in a cell, preferably for conferring signal transmission in a cell.

In certain preferred embodiments herein, the method of treatment comprises administering an antigen binding molecule according to the invention to a patient, particularly a human patient. Preferably, the patient is characterized as described elsewhere herein.

In certain preferred embodiments herein, the method of treatment is a method for treating or preventing cancer or an immune (preferably autoimmune) disease, in particular wherein the method is a method of treating or preventing cancer, in particular wherein the method is a method of treating cancer.

Thus, in a particularly preferred embodiment, the present invention relates to an antigen binding molecule for use in a method of treating cancer, said antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a Damage Associated Molecular Pattern (DAMP),

wherein the second antigen is different from the first antigen, and

In a particularly preferred embodiment, the present invention relates to an antigen binding molecule for use in a method of treating cancer, the antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a Damage Associated Molecular Pattern (DAMP),

the first moiety is an Ig-type binding moiety,

wherein the second antigen is different from the first antigen,

wherein the second antigen is a membrane protein of an immune cell, and

wherein the second moiety is an Ig type binding moiety.

In a further particularly preferred embodiment, the present invention relates to an antigen binding molecule for use in a method of treating cancer, the antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a Damage Associated Molecular Pattern (DAMP),

wherein the first part is Clec9a or mClec4e,

wherein the second antigen is different from the first antigen,

wherein the second antigen is a membrane protein of an immune cell, and

wherein the second moiety is an Ig type binding moiety.

In a particular embodiment, the method of treatment of the invention comprises contacting the antigen binding molecule with a damage-associated molecular pattern (DAMP) that is exposed to an extracellular environment due to cellular damage, particularly wherein the DAMP is selected from the group consisting of F-actin, GRP78, phosphatidylserine, HSP90, and SAP 130.

In a specific embodiment, the method of treatment of the invention comprises contacting an antigen binding molecule with a cell, said cell being in particular selected from the group consisting of a-i) an immune cell, in particular wherein said immune cell is selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, an NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil and a basophil, a-ii) a tumor cell, and a-iii) an autoreactive cell.

In a particularly preferred embodiment, an antigen binding molecule or pharmaceutical composition of the invention is provided, for use in medicine.

In a particularly preferred embodiment, there is provided an antigen binding molecule or pharmaceutical composition of the invention for use in a method of treating or preventing a medical condition.

In a preferred general embodiment herein, the medical condition is selected from the group consisting of cancer and immune (preferably autoimmune) diseases. Thus, in certain preferred embodiments herein, the medical condition is cancer, particularly as described elsewhere herein. Thus, in certain preferred embodiments herein, the medical condition is an immune disorder, particularly as described elsewhere herein. Preferably, the immune disease is an autoimmune disease, in particular an autoimmune disease as described elsewhere herein.

A further general aspect of the invention relates to polynucleotides encoding the antigen binding molecules of the invention.

A further general aspect of the invention relates to a vector comprising a polynucleotide of the invention.

A further general aspect of the invention relates to a cell comprising a vector of the invention.

Furthermore, the present invention relates to the use of at least a first part and at least a second part for the preparation of an antigen binding molecule comprising said at least first part and said at least second part, wherein

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

The invention further relates to the use of an antigen binding molecule for targeting cells in a tissue in which cell death is observed or to be detected, wherein

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

Furthermore, the invention relates to the use of an antigen binding molecule to induce or block signal transduction in cells in a tissue in which cell death is observed, wherein

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

Furthermore, the present invention relates to a kit for generating a molecule that specifically acts in a tissue in which cell death is observed, said molecule comprising

(1) The at least one first moiety binds to a first antigen, and

(2) The at least one second moiety binds to a second antigen;

The kit comprises:

at least a first container comprising at least a first portion, and

at least a second container comprising at least a second portion.

Furthermore, all embodiments and definitions discussed above apply for the above-mentioned uses and kits of the invention.

All documents cited herein are incorporated by reference in their entirety.

The following are examples of the methods and compositions of the present disclosure. It should be understood that various other embodiments may be practiced in view of the general description provided above. These examples are provided to illustrate but not to limit the invention.

