WO2020166714A1

WO2020166714A1 - Complexed protein, antibody complex, pharmaceutical composition, and nucleic acid

Info

Publication number: WO2020166714A1
Application number: PCT/JP2020/005875
Authority: WO
Inventors: 俊元越智; 正貴安川; 克斗竹中
Original assignee: 国立大学法人愛媛大学
Priority date: 2019-02-15
Filing date: 2020-02-14
Publication date: 2020-08-20
Also published as: JPWO2020166714A1

Abstract

Provided is a complexed protein that is capable of imparting, to an antibody or an antigen-binding fragment thereof, a binding ability to a desired antigen.　The complexed protein according to the present invention contains a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain is capable of binding to immunoglobulin while the second antigen-binding domain is capable of binding to the desired antigen.

Description

Complexed protein, antibody complex, pharmaceutical composition, and nucleic acid

The present invention relates to complexed proteins, antibody complexes, pharmaceutical compositions, and nucleic acids.

In a multispecific antibody, one antibody molecule has an antigen binding domain that shows binding to different antigens. By making the multispecific antibody an antibody that binds to a tumor antigen and CD3ε, T cell-mediated cytotoxicity can be induced. Therefore, the multispecific antibody is under clinical development as an anticancer agent. .. Further, according to the multispecific antibody, it is possible to arrange a plurality of proteins in the vicinity, and therefore, a therapeutic agent for hemophilia that targets a blood coagulation factor has been developed and marketed.

However, multispecific antibodies have problems that their production efficiency is extremely low and their production is difficult. For example, bispecific antibodies are composed of two types of heavy chains (H chains) and two types of light chains (L chains). Moreover, when two types of H chains and two types of L chains are expressed to produce the bispecific antibody, the number of combinations of two H chains and two L chains in the expressed antibody is ten, and In addition to the bispecific antibody, 9 kinds of unnecessary antibodies will be produced (Patent Document 1).

International Publication No. 2013/065708

Therefore, an object of the present invention is to provide a complexed protein capable of adding a desired antigen-binding property to an antibody or an antigen-binding fragment thereof.

To achieve the above object, the complexed protein of the present invention comprises a first antigen-binding domain and a second antigen-binding domain,
The first antigen-binding domain is capable of binding an immunoglobulin,
The second antigen binding domain is capable of binding a desired antigen.

The antibody complex of the present invention comprises an antibody capable of binding to a target antigen or an antigen-binding fragment thereof, and a complexed protein,
The complexed protein is the complexed protein of the present invention,
The complexed protein binds to the antibody or the antigen-binding fragment thereof through the first antigen-binding domain to form a complex.

The pharmaceutical composition of the present invention contains the complexed protein of the present invention.

The nucleic acid of the present invention encodes the complexed protein of the present invention.

According to the complexed protein of the present invention, it is possible to add a desired antigen-binding property to an antibody or an antigen-binding fragment thereof.

FIG. 1 is a schematic diagram showing an example of the present invention, (A) is a schematic diagram showing an example of a complexed protein of the present invention, and (B) is an immunized protein with a complexed protein of the present invention. FIG. 2 is a schematic diagram showing an example of formation of a complex with a globulin and the obtained antibody complex. FIG. 2 is a histogram showing the binding between the antibody complex and cells in Example 1. FIG. 3 is a graph showing the ratio of cytokine-producing cells in Example 1. FIG. 4 is a graph showing the ratio of cytokine-producing cells in Example 1. FIG. 5 is a histogram showing the binding between the antibody complex and cells in Example 2. FIG. 6 is a graph showing the ratio of cytokine-producing cells in Example 2. FIG. 7 is a graph showing the ratio of cytokine-producing cells in Example 2. FIG. 8 is a graph showing the proportion of cells in which CFSE is attenuated in Example 3. FIG. 9 is a graph showing the ratio of activated CD4 ⁺ T cells (CD4 ⁺ CD25 ⁺ ) and regulatory T cells (CD4 ⁺ CD25 ⁺ CD45RA ⁺ FoxP3 ⁺ ) in Example 3. FIG. 10 is a graph showing the ratio of IFN-γ producing cells and the ratio of increased CD107a expression in Example 4. FIG. 11 is a graph showing the ratio of IFN-γ producing cells and the ratio of increased CD107a expression in Example 4. FIG. 12 is a graph showing activation of NK cells and T cells in Example 4. FIG. 13 is a graph showing the activation of NK cells and T cells in Example 4. FIG. 14 is a graph showing the activation of NK cells and T cells in Example 4. FIG. 15 is a graph showing the cytotoxicity in Example 4. FIG. 16 is a graph showing the relative proportion of producing cells in Example 5. FIG. 17 is a diagram regarding the antitumor activity in the living body in Example 6.

<Complexed protein>
As described above, the complexed protein of the present invention includes a first antigen-binding domain (hereinafter, also referred to as “first binding domain”), a second antigen-binding domain (hereinafter, “second binding domain”). Also referred to as “”), the first antigen-binding domain is capable of binding an immunoglobulin, and the second antigen-binding domain is capable of binding a desired antigen. The complexed protein of the present invention is characterized in that the first antigen-binding domain can bind to an immunoglobulin and the second antigen-binding domain can bind to a desired antigen. The configuration and conditions of are not particularly limited.

The complexed protein of the present invention can bind to an immunoglobulin, that is, an antibody or an antigen-binding fragment thereof by the first binding domain, as shown in FIG. 1(A). Moreover, the complexed protein of the present invention can bind to a desired antigen by the second antigen-binding domain. Therefore, as shown in FIG. 1(B), when the complexed protein of the present invention is brought into contact with an immunoglobulin, the complexed protein binds with the immunoglobulin to form a complex. As a result, the formed antibody complex contains, as an antigen-binding domain, the second binding domain of the complexed protein in addition to the antigen-binding domain of immunoglobulin. That is, the complexed protein can impart a new antigen-binding domain called the second binding domain to the immunoglobulin by complexing with the immunoglobulin. Further, the target to which the second binding domain binds can be a desired antigen. Therefore, according to the complexed protein of the present invention, binding properties for a desired antigen can be added to the antibody or its antigen-binding fragment. Therefore, according to the complexed protein of the present invention, an antibody complex having the same function as that of the multispecific antibody can be easily produced. Further, in the complexed protein of the present invention, the target of the second binding domain, that is, the desired antigen is regulated, and by using in combination with the immunoglobulin, similar to the above-described multispecific antibody. It can exert its function. As a specific example, the desired antigen to which the second binding domain binds is an antigen expressed on T cells such as CD3, and the binding target of the binding domain of the immunoglobulin is a tumor antigen, thereby forming the complex. An antibody complex of a protein and the above immunoglobulin can be used as an anticancer agent that exhibits the same function as an anticancer agent containing a multispecific antibody, for example, because it can bind to cancer cells and recruit T cells and NK cells. it can. Further, in the antibody complex, the binding target of the second binding domain and the binding target of the immunoglobulin can be arranged in the vicinity, and the antibody complex can be, for example, the above-mentioned hemophilia therapeutic agent. It can be used as a hemophilia drug that exhibits the same function as. In the antibody complex, the binding target of the second binding domain and the binding target of the immunoglobulin can be arranged in the vicinity, so that, for example, the receptor or the like forming the complex is separated, Signals mediated by receptors can be suppressed. Furthermore, since the antibody complex can bind to a plurality of antigens, it can be used to remove a plurality of antigens, for example, to remove an antigen that causes a disease.

In the present invention, the “domain” means, for example, in a polypeptide, a three-dimensionally structured or functionally integrated region. Examples of the polypeptide include peptides having a length of 10 amino acids or more.

In the present invention, “capable of binding” may mean that an antibody or the like, which is a binding substance, actually binds to a binding target that is bound to the binding substance, or a molecular docking method or the like is used. The former may be used in the simulation, but the former is preferable. The binding between the binding substance and the binding target can be detected, for example, by using an analysis method of protein-protein interaction, and for example, antibody-antigen reaction such as co-immunoprecipitation, pull-down assay, ELISA method, flow cytometry, etc. It can be detected using the method used. As a specific example, the binding between the binding product and the binding target is detected by, for example, contacting a cell expressing the binding target with a labeled binding product, and then detecting a label in the cell. it can.

In the present invention, the immunoglobulin may be an antibody or an antigen-binding fragment thereof, but is preferably an antibody containing a constant region (Fc region) or an antigen-binding fragment thereof, since it has high stability in vivo. , And more preferably an antibody. The immunoglobulin can also be referred to as a soluble immunoglobulin, for example. Hereinafter, unless otherwise specified, the antibody or antigen-binding fragment thereof to which the complexed protein of the present invention is bound is also referred to as “antibody or the like”. The binding target such as the antibody is not particularly limited and can be any antigen. The antibody or the like can bind to, for example, one or more antigens. In the latter case, the antibody and the like can be referred to as, for example, a multispecific antibody such as a bispecific antibody or an antigen-binding fragment thereof. Examples of the type of the antibody include IgA, IgD, IgE, IgG, or IgM. Examples of IgA include IgA1 and IgA2. Examples of IgG include IgG1, IgG2, IgG3, and IgG4. The antigen-binding fragment is, for example, a part of the antibody, more specifically, a polypeptide containing the binding region or the variable region of the antibody. Examples of the antigen-binding fragment include Fab, Fab′, F(ab′)2, Fv fragment, rIgG (half IgG) fragment, single chain antibody (scFv), and double variable domain antibody (DVD-Ig™). ), diabody, triabody, tetrabody, tanda, flexibody which is a combination of scFv and diabody, tandem scFv (for example, BiTE (Registered trademark), Micromet), DART (trademark) (MacroGenics), Fcab (trademark) or mAb ² (trademark) (F-star), Fc engineering antibody (Xencor) or DuoBody (trademark) (Genmab). Company) etc. As the immunoglobulin, a known immunoglobulin, that is, a known antibody or an antigen-binding fragment thereof may be used, or a new antibody or an antigen-binding fragment thereof obtained by immunizing with an arbitrary antigen may be used. Good.