Examples

Example 1Expression and purification of recombinant antibodies

Recombinant antibodies were transiently expressed using an Expi293 cell line (Thermo Fisher, carlsbad, CA, USA). Antibody purification was performed using protein G or a affinity chromatography and gel filtration. Table 1 summarizes the combinations of genes encoding the heavy and light chains of each antibody for co-transfection. Each expression vector encoding an antibody sequence is designed for use in a mammalian expression system. Bispecific antibody preparation was performed using Fab Arm Exchange (FAE) according to the described method (Proc Natl Acad Sci U S A.2013Mar26; 110 (13): 5145-5150). For the concentration of purified antibodies, their absorbance at 280nm was measured using a spectrophotometer. From the values obtained, the concentration of antibody was calculated using the extinction coefficient calculated by PACE et al (Protein Science 1995; 4:2411-2423).

TABLE 1

[ Table 1-1]

/>

Example 2Clec9A conjugated antibodies that bind living and dead cells

Clec9A mediated binding of living and dead cells was detected by FACS analysis. Cell death of Ba/F3 cells was induced by treatment with 20. Mu.M Mitomycin C (MMC) at 37℃for 24 hours, followed by 2.5X10 ⁵ Each cell was stained for each sample. Briefly, cells were incubated with 1000x diluted Zombie Violet (ZV) dye (BioLegend, # 423114) for 20 minutes at RT, washed and blocked with anti-mouse CD16/32 antibody (BioLegend # 101320) at a final concentration of 2 μg/ml for 10 minutes on ice. Surface staining was performed with an unconjugated or conjugated Clec9A-ECD (SEQ ID NO: 9) or Celc-9A-CTLD (SEQ ID NO: 10) AF 488-labeled anti-Keyhole Limpet Hemocyanin (KLH) antibody (α -KLH) at a final concentration of 10 μg/mL on ice for 30 min. Annexin V (AnnV) staining was performed using a commercial kit according to the manufacturer's instructions (BioLegend # 640930). Data at BDLSRFortessa ^TM Collected on an X-20 flow cytometer and analyzed using Flowjo v10.4.2 software.

Binding of Clec9A conjugated antibodies was observed mainly on zv+anv+ necrotic cells, while minimal to no binding was observed on ZV-annv+ apoptotic cells (fig. 4). The ZV-AnnV-viable cell population did not show binding. Taken together, the data indicate that necrotic cells or components thereof specifically bind Clec9A and mediate its clustering of binding antibodies.

Example 3CD3 activation by Clec9A mediated clustering

In the mouse B cell line Ba/F3, the concentration was 1X10 at 37℃by using 5. Mu.M Methotrexate (MTX) (Sigma, #M 2305000) ⁶ Individual cells/mL, 5% CO ₂ The lower treatment was carried out for 24 hours to induce cell death. Untreated or MTX-treated Ba/F3 cells were then harvested, washed in fresh medium, and co-cultured at a ratio of 1 Ba/F3 to 5 Jurkat luciferase reporter T cells (Promega, # J1601). To evaluate the effect of Clec 9A-mediated T cell activation, the Extracellular (ECD) domain of Clec9A (SEQ ID NO: 9) or the C-type lectin domain of Clec9A (CTLD) (SEQ ID NO: 10) was conjugated to the C-terminus of the Fc region of an anti-human CD3 antibody (αcd3). Clec9A conjugated or unconjugated control anti-CD 3 antibody was added to co-cultures of BaF3 and Jurkat T cell cultures for 24 hours and Bio-Glo was used ^TM The luciferase assay system (Promega, #G7941) evaluates the luciferase activity of the supernatants. Data and charts were analyzed using GraphPad Prism software v 8.4.2. Fold induction was calculated relative to the negative control for each condition.

In the presence of "live" Ba/F3 cells not treated with MTX, unconjugated anti-CD 3 antibodies induced a maximum of about 22-fold relative luciferase signal, whereas only about 5-fold induction was observed with CD3 targeting antibodies conjugated to Clec9A (fig. 5). However, clec9A-ECD and Clec9A-CTLD conjugated antibodies induced a maximum of about 54-fold and about 46-fold relative luciferase activity, respectively, in the presence of "dead" Ba/F3 cells treated with MTX. This is significantly higher than about 20 relative fold induction observed with unconjugated anti-CD 3 antibodies. These data indicate that the presence of dead cells mediates clustering of Clec9A conjugated anti-CD 3 antibodies, resulting in immune T cell stimulation.