The origin of the antibody and the like is not particularly limited, and examples thereof include human and non-human animals. Examples of the non-human animals include mammals such as mice, rats, dogs, monkeys, rabbits, sheep, horses and pigs; birds such as chicken and Emu; and the like. The antibody and the like may be derived from a plurality of animals, for example. That is, the antibody or the like may be a chimeric antibody or an antigen-binding fragment thereof, or a humanized antibody or an antigen-binding fragment thereof.

The first binding domain only needs to be able to bind to the immunoglobulin (antibody or the like). The binding position of the first binding domain in the antibody or the like, that is, the position of the epitope of the first binding domain in the antibody or the like is not particularly limited and inhibits the binding between the antibody and the antigen such as the antibody. It can be set arbitrarily within the range that does not. The first binding domain has, for example, an epitope in a region other than the binding region or the variable region of the antibody or the like, and preferably has a constant (Fc) region. The Fc region may be a heavy chain Fc region or a light chain Fc region. Examples of the Fc region of the heavy chain include CH1 region, CH2 region, or CH3 region. Examples of the Fc region of the light chain include CL region. The first binding domain preferably has an epitope in a region other than the binding region of the Fc receptor in the immunoglobulin, since it can suppress a decrease in complement activity or cytotoxic activity mediated by an antibody, for example. A specific example is the CH1 region or CH3 region. When the antibody or the like is IgG or an antigen-binding fragment thereof, the first binding domain has, for example, an epitope in a region other than positions 232 to 236 in the region represented by EU numbering of the Fc region, Is 237-447th, 237-340th, 341-447th, 250-447th, 260-447th, 270-447th, 280-447th, 290-447th, 300-447th, 310-447th Position, 320-447th, 330-447th, 340-447th, 350-447th, 360-447th, 370-447th, 380-447th, 390-447th, 400-447th, 410-447th Position, 420-447, or 430-447. The EU numbering can be determined according to Reference Document 1 below.
Reference 1: Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

The Fc receptor binding region of the immunoglobulin is, for example, a region that binds via the Fc receptor when NK cells exhibit antibody-dependent cellular cytotoxicity (ADCC) activity. The region of the immunoglobulin other than the Fc receptor binding region can be referred to as a region in the Fc region that does not inhibit NK cell ADCC activity. In addition, the region of the immunoglobulin other than the Fc receptor binding region can be examined using, for example, whether or not the ADCC activity of NK cells is suppressed. As a specific example, it is shown when an antibody, a cell expressing an antigen to which the antibody binds (target cell), a complexed protein having a first binding domain of interest, and NK cells are co-cultured. The NK cell cytotoxicity is significantly different from the NK cell cytotoxicity shown when the antibody, cells expressing the antigen to which the antibody binds (target cells), and NK cells are co-cultured. If not, it can be said that the first binding domain of interest in the complexed protein binds to a region of the immunoglobulin other than the Fc receptor binding region. The binding region of the Fc receptor in the immunoglobulin is, for example, among the regions represented by EU numbering of the Fc region, positions 233 to 237 (ELLGG), 239 (S), 265 (D), 269 ( E), 294th (E), 296-298th (YNS), 328th (L) and 329th (P). The number of target cells and the number of NK cells, and the culture conditions can be set, for example, according to the examples described below.

As described above, the first binding domain only needs to be able to bind to an immunoglobulin, and for example, the structure of a polypeptide capable of binding to an immunoglobulin can be adopted. Specific examples of the first binding domain include a receptor that binds to the antibody and the like, and an antibody or an antigen-binding fragment thereof that binds to the antibody and the like. In the present invention, “binding” refers to, for example, contacting an antibody or the like to be measured with an antigen, and then measuring the resulting complex by flow cytometry, as compared with a control antibody that does not bind to the antigen, This means that the fluorescence intensity is significantly increased.

When the first binding domain is a receptor that binds to an antibody or the like, the first binding domain may be, for example, an Fc receptor. The type of the Fc receptor is not particularly limited and can be appropriately set depending on, for example, the type of the immunoglobulin. The Fc receptor includes, for example, FcαR (CD89), FcαR such as Fcα/μR; FcεR such as FcεRI; FcγRIa (CD64), FcγRIIa (CD32), FcγRIIb1 (CD32), FcγRIIb2 (CD32), FcγRIIIa (CD16), FcγR of FcγRIIIb; FcμR such as Fcα/μR; FcRn (neonatal Fc receptor); and the like. When the immunoglobulin is IgG, the Fc receptor is, for example, FcγR. The first binding domain may be a polypeptide consisting of the entire amino acid sequence of the Fc receptor or a partial sequence thereof. In the latter case, the polypeptide consisting of said subsequence is capable of binding to said immunoglobulin, ie comprises the ligand binding domain of said Fc receptor.

When the first binding domain is an antibody or an antigen-binding fragment thereof that binds to an antibody or the like, the first binding domain can adopt a structure similar to that of the antibody or the antigen-binding fragment thereof described above. It has a chain variable region and a light chain variable region. Hereinafter, the heavy chain variable region and the light chain variable region of the first binding domain are also referred to as the first heavy chain variable region and the first light chain variable region, respectively. The first heavy chain variable region and the first light chain variable region have the same structure as the heavy chain variable region and the light chain variable region in the antibody molecule. Generally, the heavy chain variable region and the light chain variable region of an antibody molecule each have three complementarity determining regions (CDRs). CDR is also called a hypervariable domain. Even in the variable regions of the heavy chain and the light chain, CDR is a region in which the primary structure has particularly high variability, and it is usually separated at three positions on the primary structure. In the present invention, three CDRs in the heavy chain variable region are determined from the amino terminal (N-terminal) side in the amino acid sequence of the heavy chain variable region, heavy chain CDR1 (CDRH1), heavy chain CDR2 (CDRH2), and heavy chain CDR3. (CDRH3), and the three CDRs in the light chain variable region are represented by the light chain CDR1 (CDRL1), the light chain CDR2 (CDRL2), and the light chain CDR3 (CDRL3 from the amino-terminal side in the amino acid sequence of the light chain variable region. ). These sites are conformationally close to each other and determine their specificity for binding antigen. The above description can be applied to the antigen-binding fragment. The first binding domain preferably comprises an scFv, ie an scFv capable of binding the immunoglobulin. The scFv can be prepared, for example, by linking the heavy chain variable region and the light chain variable region with a linker peptide. The amino acid sequence of the linker peptide is not particularly limited.

When the first binding domain has the first heavy chain variable region and the first light chain variable region, the first heavy chain variable region and the first light chain variable region are, for example, the antibody and the like. May be derived from the heavy chain variable region and the light chain variable region of the antibody that binds to, or the heavy chain variable region and the light chain variable region of the antibody capable of binding to the obtained antibody etc. It may be derived, or may be derived from the heavy chain variable region and the light chain variable region of an antibody capable of binding to an antibody etc. obtained by a screening method such as phage display.

As described above, the complementarity determining regions (CDRs) in the heavy chain variable region and the light chain variable region are important for the binding of the antibody to the antigen. Therefore, the CDRH1, CDRH2, and CDRH3 of the first heavy chain variable region may be polypeptides each consisting of the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region of the antibody that binds to the antibody or the like. However, it may be a polypeptide containing the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region of the antibody that binds to the above-mentioned antibody or the like. Further, CDRL1, CDRL2, and CDRL3 of the first light chain variable region may each be a polypeptide comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the light chain variable region of the antibody that binds to the antibody or the like. Alternatively, it may be a polypeptide containing the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the light chain variable region of an antibody that binds to the above-mentioned antibody or the like.

In the first heavy chain variable region, the region other than CDRH1, CDRH2, and CDRH3, that is, the framework region (FR) may include, for example, the FR of the heavy chain variable region in the antibody that binds to the antibody or the like. .. The FRs are usually separated in four places on the primary structure. In the present invention, four FRs in the heavy chain variable region are labeled from the N-terminal side in the amino acid sequence of the heavy chain variable region to heavy chain FR1 (FRH1), heavy chain FR2 (FRH2), heavy chain FR3 (FRH3) , And heavy chain FR4 (FRH4). The CDRHs and the FRHs are arranged in the order of FRH1, CDRH1, FRH2, CDRH2, FRH3, CDRH3, and FRH4 from the N-terminal side in the amino acid sequence of the heavy chain variable region. .. FRH1, FRH2, FRH3, and FRH4 in the first heavy chain variable region may be, for example, a polypeptide consisting of the amino acid sequences of FRH1, FRH2, FRH3, and FRH4 of the antibody that binds to the antibody or the like, respectively, It may be a polypeptide containing the amino acid sequences of FRH1, FRH2, FRH3, and FRH4 of the antibody that binds to the above-mentioned antibody or the like. The “antibody that binds to an antibody or the like” in the description of each CDRH and the “antibody that binds to an antibody or the like” in the description of each FRH are preferably the same antibody and the like.

In the first light chain variable region, the region other than CDRL1, CDRL2, and CDRL3, that is, the framework region (FR) may include, for example, the FR of the light chain variable region in the antibody that binds to the antibody or the like. .. The FRs are usually separated in four places on the primary structure. In the present invention, four FRs in the light chain variable region are labeled from the N-terminal side in the amino acid sequence of the light chain variable region, ie, light chain FR1 (FRL1), light chain FR2 (FRL2), light chain FR3 (FRL3). , And light chain FR4 (FRL4). The CDRLs and the FRLs are arranged, for example, in the order of FRL1, CDRL1, FRL2, CDRL2, FRL3, CDRL3, and FRL4 from the N-terminal side in the amino acid sequence of the light chain variable region. .. FRL1, FRL2, FRL3, and FRL4 in the first light chain variable region may be, for example, polypeptides each comprising the amino acid sequences of FRL1, FRL2, FRL3, and FRL4 of the antibody that binds to the antibody or the like, It may be a polypeptide containing the amino acid sequences of FRL1, FRL2, FRL3, and FRL4 of the antibody that binds to the antibody or the like. The “antibody that binds to an antibody or the like” in the description of each CDRL and the “antibody that binds to an antibody or the like” in the description of each FRL are preferably the same antibody and the like. In addition, the “antibody that binds to an antibody or the like” in the description of each CDRH and the “antibody that binds to an antibody or the like” in the description of each CDRL are preferably the same antibody and the like.

The antibody that binds to the antibody or the like is not particularly limited and can be appropriately selected depending on the type of the antibody or the like. The antibody that binds to the antibody or the like may be an antibody that binds to a known antibody or the like, or an antibody capable of binding to the antibody or the like obtained by immunizing with the antibody or the like. When the antibody is human IgG, examples of the antibody that binds to human IgG include the antibodies described in

References

2 and 3 below. As a specific example, when the antibody is human IgG, the antibody (clone name) that binds to human IgG is, for example, HP6017, HP6000, HP6018, HP6043, HP6052, HP6064, HP6065, HP6087, HP6092, HP6044, HP6046, HP6063, etc. Can be given. Hybridomas producing antibodies of the various clones can be obtained from depository institutions such as American Type Culture Collection (ATCC).
Reference 2: Jefferis, R et.al., “Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.”, Immunol Lett., 1985, volume 10, Issues 3-4 , pages 223-52
Reference 3: Reimer CB et.al, “Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes.”, Hybridoma., 1984, volume 3, Issue 3, pages 263-75.

When the antibody or the like is human IgG, the first binding domain is, for example, when an antibody complex is formed with a human IgG antibody, human NK cells bind to the antibody complex via an Fcγ receptor, Since the human NK cells can exhibit ADCC activity, it preferably contains the following first heavy chain variable region and first light chain variable region.
(First heavy chain variable region)
Including CDRH1, CDRH2, and CDRH3,
CDRH1 is a polypeptide containing the following amino acid sequence (H1-A),
CDRH2 is a polypeptide containing the following amino acid sequence (H2-A),
CDRH3 is a polypeptide containing the amino acid sequence of (H3-A) below.
(First light chain variable region)
Includes CDRL1, CDRL2, and CDRL3,
CDRL1 is a polypeptide containing the following amino acid sequence (L1-A),
CDRL2 is a polypeptide containing the amino acid sequence of (L2-A) below,
CDRL3 is a polypeptide containing the following (L3-A) amino acid sequence.

(H1-A) The following (H1-A1), (H1-A2) or (H1-A3) amino acid sequence (H1-A1) SEQ ID NO: 1 (GYTFTNYW) amino acid sequence (H1-A2) SEQ ID NO: 1 Amino acid sequence having 80% or more identity to the sequence (H1-A3) In the amino acid sequence of SEQ ID NO: 1, one or several amino acids are deleted, substituted, inserted and/or added (H2-A) Amino acid sequence of (H2-A1), (H2-A2) or (H2-A3) below (H2-A1) SEQ ID NO: 2 (IYPGGGIT) (H2-A2) Amino acid of SEQ ID NO: 2 Amino acid sequence having 80% or more identity to the sequence (H2-A3) In the amino acid sequence of SEQ ID NO: 2, one or several amino acids are deleted, substituted, inserted and/or added (H3-A) Amino acid sequence of (H3-A1), (H3-A2) or (H3-A3) below (H3-A1) SEQ ID NO: 3 (SRSYGKYFDY) (H3-A2) Amino acid of SEQ ID NO: 3 Amino acid sequence having 80% or more identity to the sequence (H3-A3) In the amino acid sequence of SEQ ID NO: 3, one or several amino acids have been deleted, substituted, inserted and/or added

(L1-A) Amino acid sequence of the following (L1-A1), (L1-A2) or (L1-A3) (L1-A1) SEQ ID NO: 4 (QDIKSY) (L1-A2) Amino acid of SEQ ID NO: 4 Amino acid sequence having 80% or more identity to the sequence (L1-A3) In the amino acid sequence of SEQ ID NO: 4, one or several amino acids are deleted, substituted, inserted and/or added (L2-A) The following (L2-A1), (L2-A2) or (L2-A3) amino acid sequence (L2-A1) SEQ ID NO:5 (YST) amino acid sequence (L2-A2) SEQ ID NO:5 amino acid Amino acid sequence having 80% or more identity to the sequence (L2-A3) SEQ ID NO: 5 amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added (L3-A) Amino acid sequence of the following (L3-A1), (L3-A2) or (L3-A3) (L3-A1) SEQ ID NO:6 (LQHDESPFT) (L3-A2) Amino acid of SEQ ID NO:6 Amino acid sequence having 80% or more identity to the sequence (L3-A3) In the amino acid sequence of SEQ ID NO: 6, one or several amino acids are deleted, substituted, inserted and/or added ..

In each of the CDRs, “identity” is, for example, the degree of identity when the sequences to be compared are properly aligned, and means the occurrence rate (%) of exact matches of amino acids between the sequences. The “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, respectively. Is. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (the same applies hereinafter).

In each of the CDRs, “1 or several” regarding substitution and the like means, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 or 2 It is one.

The substitution of the amino acid may be, for example, a conservative substitution (hereinafter the same). The conservative substitution means substituting one or several amino acids with other amino acids and/or amino acid derivatives so that the function of the protein is not substantially changed. It is preferable that the “substituted amino acid” and the “substituted amino acid” have similar properties and/or functions, for example. Specifically, for example, it is preferable that the hydrophobic and hydrophilic indices (hydropathy), chemical properties such as polarity and charge, or physical properties such as secondary structure are similar. Amino acids or amino acid derivatives having similar properties and/or functions are known in the art, for example. Specific examples include non-polar amino acids (hydrophobic amino acids) such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar amino acids (neutral amino acids) include glycine, serine, and threonine. , Tyrosine, glutamine, asparagine, cysteine, etc., positively charged amino acids (basic amino) acids include arginine, histidine, lysine, etc., negatively charged amino acids (acidic amino acids) include aspartic acid, Examples thereof include glutamic acid.

The first heavy chain variable region preferably contains, for example, a polypeptide consisting of the following (H-A) amino acid sequence. The second light chain variable region preferably contains, for example, a polypeptide having the following amino acid sequence (LA).

(HA) following (H-A1), (H -A2) or (H-A3) of the amino acid sequence (H-A1) SEQ ID NO: 13 amino acid sequence SEQ ID NO 13: QVQLQQPGAELVKPGASVKMSCKAS GYTFTNYW INWVKQRPGQGLEWIGD IYPGGGIT NYNEKFKTKATLTLDTSSSTVYMQLSSLTSEDSAVYYC SRSYGKYFDY WGQGTTLIVSS
(H-A2) Amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 13 (H-A3) In the amino acid sequence of SEQ ID NO: 13, one or several amino acids are deleted or substituted , Inserted and/or added amino acid sequence

(LA) the following (L-A1), (L -A2) or (L-A3) of the amino acid sequence (L-A1) SEQ ID NO: 14 amino acid sequence SEQ ID NO 14: DIKMTQSPSSMYASVGERVTITCKAS QDIKSY LTWYQKKPWKSPRTLIF YST RLADGVPSRFSGSGSGQDFSLTISSLESDDTATYYC LQHDESPFT FGGGTKLEIK
(L-A2) Amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 14 (L-A3) In the amino acid sequence of SEQ ID NO: 14, one or several amino acids are deleted or substituted , Inserted and/or added amino acid sequence

The above-mentioned (H-A1) amino acid sequence is, for example, a sequence including the (H1-A1) of CDRH1, the (H2-A1) of CDRH2, and the (H3-A1) of CDRH3. The amino acid sequence of (H-A2) includes, for example, the amino acid sequences of (H1-A1) of CDRH1, (H2-A1) of CDRH2, and CDRH3 (H3-A1), and the amino acid sequence of SEQ ID NO: 13. The amino acid sequence may have an identity of 70% or more. The amino acid sequence of (H-A3) includes, for example, the amino acid sequences of CDRH1 (H1-A1), CDRH2 (H2-A1), and CDRH3 (H3-A1), and the amino acid sequence of SEQ ID NO: 13. In, it may be an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added.

The above-mentioned (L-A1) amino acid sequence is, for example, a sequence including the CDL1 (L1-A1), CDRL2 (L2-A1), and CDRL3 (L3-A1) amino acid sequences. The amino acid sequence of (L-A2) includes, for example, the amino acid sequences of (L1-A1) of CDRL1, (L2-A1) of CDRL2, and (L3-A1) of CDRL3, and the amino acid sequence of SEQ ID NO:14. It may be an amino acid sequence having 70% or more identity with the sequence. The amino acid sequence of (L-A3) includes, for example, the amino acid sequences of (L1-A1) of CDRL1, (L2-A1) of CDRL2, and (L3-A1) of CDRL3, and the amino acid of SEQ ID NO:14. It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the sequence.

In the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region, the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

In the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region, “1 or several” regarding substitution and the like are, for example, 1 to 30, 1 to 20, 1 to 15, respectively. It is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1.

When the antibody or the like is human IgG, the first binding domain is, for example, when an antibody complex is formed with a human IgG antibody, human NK cells bind to the antibody complex via an Fcγ receptor, Since the human NK cells can exhibit ADCC activity, it preferably contains a polypeptide comprising the amino acid sequence of the first scFv below. The polypeptides having the amino acid sequences of (S-A2) and (S-A3) below can bind to human IgG, for example.