Example 4Anti-phosphatidylserine and anti-HSP 90 binding to the surface of living and dead cells

Phosphatidylserine (PS) and heat shock protein 90 (HSP 90) are sequestered in normal cells, but are exposed to the extracellular environment during cellular stress. To confirm this, anti-PS antibodies (immunotarget Ther.2018Jan 23;7:1-14) And anti-HSP 90 antibody clones 1.5.1 (US 9068987B 2) and 6H8 (WO 2011/129379) were used to detect individual targets on living and dead cells by flow cytometry. To test PS, ba/F3 mouse pro-B cell lines were first treated with 20. Mu.M Mitomycin C (MMC) for 48 hours to induce cell death. To detect HSP90, freeStyle was used ^TM 293-F (Thermofisher Scientific #R790-07) human embryonic kidney cell lines were pretreated with 100. Mu.M MMC for 72 hours. In subsequent staining, cells were incubated with 1000x diluted Zombie Violet (ZV) dye (BioLegend, # 423114) for 20 minutes at Room Temperature (RT), washed and blocked with anti-mouse CD16/32 antibody (BioLegend # 101320) at a final concentration of 2 μg/mL or 20-fold diluted human FcR blocking reagent (# 130-059-901) for 10 minutes at RT. Surface staining was performed with anti-PS (3G 4)) or anti-HSP 90 (1.5.1) and anti-HHSP 90 (6H 8)) at a final antibody concentration of 10 μg/mL for 30 min at RT. Data in BD LSRFortessa ^TM Collected on an X-20 flow cytometer and analyzed using Flowjo v10.4.2 software.

Binding of anti-PS and anti-HSP 90 antibodies was observed on zv+ gated dead cells treated with MMC, whereas no binding was detected on ZV-living cells (fig. 6). Thus, surface expression of PS or HSP90 is specific only for dead cells.

Example 5Activation of CD3 by anti-PS and anti-HSP 90 antibodies

To investigate whether dead cell-specific surface expression of PS or HSP90 was converted to dead cell-specific immune cell activation, live or dead cells were co-cultured with Jurkat reporter T cells expressing CD3 (Promega, # J1600) in the presence of anti-PS// CD3 (anti-PS (3G 4)// CD 3) or anti-HSP 90// CD3 (anti-HSP 90 (1.5.1)// CD3 or anti-HSP 90 (6H 8)// CD 3) bispecific antibodies. To evaluate anti-PS// CD3 activity, ba/F3 cells were pretreated with 5 μm Mitoxantrone (MTX) (Sigma, #m 2305000) for 48 hours to induce cell death. The untreated or MTX treated Ba/F3 cells were then co-cultured with 5-fold numbers of Jurkat reporter T cells. To assess anti-PS// HSP90 activity, untreated or MMC treated FreeStyle ^TM 293-F cells were co-cultured with equal amounts of Jurkat reporter T cells. 24 hours after antibody stimulation, bio-Glo was used ^TM Luciferase assay System (Promega, #G7941) and GloMax (registered) Trademark) Explorer (#gm3500) detects luciferase activity. Data and charts were analyzed using GraphPad Prism software v9.0.2. Fold induction was calculated relative to no antibody treatment control for each co-culture condition.

In the presence of dead cells, anti-PS// CD3 antibodies induced 16-fold and 70-fold luciferase signals at 5 and 25nM antibody concentrations, respectively (FIG. 7). This activity induction was significantly higher compared to living cell co-culture at the same antibody concentration. Similarly, at 5 and 25nM antibody concentrations, anti-HSP 90// CD3 antibodies showed induction of luciferase activity in co-culture with dead cells, but no relative fold induction when co-cultured with living cells (fig. 7). This phenomenon is independent of antibody clonality, as both anti-HSP 90 clones only induce similar luciferase reporter activity in the presence of dead cells. Taken together, these results are consistent with the death-dependent manner of CD3 activation and suggest that different death-related molecules can be targeted for immune cell modulation.