(First scFv)
(SA) the following (S-A1), (S-A2) or (S-A3) of the amino acid sequence (S-A1) SEQ ID NO: 15 amino acid sequence SEQ ID NO 15: DiaikeiemutikyuesuPiesuesuemuwaieiesubuijiiarubuitiaitishikeieiesukyudiaikeiesuwaierutidaburyuwaikyukeikeiPidaburyukeiesuPiarutieruaiefuwaiesutiaruerueidijibuiPiesuaruefuesujiesujiesujikyudiefuesuerutiaiesuesueruiesudiditieitiwaiwaishierukyueichidiiesuPiefutiefujijijitikeieruiaikeijijijijiesujijijijiesujijijijiesukyubuikyuerukyukyuPijieiierubuikeiPijieiesubuikeiemuesushikeieiesujiwaitiefutienuwaidaburyuaienudaburyubuikeikyuaruPijikyujieruidaburyuaijidiaiwaiPijijijiaitienuwaienuikeiefukeitikeieitierutieruditiesuesuesutibuiwaiemukyueruesuesuerutiesuidiesueiVYYCSRSYGKYFDYWGQGTTLIVSS
(S-A2) Amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 15 (S-A3) In the amino acid sequence of SEQ ID NO: 15, one or several amino acids are deleted or substituted , Inserted and/or added amino acid sequence

The above-mentioned (S-A1) amino acid sequence is, for example, a sequence including the (H1-A1) of CDRH1, (H2-A1) of CDRH2, and (H3-A1) of CDRH3. The amino acid sequence of (S-A2) includes, for example, (H1-A1) of CDRH1, (H2-A1) of CDRH2, and CDRH3 (H3-A1), and the amino acid sequence of SEQ ID NO:15. The amino acid sequence may have an identity of 70% or more. The amino acid sequence of (S-A3) includes, for example, (H1-A1) of CDRH1, (H2-A1) of CDRH2, and CDRH3 (H3-A1), and the amino acid sequence of SEQ ID NO:15. In, it may be an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added.

The (S-A1) amino acid sequence is, for example, a sequence including the (H-A1) amino acid sequence of the first heavy chain variable region. The amino acid sequence of (S-A2) includes, for example, the amino acid sequence of (H-A1) of the first heavy chain variable region and has 70% or more identity to the amino acid sequence of SEQ ID NO: 15. May be an amino acid sequence having The amino acid sequence of (S-A3) includes, for example, the amino acid sequence of (H-A1) of the first heavy chain variable region, and in the amino acid sequence of SEQ ID NO: 15, one or several amino acids are It may be a deleted, substituted, inserted and/or added amino acid sequence.

The above-mentioned (S-A1) amino acid sequence is, for example, a sequence including the CDL1 (L1-A1), CDRL2 (L2-A1), and CDRL3 (L3-A1) amino acid sequences. The amino acid sequence of (S-A2) includes, for example, (L1-A1) of CDRL1, (L2-A1) of CDRL2, and (L3-A1) of CDRL3, and the amino acid sequence of SEQ ID NO:15. It may be an amino acid sequence having 70% or more identity with the sequence. The amino acid sequence of (S-A3) includes, for example, the amino acid sequences of (L1-A1) of CDRL1, (L2-A1) of CDRL2, and (L3-A1) of CDRL3, and the amino acid sequence of SEQ ID NO:15. It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the sequence.

The above-mentioned (S-A1) amino acid sequence is, for example, a sequence including the (L-A1) amino acid sequence of the first light chain variable region. The amino acid sequence of (S-A2) includes, for example, the amino acid sequence of (L-A1) of the first light chain variable region, and has 70% or more identity to the amino acid sequence of SEQ ID NO: 15. May be an amino acid sequence having The amino acid sequence of (S-A3) includes, for example, the amino acid sequence of (L-A1) of the first light chain variable region, and in the amino acid sequence of SEQ ID NO: 15, one or several amino acids are It may be a deleted, substituted, inserted and/or added amino acid sequence.

In the amino acid sequence of (S-A2), the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, It is 97% or more, 98% or more, and 99% or more.

In the amino acid sequence of (S-A3), “one or several” regarding substitution and the like is, for example, 1 to 72, 1 to 60, 1 to 48, 1 to 40, 1 to 24, respectively. 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 One, two or one.

The second binding domain can bind to a desired antigen as described above. Examples of the desired antigen include antigens (proteins) expressed in immune cells, antigens (proteins) in body fluids, tumor antigens, virus antigens, bacterial antigens, parasite antigens, antigens associated with autoimmune diseases, sugar chain antigens, etc. Can be given. The tumor antigen means, for example, a biomolecule having antigenicity such as an antigen newly expressed by a canceration of cells or an antigen whose expression level is increased. The tumor antigen may be, for example, a tumor-specific antigen or a tumor-associated antigen. The desired antigen is preferably different from the antigen to which the antibody or the like can bind, for example.

Examples of the antigens expressed on the immune cells include antigens expressed on T cells, NK cells, or NKT cells. Examples of the antigen expressed on the T cell include a T cell activation protein or a T cell activation suppression protein. Examples of the T cell activating protein include CD2, CD3, CD4, CD5, CD8α, CD8β, CD27, CD28, CD134 (OX40), CD137 (4-1BB), CD154, GITR, ICOS, etc., and are preferred. Is CD3. Examples of the CD3 include CD3γ, CD3δ, CD3ε, CD3ζ, and CD3η, and preferably CD3ε. Examples of the T cell activation inhibitory protein include CTLA4, PD-1, LAG3, B7-H3, TIM3, and TIGIT. Examples of the antigens expressed on the NK cells include NK cell activation proteins and NK cell activation suppression proteins. Examples of the NK cell activating protein include CD94/NKG2C, CD94/NKG2E, NKG2D/NKG2D and the like. Examples of the NK cell activation suppressing protein include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR2DL4, KIR2DL5 and KIR3DL3. The antigen expressed in the NKT cell is, for example, an NKT cell activation protein or an NKT cell activation inhibitory protein, and specific examples include the description of the T cell-expressed antigen and the NK cell-expressed antigen. Can be used.

Examples of the antigen in the body fluid include cytokines and blood coagulation factors. Examples of the cytokine include TNF (Tumor Necrosis Factor) such as TNF-α and TNF-β; lymphotoxin; IL-1 to IL-38 (eg, IL-1α, IL-1β, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-17, IL-18, IL-23, IL-33, IL-36 and the like); chemokines; hematopoietic factors; cell growth factors; adipokines; growth factors such as VEGF; and the like.

The tumor antigens include, for example, 5T4, α5β1-integrin, 707-AP, α-fetoprotein (AFP), lectin-reactive AFP, ART-4, AURKA (AURORA A), B7H4, BAGE, β-catenin, BCMA, Bcr-. abl, BTAA, MN/CA IX antigen, CA125, CA19-9, CA72-4, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD47, CD52, CD56, CD80, CD96, CD123, CDK4, carcinoembryonic antigen (CEA), CLL1, CT, Cyclin A1, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE (GAGE). -1, GAGE-2, etc.), GD2 (GangliosideG2), GnT-V, Gp100, GPA33, GPC3, HAGE, β-human chorionic gonadotropin (HCG), HER2/neu, HLA-A*0201-R170I, HPV -E7, HSP70-2M, HST-2, iCE, insulin growth factor (IGF)-1, IGF-2, IGF-1R, IL-2R, IL-5, KIAA0205, K-Ras, LAGE, LDLR/FUT, MAGE (MAGE-3, MAGE-4, MAGE-5, MAGE-6 etc.), MART-1/melan-A, MART-2/Ski, MC1R, mesothelin (MSLN), myosin, MUC1, MUM-1, MUM -2, MUM-3, NA88-A, prostatic acid phosphatase (PAP), proteinase-3, PRAME (Melanoma antigen preferentially expressed intumors), p53, p190 minor bcr-abl, Pml/RARα, prostate tumor antigen-1 ( PCTA-1), PRAME, prostate specific antigen (PSA), PSM, PSMA, RAGE, RAS, RHAMM (CD168), RU1, RU2, SAGE, SART-1, SART-3, thyroglobulin, survivin, telomerase reverse transcriptase. (TERT or TRT), TEL/AML1, TGFβ, TIM3, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1, NY-Eso-B, etc. To be

The viral antigens are, for example, adenovirus (adenovirus) adenoviridae such as (Adenoviridae); coronavirus (coronavirus) coronavirus family, such as (Coronaviridae); Ebola virus (Ebolavirus) Filoviridae such as (Filoviridae); Flaviviridae such as hepatitis C virus (HCV: hepatitis C virus), dengue virus (Dengue virus), Japanese encephalitis virus (Japanese encephalitis virus), west Nile virus (west Nile virus), yellow fever virus (yellow fever virus) ( Flaviviridae); B hepatitis virus (HVB: hepatitis B virus) hepadnaviridae such as (Hepadnaviridae); herpes simplex virus type 1 (HSV-1: herpes simplex virus -1), herpes simplex virus type 2 (HSV-2 : Herpes simplex virus-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), EB virus (EBV: Epstein-Barr virus), Kaposi's sarcoma-related herpes virus (KSHV) : Kaposi's sarcoma-associated herpesvirus) and other herpesviruses ( Herpesviridae ); orthomyxoviruses such as influenza A virus genus (Influenzavirus A), influenza B virus genus (Influenzavirus B), influenza C virus genus (Influenzavirus C) Family ( Orthomyxoviridae ); Paramyxoviridae family such as measles virus (measles virus), human parainfluenza virus types 1 to 4 (Human parainfluenza virus 1 to 4), mumps virus (Respiratory syncytial virus), etc. Paramyxoviridae ); Parvovirus such as Parvovirus B19 Viruses ( Parvoviridae ); enterovirus (enterovirus), poliovirus (poliovirus), human rhinovirus AB (human rhinovirus AB), hepatitis A virus (hepatitis A virus), coxsackievirus (coxsackievirus), echovirus (echo) virus) etc.; Picornaviridae ); smallpox virus (variola virus); vaccinia virus (Vaccinia virus) and other poxviridae ( Poxviridae ); human immunodeficiency virus (HIV: human immunodeficiency virus)-1, 2, human T-lymphotropic virus (HTLV: human T-lymphocytropic virus ) -I, Retroviridae of II such as (Retroviridae); rabies virus (rabies virus), vesicular stomatitis virus (vesicular stomatitis virus), etc. rhabdovirus of Family ( Rhabdoviridae ); Rubella virus (Rubella virus), Chikungunya virus (Chikungunya virus), etc. Togaviridae ( Togaviridae );