Example 6CD137 activation by Clec9A mediated clustering

To examine whether the concept of dead cell immunomodulation could be extended to effector targets other than CD3, the extracellular domain of Clec9A was conjugated to the C-terminus of the Fc region on an anti-human CD137 agonist antibody (CAS: 1417318-27-4) (anti-CD 137-Clec 9A). Dead cell-dependent activation of CD137 was assessed in live or MTX treated dead Ba/F3 cells, which were associated with CD137Jurkat reporter T cells (promega#j 2332) at 1:5 effective target ratio co-culture. Cells were treated with Clec9A conjugated anti-CD 137-Clec9A or control unconjugated anti-human CD137 antibody (anti-CD 137) for 24 hours, then luciferase activity was detected and analyzed.

Induction of CD137 was observed under conditions of anti-CD 137-Clec9A antibody treatment in the presence of MTX treated dead Ba/F3 cells (fig. 8). This induction is Clec9A dependent in that unconjugated anti-CD 137 antibodies do not result in a relative fold induction of luciferase activity. Notably, fold induction of anti-CD 137-Clec9A antibodies was not observed in the presence of live Ba/F3 either. These results indicate that conjugation of a dead cell binding moiety, such as Clec9A, to a CD137 agonist antibody leads to a lethal cell-dependent immune activation, similar to that observed with a dead cell binding CD3 agonist antibody. Thus, dead cell specificity may be conferred to different effector molecules by binding to the dead cell binding moiety.

Example 7PS-resistant CD137 activation

To investigate whether the dead cell specific surface expression of PS or HSP90 was converted to dead cell specific immune cell activation by CD137, anti-PS// CD137 bispecific antibodies were used in a similar assay as described for anti-CD 137-Clec9A and luciferase reporter activity was assessed. anti-PS// CD137 induced enhanced reporter cell activity in the presence of dead cells compared to living cell co-culture. In contrast, anti-KLH// CD137 antibodies that did not bind to PS did not show any difference in reporter activity between the live and dead cell co-cultures (fig. 9). In summary, the data indicate that the concept of dead cell specific modulation can be applied to different effector targets and by binding to different dead cell related molecules.

Example 8Clec9A fusion antibodies activate CD3 in an F-actin dependent manner

In the present invention, dead cell-dependent modulation of Clec9A conjugated antibodies is described. To confirm that this phenomenon occurs through binding of filiform actin (F-actin) to dead cells (Immunity. 2012Apr 20;36 (4): 646-57), activation of CD3 was detected in the presence or absence of F-actin. Briefly, recombinant F-actin (Cytoskeleton, # AKF99) reconstituted to 0.4mg/mL was coated on Nunc Maxisorp ^TM Wells of plate (Thermofisher Scientific # 442404) were left overnight at 4 ℃. Uncoated wells were filled with 50uL of universal actin buffer (Cytoskeleton, #bsa 01). The next day, the wells were washed and blocked with Dulbecco's Phosphate Buffered Saline (DPBS) supplemented with 5% bovine serum albumin (BSA, sigma-Aldrich #A7888) at Room Temperature (RT) for 1 hour. After the blocking step, anti-CD 3 antibodies unconjugated (anti-CD 3) or conjugated to Clec9A C type lectin domain (anti-CD 3-Clec 9A) were titrated and added to F-actin coated or uncoated wells for 15 min at 37℃and then per wellAdding 5x10 ⁴ Jurkat luciferase reported T cells (Promega, # J1601). At 37℃with 5% CO ₂ After incubation for 24 hours, bio-Glo was used ^TM Luciferase assay System (Promega, #G7940) and GloMax (registered trademark) Explorer System (Promega #GM 3500) were used to evaluate luciferase activity. The results were analyzed using GraphPad Prism software v9.0.2.