The bacterial antigen can be, for example, tetanus (Clostridium tetani) Clostridium genus, etc. (Clostridium); E. (Escherichia coli) Esukerikia genus such as (Escherichia); Helicobacter pylori (Helicobacter pyloris) Helicobacter, such as (Helicobacter); Legionella・Legionella genus ( Legionella ) such as Pneumophilia ( Legionella pneumophila ); Listeria genus ( Listeria ) such as Listeria monocytogenes ( Mycobacterium tuberculosis ), Mycobacterium leprae , Mycobacterium avium (Mycobacterium avium), Mycobacterium intracellulare et (Mycobacterium intracellulare), Mycobacterium kansasii (Mycobacterium kansasii), Mycobacterium Gorudone (Mycobacterium gordonae) Mycobacterium such as (Mycobacterium); Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria, such as Neisseria meningitidis (Neisseria meningitidis) (Neisseria); Pseudomonas aeruginosa (Pseudomonas aeruginosa) Pseudomonas such as (Pseudomonas); Salmonella typhi (Salmonella enterica serovar typhi), paratyphoid bacteria (Salmonella enterica serovar Paratyphi a) Salmonella such as (Salmonella); Staphylococcus aureus (Staphylococcus aureus) Staphylococcus etc. (Staphylococcus); pneumococcal (Streptococcus pneumoniae), Streptococcus, such as Streptococcus pyogenes (Streptococcus pyogenes) (Streptococcus); Antigens derived from bacteria such as

The parasite antigens include, for example, liver fluke ( Clonorchis sinensis ), Schistosoma japonicum ( Schistosoma japonicum ), human carnivore ( Ascaris lumbricoides ), human pinworm ( Enterobius vermicularis ), Cysticercus cellulosae , and broad-segment cleft head. Antigens derived from parasites such as insects ( Diphyllobothrium latum ), Echinococcus ( Echinococcus ), Entamoeba histolytica ), and Plasmodium malaria ( Plasmodium ).

The second binding domain may be, for example, a receptor or a ligand that binds to the desired antigen, or an antibody or an antigen-binding fragment thereof that binds to the desired antigen.

When the second binding domain is a ligand or receptor that binds to a desired antigen, the first binding domain includes, for example, a receptor that binds to the ligand or a ligand that binds to the receptor, It can be appropriately set depending on the type of ligand or receptor. As a specific example, when the ligand is a CTLA4 ligand, the receptor is, for example, CTLA4. When the ligand is PD-1 ligand, the receptor is PD-1.

When the second binding domain is an antibody or an antigen-binding fragment thereof that binds to a desired antigen, the second binding domain can adopt the same structure as the above-mentioned antibody or an antigen-binding fragment thereof. It has a chain variable region and a light chain variable region. Hereinafter, the heavy chain variable region and the light chain variable region of the second binding domain are also referred to as the second heavy chain variable region and the second light chain variable region, respectively. The above description can be applied to the antigen-binding fragment. The second binding domain preferably comprises an scFv, ie an scFv capable of binding the desired antigen.

When the second binding domain has the second heavy chain variable region and the second light chain variable region, the second heavy chain variable region and the second light chain variable region are, for example, The heavy chain variable region and the light chain variable region of the antibody, which may be derived from the heavy chain variable region and the light chain variable region of the antibody that bind to the antigen, may be immunized with the desired antigen and bind to the obtained desired antigen. It may be derived from the variable region, or may be derived from the heavy chain variable region and the light chain variable region of the antibody capable of binding to the antibody etc. obtained by a screening method such as phage display.

As described above, the complementarity determining regions (CDRs) in the heavy chain variable region and the light chain variable region are important for the binding of the antibody to the antigen. Therefore, the CDRH1, CDRH2, and CDRH3 of the second heavy chain variable region are each a polypeptide consisting of the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region of the antibody that binds to the desired antigen. Alternatively, it may be a polypeptide containing the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region of the antibody that binds to the desired antigen. Further, CDRL1, CDRL2, and CDRL3 of the second light chain variable region may each be a polypeptide comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the light chain variable region of the antibody that binds to the desired antigen. However, it may be a polypeptide containing the amino acid sequences of CDRL1, CDRL2, and CDRL3 of the light chain variable region of an antibody that binds to the desired antigen.

In the second heavy chain variable region, the region other than CDRH1, CDRH2, and CDRH3, that is, the framework region (FR) may include, for example, the FR of the heavy chain variable region in the antibody that binds to the desired antigen. Good. FRH1, FRH2, FRH3, and FRH4 in the second heavy chain variable region may be, for example, a polypeptide comprising the amino acid sequences of FRH1, FRH2, FRH3, and FRH4 of the antibody that binds to the desired antigen, respectively. Alternatively, it may be a polypeptide containing the amino acid sequences of FRH1, FRH2, FRH3, and FRH4 of an antibody that binds to the desired antigen. The “antibody that binds to the desired antigen” in the description of each CDRH and the “antibody that binds to the desired antigen” in the description of each FRH are preferably the same antibody and the like.

In the second light chain variable region, a region other than CDRL1, CDRL2, and CDRL3, that is, the framework region (FR) may include, for example, the FR of the light chain variable region in the antibody that binds to the desired antigen. Good. FRL1, FRL2, FRL3, and FRL4 in the second light chain variable region may be, for example, a polypeptide comprising the amino acid sequences of FRL1, FRL2, FRL3, and FRL4 of the antibody that binds to the desired antigen, respectively. The polypeptide may include the amino acid sequences of FRL1, FRL2, FRL3, and FRL4 of the antibody that binds to the desired antigen. The “antibody that binds to the desired antigen” in the description of each CDRL and the “antibody that binds to the desired antigen” in the description of each FRL are preferably the same antibody and the like. The “antibody that binds to the desired antigen” in the description of each CDRH and the “antibody that binds to the desired antigen” in the description of each CDRL are preferably the same antibody and the like.

When the second binding domain has the second heavy chain variable region and the second light chain variable region, the second heavy chain variable region and the second light chain variable region are, for example, The heavy chain variable region and the light chain variable region of the antibody that bind to the antigen may be used, or the heavy chain variable region and the light chain variable region of the desired antibody capable of binding to the desired antigen obtained by immunizing with the desired antigen Areas may be used. As a specific example, when the desired antigen is human CD3ε, examples of the antibody (clone name) that binds to human CD3ε include OKT3 antibody, UCHT1 antibody, L2K antibody, HIT3a antibody, 28F11 antibody, and 27H5 antibody.

When the desired antigen is CD3ε, the second binding domain binds to T cells via the second binding domain when the second binding domain forms an antibody complex with the antibody or the like, for example. However, since the T cells can exert cytotoxic activity, it is preferable to include the following second heavy chain variable region and second light chain variable region.
(Second heavy chain variable region)
Including CDRH1, CDRH2, and CDRH3,
CDRH1 is a polypeptide containing the following amino acid sequence (H1-B),
CDRH2 is a polypeptide containing the following amino acid sequence (H2-B),
CDRH3 is a polypeptide containing the following amino acid sequence (H3-B).
(Second light chain variable region)
Includes CDRL1, CDRL2, and CDRL3,
CDRL1 is a polypeptide containing the following amino acid sequence (L1-B),
CDRL2 is a polypeptide containing the amino acid sequence of (L2-B) below,
CDRL3 is a polypeptide containing the following amino acid sequence (L3-B).

(H1-B) The following (H1-B1), (H1-B2) or (H1-B3) amino acid sequence (H1-B1) SEQ ID NO: 7 (GYTFTRYT) amino acid sequence (H1-B2) SEQ ID NO: 7 amino acid Amino acid sequence having 80% or more identity to the sequence (H1-B3) In the amino acid sequence of SEQ ID NO: 7, one or several amino acids are deleted, substituted, inserted and/or added (H2-B) Amino acid sequence of (H2-B1), (H2-B2) or (H2-B3) below (H2-B1) SEQ ID NO: 8 (INPSRGYT) amino acid sequence (H2-B2) SEQ ID NO: 8 Amino acid sequence having 80% or more identity to the sequence (H2-B3) Amino acid sequence of SEQ ID NO: 8 with one or several amino acids deleted, substituted, inserted and/or added (H3-B) The following (H3-B1), (H3-B2) or (H3-B3) amino acid sequence (H3-B1) SEQ ID NO: 9 (ARYYDDHYCLDY) amino acid sequence (H3-B2) SEQ ID NO: 9 Amino acid sequence having 80% or more identity to the sequence (H3-B3) In the amino acid sequence of SEQ ID NO: 9, one or several amino acids are deleted, substituted, inserted and/or added

(L1-B) Amino acid sequence of the following (L1-B1), (L1-B2) or (L1-B3) (L1-B1) SEQ ID NO: 10 (SSVSY) (L1-B2) Amino acid of SEQ ID NO: 10 Amino acid sequence having 80% or more identity to the sequence (L1-B3), wherein the amino acid sequence of SEQ ID NO: 10 has one or several amino acids deleted, substituted, inserted and/or added (L2-B) The following (L2-B1), (L2-B2) or (L2-B3) amino acid sequence (L2-B1) SEQ ID NO: 11 (DTS) amino acid sequence (L2-B2) SEQ ID NO: 11 Amino acid sequence (L2-B3) having an identity of 80% or more with respect to the sequence, wherein one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 11. (L3-B) Amino acid sequence of (L3-B1), (L3-B2) or (L3-B3) below (L3-B1) SEQ ID NO: 12 (QQWSSNPFT) amino acid sequence (L3-B2) SEQ ID NO: 12 Amino acid sequence having 80% or more identity to the sequence (L3-B3) SEQ ID NO: 12 amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added ..

In each of the CDRs, the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98%, respectively. As a result, it is 99% or more.

The second heavy chain variable region preferably contains, for example, a polypeptide consisting of the following (H-B) amino acid sequence. The second light chain variable region preferably contains, for example, a polypeptide having the amino acid sequence of (LB) below.

(HB) below (H-B1), (H -B2) or (H-B3) of the amino acid sequence (H-B1) SEQ ID NO: 16 amino acid sequence SEQ ID NO 16: QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTLTVSS
(H-B2) Amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 16 (H-B3) In the amino acid sequence of SEQ ID NO: 16, one or several amino acids are deleted or substituted , Inserted and/or added amino acid sequence

(LB) following (L-B1), (L -B2) or (L-B3) of the amino acid sequence (L-B1) of SEQ ID NO: 17 amino acid sequence SEQ ID NO: 17: QIVLTQSPAIMSASPGEKVTMTCSAS SSVSY MNWYQQKSGTSPKRWIY DTS KLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYC QQWSSNPFT FGSGTKLEIN
(L-B2) Amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 17 (L-B3) In the amino acid sequence of SEQ ID NO: 17, one or several amino acids are deleted or substituted , Inserted and/or added amino acid sequence

The above-mentioned (H-B1) amino acid sequence is, for example, a sequence including the (H1-B1) of CDRH1, (H2-B1) of CDRH2, and (H3-B1) of CDRH3. The amino acid sequence of (H-B2) includes, for example, (H1-B1) of CDRH1, (H2-B1) of CDRH2, and CDRH3 (H3-B1), and the amino acid sequence of SEQ ID NO:16. The amino acid sequence may have an identity of 70% or more. The amino acid sequence of (H-B3) includes, for example, the amino acid sequences of (H1-B1) of CDRH1, (H2-B1) of CDRH2, and CDRH3 (H3-B1), and the amino acid sequence of SEQ ID NO: 16. In, it may be an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added.

The above-mentioned (L-B1) amino acid sequence is, for example, a sequence including the CDL1 (L1-B1), CDRL2 (L2-B1), and CDRL3 (L3-B1) amino acid sequences. The amino acid sequence of (L-B2) includes, for example, the amino acid sequences of (L1-B1) of CDRL1, (L2-B1) of CDRL2, and (L3-B1) of CDRL3, and the amino acid sequence of SEQ ID NO:17. It may be an amino acid sequence having 70% or more identity with the sequence. The amino acid sequence of (L-B3) includes, for example, the amino acid sequences of (L1-B1) of CDRL1, (L2-B1) of CDRL2, and (L3-B1) of CDRL3, and the amino acid sequence of SEQ ID NO:17. It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the sequence.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, respectively. 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, “one or several” regarding substitution and the like are, for example, 1 to 30, 1 to 20, 1 to 15, respectively. It is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1.

When the desired antigen is CD3ε, the second binding domain binds to T cells via the second binding domain in the antibody complex when forming an antibody complex with the antibody or the like. However, since the T cells can exhibit cytotoxic activity, it is preferable to include a polypeptide having the amino acid sequence of the second scFv below. A polypeptide comprising the amino acid sequences of (S-B2) and (S-B3) below can bind to CD3ε, for example.

(Second scFv)
(SB) following (S-B1), (S-B2) or (S-B3) of the amino acid sequence (S-B1) of SEQ ID NO: 18 amino acid sequence SEQ ID NO: 18: KyubuikyuerukyukyuesujieiierueiaruPijieiesubuikeiemuesushikeieiesujiwaitiefutiaruwaitiemueichidaburyubuikeikyuaruPijikyujieruidaburyuaijiwaiaienuPiesuarujiwaitienuwaienukyukeiefukeidikeieitierutitidikeiesuesuesutieiwaiemukyueruesuesuerutiesuidiesueibuiwaiwaishieiaruwaiwaididieichiwaishierudiwaidaburyujikyujititierutibuiesuesujijijijiesujijijijiesujijijijiesukyuaibuierutikyuesuPieiaiemuesuEiesupijiikeibuitiemutishiesueiesuesuesubuiesuwaiemuenudaburyuwaikyukyukeiesujitiesuPikeiarudaburyuaiwaiditiesukeierueiesujibuiPieieichiefuarujiesujiesujitiesuwaiesuerutiaiesujiemuieiidiAATYYCQQWSSNPFTFGSGTKLEIN
(S-B2) Amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 18 (S-B3) In the amino acid sequence of SEQ ID NO: 18, one or several amino acids are deleted or substituted , Inserted and/or added amino acid sequence

The above-mentioned (S-B1) amino acid sequence is, for example, a sequence including the (H1-B1) of CDRH1, (H2-B1) of CDRH2, and (H3-B1) of CDRH3. The amino acid sequence of (S-B2) includes, for example, (H1-B1) of CDRH1, (H2-B1) of CDRH2, and CDRH3 (H3-B1), and the amino acid sequence of SEQ ID NO:18. The amino acid sequence may have an identity of 70% or more. The amino acid sequence of (S-B3) includes, for example, (H1-B1) of CDRH1, (H2-B1) of CDRH2, and CDRH3 (H3-B1), and the amino acid sequence of SEQ ID NO:18. In, it may be an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added.

The (S-B1) amino acid sequence is, for example, a sequence containing the (H-B1) amino acid sequence of the second heavy chain variable region. The amino acid sequence of (S-B2) includes, for example, the amino acid sequence of (H-B1) of the second heavy chain variable region, and has 70% or more identity to the amino acid sequence of SEQ ID NO: 18. May be an amino acid sequence having The amino acid sequence of (S-B3) includes, for example, the amino acid sequence of (H-B1) of the second heavy chain variable region, and in the amino acid sequence of SEQ ID NO: 18, one or several amino acids are It may be a deleted, substituted, inserted and/or added amino acid sequence.

The above-mentioned (S-B1) amino acid sequence is, for example, a sequence containing the CDL1 (L1-B1), CDRL2 (L2-B1), and CDRL3 (L3-B1) amino acid sequences. The amino acid sequence of (S-B2) includes, for example, (L1-B1) of CDRL1, (L2-B1) of CDRL2, and (L3-B1) of CDRL3, and the amino acid sequence of SEQ ID NO: 18 It may be an amino acid sequence having 70% or more identity with the sequence. The amino acid sequence of (S-B3) includes, for example, (L1-B1) of CDRL1, (L2-B1) of CDRL2, and (L3-B1) of CDRL3, and the amino acid sequence of SEQ ID NO:18. It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the sequence.

The above-mentioned (S-B1) amino acid sequence is, for example, a sequence containing the (L-B1) amino acid sequence of the second light chain variable region. The amino acid sequence of (S-B2) includes, for example, the amino acid sequence of (L-B1) of the second light chain variable region, and has 70% or more identity to the amino acid sequence of SEQ ID NO: 18. May be an amino acid sequence having The amino acid sequence of (S-B3) includes, for example, the amino acid sequence of (L-B1) of the second light chain variable region, and has one or several amino acids in the amino acid sequence of SEQ ID NO: 18. It may be a deleted, substituted, inserted and/or added amino acid sequence.

In the amino acid sequence of (S-B2), the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, respectively. It is 97% or more, 98% or more, and 99% or more.

In the amino acid sequence of (S-B3), “one or several” regarding substitution and the like is, for example, 1 to 72, 1 to 60, 1 to 48, 1 to 40, 1 to 24, respectively. 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 One, two or one.

The first binding domain and the second binding domain may be directly or indirectly linked, or when the first binding domain and the second binding domain coexist, both are It may be formed so as to be associated (bonded). In the case of the indirect linkage, it is preferable that the first binding domain and the second binding domain are linked by a linker peptide (domain linker). The domain linker is composed of, for example, 1 to 40, 1 to 18, 1 to 15, 1 to 7, 1 to 3, 1 or 2 amino acids. The domain linker is composed of, for example, amino acids such as glycine and serine, and a specific example thereof is (G _m S) _n . The m is a positive integer, for example, an integer of 1 to 6. The n is a positive integer, for example, an integer of 1 to 6. The m and n may be the same or different. A specific example of the domain linker is a polypeptide having the amino acid sequence of SEQ ID NO: 19 below. For the association, for example, charges of amino acid side chains can be used. As a specific example, the association can be induced, for example, by adding a domain of an amino acid having a positive charge to one binding domain and adding a domain of an amino acid having a negative charge to the other binding domain. Further, for the association, for example, a combination of a tag sequence and a polypeptide that recognizes the tag sequence can be used.
Domain linker (SEQ ID NO: 19): SGSG

Since the complexed protein of the present invention can be used in combination with an antibody capable of binding to a tumor antigen to recruit NK cells and T cells and induce cytotoxic activity against tumors, the following (B) A polypeptide consisting of an amino acid sequence is preferred. The polypeptides having the amino acid sequences of (B2) and (B3) below can bind to human IgG and/or CD3ε, for example.

(B) below (B1), (B2), amino acid sequence (B1) SEQ ID NO: 20 amino acid sequence SEQ ID NO: 20 (B3): MMRPIVLVLLFATSAAADIKMTQSPSSMYASVGERVTITCKASQDIKSYLTWYQKKPWKSPRTLIFYSTRLADGVPSRFSGSGSGQDFSLTISSLESDDTATYYCLQHDESPFTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTNYWINWVKQRPGQGLEWIGDIYPGGGITNYNEKFKTKATLTLDTSSSTVYMQLSSLTSEDSAVYYCSRSYGKYFDYWGQGTTLIVSSSGSGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN
(B2) Amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 20. (B3) In the amino acid sequence of SEQ ID NO: 20, one or several amino acids are deleted, substituted, inserted and/or Or added amino acid sequence

The above-mentioned (B1) amino acid sequence is, for example, a sequence including the CDRH1 (H1-A1), CDRH2 (H2-A1), and CDRH3 (H3-A1) amino acid sequences. The amino acid sequence of (B2) includes, for example, the amino acid sequences of CDRH1 (H1-A1), CDRH2 (H2-A1), and CDRH3 (H3-A1), and And may be an amino acid sequence having 70% or more identity. The amino acid sequence of (B3) includes, for example, (H1-A1) of CDRH1, (H2-A1) of CDRH2, and CDRH3 (H3-A1), and in the amino acid sequence of SEQ ID NO: 20, It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added.

The above-mentioned (B1) amino acid sequence is, for example, a sequence including the (H-A1) amino acid sequence of the first heavy chain variable region. The amino acid sequence of (B2) includes, for example, the amino acid sequence of (H-A1) of the first heavy chain variable region, and has 70% or more identity to the amino acid sequence of SEQ ID NO:20. It may be an amino acid sequence. The amino acid sequence of (B3) includes, for example, the amino acid sequence of (H-A1) of the first heavy chain variable region, and one or several amino acids are deleted in the amino acid sequence of SEQ ID NO:20. , A substituted, inserted and/or added amino acid sequence.