Clec9A conjugated CD3 antibody induced significantly higher T cell reporter activation in the presence of F-actin than in wells not coated with F-actin (fig. 10). This enhanced activation is dependent on Clec9A, as unconjugated CD3 antibodies did not show any difference in reporter activity between F-actin coated and uncoated wells. Thus, it is conceivable that modulation of CD3 activity by anti-CD 3-Clec 9A antibodies depends on the interaction between Clec9A and F-actin. This result demonstrates the mechanism of dead cell-dependent activation of Clec9A conjugated antibodies.

INDUSTRIAL APPLICABILITY

The present invention provides a molecule that binds to a component of a cell exposed to an extracellular environment due to cell death (e.g., a filament or histone forming the cytoskeleton or nuclear framework) or a portion thereof that is specifically observed in an affected tissue (e.g., TME), while simultaneously binding to a target molecule (e.g., a membrane protein) on or within a cell (e.g., an immune cell, an affected cell such as a tumor cell, an autoreactive cell, and a virus-infected cell), the molecule specifically functioning in a tissue in which cell death is observed such as an affected tissue, an abnormal tissue, and the like.

Sequence listing

<110> Chinese and foreign pharmaceutical Co Ltd

<120> molecules that specifically act in tissues where cell death is observed

<130> C1-A2033P

<150> JP 2021-013586

<151> 2021-01-29

<150> EP 21211440.9

<151> 2021-11-30

<150> EP 22151665.1

<151> 2022-01-14

<160> 57

<170> PatentIn version 3.5

<210> 1

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 1

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Tyr Ser Glu Thr Arg Leu 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

Ala Leu Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 2

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Asp Lys Ser Gln Asn Tyr Ala Thr Tyr Val Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Ala Asp Ser Lys Asn Ser

65 70 75 80

Ile Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Tyr Val His Tyr Ala Ala Gly Tyr Gly Val Asp Ile Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 3

<211> 324

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 3

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 4

<211> 540

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 4

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Lys Phe Phe Gln

325 330 335

Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu Ile Gln Gln Asp

340 345 350

Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys Tyr Thr Leu Glu

355 360 365

Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser Gly Thr Asp Ala

370 375 380

Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met Val Pro Gln Thr

385 390 395 400

Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser Pro Cys Pro His Asn

405 410 415

Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu

420 425 430

Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu

435 440 445

Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe Ile Ser Ser Ile Gly

450 455 460

Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly Val Phe Gln Asp Gly

465 470 475 480

Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp

485 490 495

Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr

500 505 510

Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr

515 520 525

Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser Cys Ile

530 535 540

<210> 5

<211> 466

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 5

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Asp Cys

325 330 335

Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr

340 345 350

Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu

355 360 365

Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu

370 375 380

Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val

385 390 395 400

Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly

405 410 415

Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala

420 425 430

Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys

435 440 445

Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser

450 455 460

Cys Ile

465

<210> 6

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 6

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Pro Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 7

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 7

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

100 105

<210> 8

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 8

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

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

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

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 9

<211> 208

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 9

Lys Phe Phe Gln Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu

1 5 10 15

Ile Gln Gln Asp Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys

20 25 30

Tyr Thr Leu Glu Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser

35 40 45

Gly Thr Asp Ala Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met

50 55 60

Val Pro Gln Thr Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser Pro

65 70 75 80

Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val Phe

85 90 95

Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys Glu

100 105 110

Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe Ile

115 120 125

Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly Val

130 135 140

Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser Ser

145 150 155 160

Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly Gln

165 170 175

Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys Asp

180 185 190

Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser Cys Ile

195 200 205

<210> 10

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 10

Gly Ser Asp Cys Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys

1 5 10 15

Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys

20 25 30

Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys

35 40 45

Glu Glu Met Glu Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn

50 55 60

Lys Tyr Trp Val Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe

65 70 75 80

Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg

85 90 95

Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu

100 105 110

Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys

115 120 125

Ala Phe Gly Ser Cys Ile

130

<210> 11

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 11

Gly Gly Gly Ser

1

<210> 12

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 12

Ser Gly Gly Gly

1

<210> 13

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 13

Gly Gly Gly Gly Ser

1 5

<210> 14

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 14

Ser Gly Gly Gly Gly

1 5

<210> 15

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 15

Gly Gly Gly Gly Gly Ser

1 5

<210> 16

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 16

Ser Gly Gly Gly Gly Gly

1 5

<210> 17

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 17

Gly Gly Gly Gly Gly Gly Ser

1 5

<210> 18

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 18

Ser Gly Gly Gly Gly Gly Gly

1 5

<210> 19

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Asp Lys Ser Gln Asn Tyr Ala Thr Tyr Val Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Ala Asp Ser Lys Asn Ser