The above-mentioned (B1) amino acid sequence is, for example, a sequence including the CDL1 (L1-A1), CDRL2 (L2-A1), and CDRL3 (L3-A1) amino acid sequences. The amino acid sequence of (B2) above includes, for example, the amino acid sequences of (L1-A1) of CDRL1, (L2-A1) of CDRL2, and (L3-A1) of CDRL3, and On the other hand, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (B3) includes, for example, (L1-A1) of CDRL1, (L2-A1) of CDRL2, and (L3-A1) of CDRL3, and in the amino acid sequence of SEQ ID NO: 20, It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added.

The aforementioned (B1) amino acid sequence is, for example, a sequence containing the (L-A1) amino acid sequence of the first light chain variable region. The amino acid sequence of (B2) includes, for example, the amino acid sequence of (L-A1) of the first light chain variable region, and has 70% or more identity with the amino acid sequence of SEQ ID NO:20. It may be an amino acid sequence. The amino acid sequence of (B3) includes, for example, the amino acid sequence of (L-A1) of the first light chain variable region, and one or several amino acids are deleted in the amino acid sequence of SEQ ID NO:20. , A substituted, inserted and/or added amino acid sequence.

The amino acid sequence of (B1) is a sequence containing the amino acid sequences of (H1-B1) of CDRH1, (H2-B1) of CDRH2, and (H3-B1) of CDRH3, for example. The amino acid sequence of (B2) includes, for example, the amino acid sequences of CDRH1 (H1-B1), CDRH2 (H2-B1), and CDRH3 (H3-B1), and And may be an amino acid sequence having 70% or more identity. The amino acid sequence of (B3) includes, for example, (H1-B1) of CDRH1, (H2-B1) of CDRH2, and CDRH3 (H3-B1), and in the amino acid sequence of SEQ ID NO: 20, It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added.

The above-mentioned (B1) amino acid sequence is, for example, a sequence including the (H-B1) amino acid sequence of the second heavy chain variable region. The amino acid sequence of (B2) includes, for example, the amino acid sequence of (H-B1) of the second heavy chain variable region, and has 70% or more identity to the amino acid sequence of SEQ ID NO:20. It may be an amino acid sequence. The amino acid sequence of (B3) includes, for example, the amino acid sequence of (H-B1) of the second heavy chain variable region, and one or several amino acids are deleted in the amino acid sequence of SEQ ID NO:20. , A substituted, inserted and/or added amino acid sequence.

The above-mentioned (B1) amino acid sequence is, for example, a sequence including the CDL1 (L1-B1), CDRL2 (L2-B1), and CDRL3 (L3-B1) amino acid sequences. The amino acid sequence of (B2) includes, for example, the amino acid sequences of (L1-B1) of CDRL1, (L2-B1) of CDRL2, and (L3-B1) of CDRL3, and in the amino acid sequence of SEQ ID NO: 20. On the other hand, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (B3) includes, for example, (L1-B1) of CDRL1, (L2-B1) of CDRL2, and (L3-B1) of CDRL3, and in the amino acid sequence of SEQ ID NO: 20, It may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added.

The above-mentioned (B1) amino acid sequence is, for example, a sequence containing the (L-B1) amino acid sequence of the second light chain variable region. The amino acid sequence of (B2) includes, for example, the amino acid sequence of (L-B1) of the second light chain variable region, and has 70% or more identity with the amino acid sequence of SEQ ID NO:20. It may be an amino acid sequence. The amino acid sequence of (B3) includes, for example, the amino acid sequence of (L-B1) of the second light chain variable region, and one or several amino acids are deleted in the amino acid sequence of SEQ ID NO:20. , A substituted, inserted and/or added amino acid sequence.

In the amino acid sequence of (B2), the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97%. As described above, it is 98% or more and 99% or more.

In the amino acid sequence of (B3), “one or several” regarding substitution and the like is, for example, 1 to 72, 1 to 60, 1 to 48, 1 to 40, 1 to 24, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, One or two and one.

The complexed protein of the present invention has one or more of the second binding domains. In the latter case, each second binding domain may bind the same antigen or different antigens. Further, when each second binding domain binds to the same antigen, each second binding domain may bind to the same epitope of the same antigen, or may bind to different epitopes of the same antigen.

The complexed protein of the present invention preferably has a cross-linking portion capable of cross-linking with the immunoglobulin. With such a constitution, the complexed protein of the present invention can stabilize the obtained antibody complex by forming a complex with the immunoglobulin and then cross-linking with the immunoglobulin. Therefore, the complexed protein having the cross-linked portion can prevent dissociation of the immunoglobulin and the complexed protein when the antibody complex is administered to a living body, and is effective as a pharmaceutical composition. Can be used for The cross-linking portion is preferably a photo-reactive cross-linking portion that undergoes a cross-linking reaction upon irradiation with light. When the complexed protein of the present invention has a photoreactive cross-linking moiety, some or all of the amino acids constituting the complexed protein, for example, as the photo-reactive cross-linking moiety, a photoactive reactive group It is preferable that a part or all of the amino acids constituting the first binding domain have a photoactive reactive group as the photoreactive crosslinker. Examples of the amino acid having a photoactive type reactive group include L-photo-leucine, L-photo-isoleucine, L-photo-methionine and the like. The complexed protein having an amino acid having a photoactive reactive group is, for example, an amino acid having a photoactive reactive group in addition to or in place of the corresponding amino acid in the expression system of the complexed protein. Can be prepared by adding to the expression system to express the complexed protein. For the amino acid having a photoactive reactive group and the crosslinking method using the same, refer to Reference Document 4 below.
Reference 4: Monika Suchanek et.al., “Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells.”, Nature Method, 2005, DOI:10.1038/NMETH752

In the present invention, the first binding domain of the complexed protein binds to an antibody or the like, but when the antibody or the like has an additional sequence, the first binding domain may be capable of binding to the additional sequence. .. Examples of the additional sequence include tags such as peptide tags and protein tags. Examples of the tag include FLAG (registered trademark) tag, HA tag, His tag, Myc tag, V5 tag, and truncated NGFR (nerve growth factor receptor). The additional sequence is added to at least one of the N-terminus and the C-terminus of the complexed protein of the present invention, for example. When the additional sequence is a His tag, the His tag is added to the C-terminus of the complexed protein of the present invention, for example. The complexed protein of the present invention and the additional sequence may be linked by the above-mentioned linker peptide.

The method for producing the complexed protein of the present invention is not particularly limited, and for example, it can be produced by genetic engineering based on the above-mentioned amino acid sequence information. Specifically, for example, it can be performed as follows. The present invention is not limited to this example.

First, an expression vector containing a nucleic acid encoding the complexed protein of the present invention is introduced into a host to obtain a transformant. Then, the transformant is cultured to obtain a transformant expressing the complexed protein. Then, the transformant is cultured, the fraction containing the complexed protein is collected, and the complexed protein is isolated or purified from the obtained collected fraction.

Examples of the expression vector include a vector containing a nucleic acid encoding the complexed protein. The expression vector can be prepared, for example, by ligating a nucleic acid encoding the complexed protein to a ligation vector. The type of the ligation vector is not particularly limited, and examples thereof include retrovirus vectors such as oncoretrovirus vector, lentivirus vector, pseudotype vector; adenovirus vector, adeno-associated virus (AAV) vector, simian virus vector, vaccinia. Examples thereof include viral vectors such as viral vectors, Sendai viral vectors, Epstein-Barr virus (EBV) vectors and HSV vectors. Specific examples of the ligation vector include pUC, pCMV, pMX, and pELP. The ligation vector can be appropriately set, for example, according to the host into which the expression vector is introduced. The host is not particularly limited, and examples thereof include cultured cells of mammalian origin such as HEK cells, CHO cells, COS cells and NSO cells.

The expression vector preferably has a regulatory sequence that regulates the expression of the nucleic acid encoding the complexed protein, for example. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, an origin of replication sequence (ori), and the like. The arrangement of the regulatory sequences in the expression vector is not particularly limited. In the expression vector, the regulatory sequence may be arranged, for example, so that the expression of the complexed protein can be functionally regulated, and can be arranged based on a known method. As the regulatory sequence, for example, a sequence that the ligation vector has in advance may be used, the ligation vector may further have the regulatory sequence inserted, or the ligation vector may have other regulatory sequences. May be replaced with the regulatory sequence of.

The expression vector may further have a selection marker coding sequence, for example. Examples of the selection marker include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers, and the like.

The method for culturing the transformant is not particularly limited and can be appropriately determined according to the type of the host. The fraction containing the complexed protein can be collected, for example, as a liquid fraction by crushing the cultured transformant. The isolation or purification of the complexed protein is not particularly limited, and a known method can be adopted.

The complexed protein of the present invention can be used for producing the antibody complex of the present invention described below. In addition, the complexed protein of the present invention can be used as a pharmaceutical composition by forming a complex with the antibody or the like.

<Antibody complex>
As described above, the antibody complex of the present invention includes an antibody capable of binding to a target antigen or an antigen-binding fragment thereof and a complexing protein, and the complexing protein is the complexing protein of the present invention. And the complexed protein binds to the antibody or the antigen-binding fragment thereof through the first antigen-binding domain to form a complex. The antibody complex of the present invention contains the complexed protein of the present invention, and the complexed protein of the present invention forms a complex with an antibody capable of binding to a target antigen or an antigen-binding fragment thereof. Is a feature, and other configurations and conditions are not particularly limited. The antibody complex of the present invention contains a second antigen binding domain of the complexed protein of the present invention in addition to the antigen binding domain of the antibody or the antigen binding fragment thereof. Therefore, the antibody complex of the present invention can exhibit the same function as, for example, the above-described multispecific antibody. As a specific example, the antibody of the present invention is obtained by using a desired antigen to which the second binding domain binds as an antigen expressed on T cells such as CD3, and a binding target of the binding domain such as the antibody as a tumor antigen. The complex can be used as an anticancer agent that exhibits the same function as an anticancer agent containing a multispecific antibody, because it can bind to cancer cells and recruit T cells and NK cells, for example. In the antibody complex of the present invention, the binding target of the second binding domain and the binding target of the immunoglobulin can be arranged in the vicinity, and the antibody complex is, for example, hemophilia described above. It can be used as a therapeutic agent for hemophilia that exhibits the same function as a therapeutic agent. In the antibody complex of the present invention, the binding target of the second binding domain and the binding target of the immunoglobulin can be arranged in the vicinity, so that, for example, the receptor forming the complex is separated. However, the signal mediated by the receptor can be suppressed. Furthermore, since the antibody complex of the present invention can bind to a plurality of antigens, it can be used to remove a plurality of antigens, for example, to remove an antigen that causes a disease. The description of the complexed protein of the present invention can be applied to the antibody complex of the present invention.