65 70 75 80

Ile Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Tyr Val His Tyr Ala Ala Gly Tyr Gly Val Asp Ile Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 20

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 20

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Tyr Ser Glu Thr Arg Leu 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

Ala Leu Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 21

<211> 324

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 21

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 22

<211> 553

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 22

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Ala Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Val Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Lys Phe Phe Gln

325 330 335

Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu Ile Gln Gln Asp

340 345 350

Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys Tyr Thr Leu Glu

355 360 365

Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser Gly Thr Asp Ala

370 375 380

Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met Val Pro Gln Thr

385 390 395 400

Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser Pro Cys Pro His Asn

405 410 415

Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu

420 425 430

Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu

435 440 445

Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe Ile Ser Ser Ile Gly

450 455 460

Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly Val Phe Gln Asp Gly

465 470 475 480

Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp

485 490 495

Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr

500 505 510

Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr

515 520 525

Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser Cys Ile Gly Gly Gly Gly

530 535 540

Ser Asp Tyr Lys Asp Asp Asp Asp Lys

545 550

<210> 23

<211> 479

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 23

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Ala Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Val Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Asp Cys

325 330 335

Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr

340 345 350

Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu

355 360 365

Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu

370 375 380

Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val

385 390 395 400

Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly

405 410 415

Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala

420 425 430

Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys

435 440 445

Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser

450 455 460

Cys Ile Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 24

<211> 540

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 24

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Lys Phe Phe Gln

325 330 335

Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu Ile Gln Gln Asp

340 345 350

Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys Tyr Thr Leu Glu

355 360 365

Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser Gly Thr Asp Ala

370 375 380

Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met Val Pro Gln Thr

385 390 395 400

Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser Pro Cys Pro His Asn

405 410 415

Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu

420 425 430

Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu

435 440 445

Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe Ile Ser Ser Ile Gly

450 455 460

Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly Val Phe Gln Asp Gly

465 470 475 480

Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp

485 490 495

Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr

500 505 510

Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr

515 520 525

Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser Cys Ile

530 535 540

<210> 25

<211> 466

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 25

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Asp Cys

325 330 335

Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr

340 345 350

Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu

355 360 365

Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu

370 375 380

Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val

385 390 395 400

Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly

405 410 415

Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala

420 425 430

Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys

435 440 445

Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser

450 455 460

Cys Ile

465

<210> 26

<211> 553

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 26

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Trp Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Lys Phe Phe Gln

325 330 335

Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu Ile Gln Gln Asp

340 345 350

Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys Tyr Thr Leu Glu

355 360 365

Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser Gly Thr Asp Ala

370 375 380

Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met Val Pro Gln Thr

385 390 395 400

Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser Pro Cys Pro His Asn

405 410 415

Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu

420 425 430

Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu

435 440 445

Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe Ile Ser Ser Ile Gly

450 455 460

Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly Val Phe Gln Asp Gly

465 470 475 480

Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp

485 490 495

Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr

500 505 510

Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr

515 520 525

Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser Cys Ile Gly Gly Gly Gly

530 535 540

Ser His His His His His His His His

545 550

<210> 27

<211> 208

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 27

Lys Phe Phe Gln Val Ser Ser Leu Val Leu Glu Gln Gln Glu Arg Leu

1 5 10 15

Ile Gln Gln Asp Thr Ala Leu Val Asn Leu Thr Gln Trp Gln Arg Lys

20 25 30

Tyr Thr Leu Glu Tyr Cys Gln Ala Leu Leu Gln Arg Ser Leu His Ser

35 40 45

Gly Thr Asp Ala Ser Thr Gly Pro Val Leu Leu Thr Ser Pro Gln Met

50 55 60

Val Pro Gln Thr Leu Asp Ser Lys Glu Thr Gly Ser Asp Cys Ser Pro

65 70 75 80

Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr Val Phe

85 90 95

Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu Lys Glu

100 105 110

Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu Phe Ile

115 120 125

Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val Gly Val

130 135 140

Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly Ser Ser

145 150 155 160

Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala Gly Gln

165 170 175

Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys Cys Asp

180 185 190

Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser Cys Ile

195 200 205

<210> 28

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 28

Gly Ser Asp Cys Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys

1 5 10 15

Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys

20 25 30

Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys

35 40 45

Glu Glu Met Glu Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn

50 55 60

Lys Tyr Trp Val Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe

65 70 75 80

Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg

85 90 95

Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu

100 105 110

Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys

115 120 125

Ala Phe Gly Ser Cys Ile

130

<210> 29

<211> 479

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 29

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Trp Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Asp Cys

325 330 335

Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys Ser Cys Tyr Tyr

340 345 350

Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys Lys Ser Cys Leu

355 360 365

Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys Glu Glu Met Glu

370 375 380

Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn Lys Tyr Trp Val

385 390 395 400

Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe Trp Glu Asp Gly

405 410 415

Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg Gln Arg Ser Ala

420 425 430

Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu Ile Ser Asp Lys

435 440 445

Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys Ala Phe Gly Ser

450 455 460

Cys Ile Gly Gly Gly Gly Ser His His His His His His His His

465 470 475

<210> 30

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 30

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Pro Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly

85 90 95

Thr Gln Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 31

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

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

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

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 32

<211> 336

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 32

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

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

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

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

325 330 335

<210> 33

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 33

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

100 105

<210> 34

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 34

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

100 105

<210> 35

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 35

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

1 5 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> 36

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 36

Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Asn Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Asp Pro Ser Tyr Ser Glu Thr Arg Leu 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

Ala Leu Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 37

<211> 336

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 37

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

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

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

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro His His His His His His His His

325 330 335

<210> 38

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 38

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

1 5 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> 39

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 39

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly His Ile Asp Pro Tyr Tyr Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Lys Gly Gly Tyr Tyr Gly His Trp Tyr Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 40

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Thr

115

<210> 41

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 41

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

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys 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 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Tyr Gly Asn Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 42

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 42

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly His Ile Asp Pro Tyr Tyr Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Lys Gly Gly Tyr Tyr Gly His Trp Tyr Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 43

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 43

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 44

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Asn Trp Leu Gln Gln Gly Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

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

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Val Ser Ser Pro Pro

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 45

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 45

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys 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 Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 46

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 46

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 47

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 47

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu

85 90 95

Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 48

<211> 324

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 48

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 49

<211> 324

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 49

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Arg Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 50

<211> 324

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 50

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Lys

100 105 110

Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

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

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Glu Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 51

<211> 328

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 51

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 52

<211> 470

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 52

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser

325 330 335

Gly Ser Asp Cys Ser Pro Cys Pro His Asn Trp Ile Gln Asn Gly Lys

340 345 350

Ser Cys Tyr Tyr Val Phe Glu Arg Trp Glu Met Trp Asn Ile Ser Lys

355 360 365

Lys Ser Cys Leu Lys Glu Gly Ala Ser Leu Phe Gln Ile Asp Ser Lys

370 375 380

Glu Glu Met Glu Phe Ile Ser Ser Ile Gly Lys Leu Lys Gly Gly Asn

385 390 395 400

Lys Tyr Trp Val Gly Val Phe Gln Asp Gly Ile Ser Gly Ser Trp Phe

405 410 415

Trp Glu Asp Gly Ser Ser Pro Leu Ser Asp Leu Leu Pro Ala Glu Arg

420 425 430

Gln Arg Ser Ala Gly Gln Ile Cys Gly Tyr Leu Lys Asp Ser Thr Leu

435 440 445

Ile Ser Asp Lys Cys Asp Ser Trp Lys Tyr Phe Ile Cys Glu Lys Lys

450 455 460

Ala Phe Gly Ser Cys Ile

465 470

<210> 53

<211> 328

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 53

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 54

<211> 328

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 54

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

290 295 300

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

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro

325

<210> 55

<211> 328

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 55

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

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

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

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

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

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 56

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 56

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

1 5 10 15

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

20 25 30

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

35 40 45

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

50 55 60

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

65 70 75 80

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

85 90 95

Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

100 105 110

Pro Cys Pro Ala Pro Glu Leu Arg Arg Gly Pro Ser Val Phe Leu Phe

115 120 125

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

130 135 140

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

145 150 155 160

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

165 170 175

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

180 185 190

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

195 200 205

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

210 215 220

Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg

225 230 235 240

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly

245 250 255

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

260 265 270

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

275 280 285

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

290 295 300

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg

305 310 315 320

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

325 330

<210> 57

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> artificially synthesized sequences

<400> 57

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

1 5 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

Claims

1. An antigen binding molecule comprising:

(1) At least one first moiety that binds to a first antigen, and

(2) At least one second moiety that binds to a second antigen;

wherein the first antigen is a damage-associated molecular pattern (DAMP), and

Wherein the second antigen is different from the first antigen,

preferably, wherein the second antigen is a membrane protein of an immune cell.

2. The antigen binding molecule of claim 1, wherein the first antigen:

ii) on any one selected from the group consisting of a filament (in particular an intermediate filament), a nucleosome, a cytoskeleton and/or a nuclear scaffold.

3. The antigen binding molecule of any one of claims 1 to 2, wherein the first antigen is selected from the group consisting of:

a) Actin, in particular F-actin;

d) A histone or a component thereof,

e) Histone deacetylase complex subunits, in particular SAP130,

f) HMGN proteins, in particular selected from HMGB1 and HMGN1,

g) A growth factor derived from a cancer of the liver cells,

H)BCL-2；

i) Calreticulin, and

j) Cyclophilin a;

preferably wherein the first antigen is F-actin.

4. The antigen binding molecule of any one of claims 1 to 3, wherein the first moiety is selected from the group consisting of:

a) An Ig-type binding moiety; or (b)

5. The antigen binding molecule of claim 4, wherein:

6. The antigen binding molecule of claim 4 or 5, wherein in the case where the first antigen is actin or SAP130, the first binding moiety is an Ig-type binding moiety.

7. The antigen binding molecule of any one of claims 1 to 6, wherein the second antigen is a membrane protein of an immune cell, in particular a membrane protein in the cell membrane of a T cell selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a co-stimulatory molecule or a co-inhibitory molecule;

8. The antigen binding molecule of any one of claims 1 to 7, wherein the second moiety is an Ig-type binding moiety.

9. The antigen binding molecule of any one of claims 1 to 8, wherein the molecule is an antibody, in particular an IgG-type antibody.

10. The antigen binding molecule of claim 9, wherein the antibody is capable of binding to a membrane protein of an immune cell, and further comprising at least one binding polypeptide, preferably linked to an Fc region,

in particular, wherein the antibody is selected from the group consisting of:

an anti-CD 3 antibody comprising a Clec9A polypeptide,

an anti-CD 137 antibody comprising a Clec9A polypeptide,

an anti-CD 3 antibody comprising a mClec4e polypeptide, and

an anti-CD 137 antibody comprising a mClec4e polypeptide.

11. The antigen binding molecule of claim 9, wherein the antibody is a bispecific antibody,

in particular, wherein the antibody is selected from the group consisting of:

Bispecific anti-HSP 90 anti-CD 3 antibodies,

bispecific anti-HSP 90 anti-CD 3 antibodies;

bispecific anti-GRP 78 anti-CD 3 antibodies,

bispecific anti-GRP 78 anti-CD 137 antibody,

bispecific anti-phosphatidylserine anti-CD 3 antibodies, and

bispecific anti-phosphatidylserine anti-CD 137 antibodies.

12. A pharmaceutical composition comprising the antigen binding molecule of any one of claims 1 to 11.

13. The antigen binding molecule or pharmaceutical composition of any one of claims 1 to 12 for use in medicine.

14. An antigen binding molecule or pharmaceutical composition according to any one of claims 1 to 13 for use in a method of treating or preventing a medical condition.

15. The antigen binding molecule or pharmaceutical composition for use according to claim 14, wherein the medical condition is selected from the group consisting of cancer and an immune disease, preferably an autoimmune disease.