The target antigen is not particularly limited and can be any antigen. The target antigen is, for example, an antigen associated with a disease, and the description of the desired antigen can be cited as a specific example. The antigen associated with the disease is, for example, an antigen related to the onset of the disease or the progression of symptoms, an antigen effective in the treatment of the disease, an antigen whose expression is increased or decreased in the disease, or a cell causing the disease. Examples include antigens that are expressed. The disease is not particularly limited, and examples thereof include tumors such as malignant lymphoma, leukemia, myeloma, lung cancer, breast cancer, kidney cancer, colorectal cancer, and ovarian cancer; immune diseases such as allergic disease or autoimmune disease; blood. Examples include genetic diseases such as friendship, idiopathic thrombocytopenic purpura, and von Willebrand disease; neurological diseases such as Alzheimer's disease, multiple sclerosis, Guillain-Barre syndrome, and the like. When the antibody complex of the present invention is used as a therapeutic drug for cancer, the target antigens include, for example, Angiopoietin, CD19, CD20, CD30, CD38, CD123, CEA, cMET, EpCam, GPA33, GPC3, Her1, Her2, Her2, Examples include IGF1R, PSMA, VEGF, EGFR, BCMA and the like. When the antibody complex of the present invention is used as a therapeutic agent for hemophilia, examples of the target antigen include blood coagulation factors such as factor IXa, factor X, vWF, IIb/IIIa and the like. When the antibody complex of the present invention is used as a therapeutic agent by removing an antigen, the target antigens are IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-13, IL-17, Examples include IL-23 and TNF-α. The target antigen is preferably different from the desired antigen.

The antibody or the like may be, for example, an antibody that binds to a known target antigen, or an antibody that can bind to the antibody or the like obtained by immunizing with the target antigen. As a specific example, when the target antigen is CD19, examples of the antibody and the like (clone name) include FMC63 antibody and SJ25C1. When the target antigen is CD20, examples of the antibody and the like include rituximab (Rituximab) and obinutuzumab (Obinutuzumab). When the target antigen is CD38, examples of the antibody and the like (clone name) include Daratumumab. The antibody or the like is preferably an antibody containing an Fc region or an antigen-binding fragment thereof, more preferably an antibody.

As mentioned above, in the complexed protein, the first binding domain is capable of binding immunoglobulin. Therefore, in the antibody complex of the present invention, the complexed protein is bound to the antibody or the antigen-binding fragment thereof by the first binding domain thereof. That is, in the antibody complex of the present invention, the antibody and the like and the complexed protein form a complex. However, the present invention is not limited to this, and the complexed protein, the antibody and the like may be unbound. In this case, the antibody complex of the present invention can be referred to as, for example, a composition for forming an antibody complex or a kit for forming an antibody complex.

In the antibody complex of the present invention, the antibody and the like and the complexed protein are preferably crosslinked. By cross-linking the antibody complex of the present invention, it is possible to prevent the antibody complex from separating into the antibody or the like and the complexed protein when administered to a living body. The crosslink can be formed using, for example, the crosslink part in the description of the complexed protein, and the description of the crosslink part can be incorporated.

The method for producing an antibody complex of the present invention includes, for example, a contact step of bringing an antibody capable of binding to the target antigen or an antigen-binding fragment thereof into contact with the complexed protein. In the contacting step, an antibody or an antigen-binding fragment thereof capable of binding to the target antigen is contacted with the complexing protein, whereby the first binding domain in the complexing protein binds to the antibody or the like. To form the antibody complex. The temperature in the contacting step is, for example, 0 to 40°C and 4 to 37°C. The contact time in the contact step is, for example, 10 to 180 minutes or 30 to 60 minutes. The moles of possible antibody or antigen binding fragment thereof binds to the target antigen in the contacting step _{(M Ab),} the ratio between the number of moles of complexing proteins _{_{_{(M CO) (M Ab:}}} M CO) is It is not particularly limited and is, for example, 1:0.01 to 100, 0.1 to 10. The ratio ( _MAb : _MCO ) is preferably, for example, after the contacting step, the free complexed protein can be reduced, and the side reaction when the antibody complex is administered to a living body can be suppressed. Is 1:1 or less, more preferably 1:0.01 to 1, 1:0.1 to 1, 1:0.1 to 0.9, 1:0.1 to 0.8, and 1. : 0.1 to 0.7, 1: 0.1 to 0.6, 1: 0.1 to 0.5.

The method for producing an antibody complex of the present invention may include a cross-linking step of cross-linking the antibody or the like and the complexed protein when the complexed protein has a cross-linked portion. For the cross-linking step, for example, refer to Reference 4 above.

The method for producing an antibody complex of the present invention may include, for example, a purification step of purifying the antibody complex after the contacting step or after the crosslinking step. The purification method in the purification step is not particularly limited. The antibody complex has a larger molecular weight than the antibody or the like and the complexed protein. Therefore, the purification step can be carried out by molecular exclusion chromatography such as gel filtration chromatography or molecular sieve chromatography. The antibody complex may be purified by affinity chromatography using the antibody or the antigen of the complexed protein.

The antibody complex of the present invention can be used as a pharmaceutical composition.

<Pharmaceutical composition>
The pharmaceutical composition of the present invention contains the complexed protein of the present invention as described above. The pharmaceutical composition of the present invention is characterized by containing the complexed protein of the present invention, and the other constitution and conditions are not particularly limited. The description of the complexed protein and antibody complex of the present invention can be applied to the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be used, for example, in the therapeutic method of the present invention described below.

The pharmaceutical composition of the present invention may include, for example, an antibody capable of binding to a target antigen or an antigen-binding fragment thereof. In this case, the complexed protein and the antibody or the like may or may not form a complex. In the former case, the complexed protein is bound to the antibody or antigen-binding fragment thereof by its first binding domain. In the latter case, the complexed protein and the antibody or the like are preferably in an unmixed state, for example. In this case, the pharmaceutical composition of the present invention can also be referred to as a pharmaceutical kit, for example.

The pharmaceutical composition of the present invention may include other components such as a pharmaceutically acceptable carrier and diluent. The other components are not particularly limited, and examples thereof include a preservative, an antioxidant, a chelating agent, a stabilizer, an emulsifier, a dispersant, a suspending agent and a thickener. Examples of the preservative include thimerosal, 2-phenoxyethanol and the like. Examples of the chelating agent include ethylenediaminetetraacetic acid and glycoletherdiaminetetraacetic acid.

The administration conditions of the pharmaceutical composition of the present invention are not particularly limited, and the administration form, administration method, administration timing, dose, etc. can be appropriately set depending on, for example, the type of target disease, the age of the patient, and the like. The method of using the pharmaceutical composition of the present invention is not particularly limited, and for example, the pharmaceutical composition of the present invention may be administered to the administration subject.

Examples of the administration target include cells, tissues or organs. Examples of the administration target include humans and non-human animals other than humans. Examples of the non-human animal include mammals such as mouse, rat, dog, monkey, rabbit, sheep, horse and pig. The administration may be, for example, in vivo or in vitro .

The administration form (dosage form) of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include injections (implanted injections, sustained injections, infusions (forms for drip infusion), freeze-dried injections, powder injections). , Filled syringes, cartridges, etc.) and liquid preparations such as injection preparations.

The administration method is not particularly limited and can be appropriately determined depending on the administration subject, for example. Examples of the administration method include parenteral administration and oral administration. Examples of the parenteral administration include local administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intralymphatic administration, intratumoral administration and the like.

In the pharmaceutical composition of the present invention, the dose of the complexed protein is not particularly limited. When the pharmaceutical composition of the present invention is used in vivo, it can be appropriately determined depending on, for example, the type of subject to be administered, symptoms, age, administration method and the like. As a specific example, when administered to humans, the dose of the complexed protein per administration is, for example, 1 to 1500 mg, 25 to 375 mg, 75 to 200 mg, or about 125 mg (eg, 100 to 150 mg). As a specific example, when administered to humans, the dose of the antibody or the like per administration is, for example, 1 to 5000 mg, 75 to 1125 mg, 225 to 600 mg, or about 375 mg (eg, 300 to 450 mg). When the antibody complex is administered as the pharmaceutical composition of the present invention, the dose of the antibody complex is not particularly limited. As a specific example, when administered to humans, the dose of the antibody complex per administration is, for example, 1 to 7500 mg, 100 to 1500 mg, 350 to 800 mg, about 500 mg (eg, 400 to 600 mg). The frequency of administration of the pharmaceutical composition of the present invention is, for example, once every 1 to 6 weeks. In the pharmaceutical composition of the present invention, the compounded amount of the complexed protein or the antibody complex is preferably contained in a container such as a vial at a concentration or amount that can realize the exemplified administration conditions.

<Nucleic acid>
The nucleic acid of the present invention encodes the complexed protein of the present invention. The nucleic acid of the present invention is characterized in that it encodes the complexed protein of the present invention, and other constitutions and conditions are not particularly limited. For the nucleic acid of the present invention, the description of the complexed protein, antibody complex, and pharmaceutical composition of the present invention can be incorporated. According to the nucleic acid of the present invention, the complexed protein of the present invention can be produced.

The nucleic acid of the present invention can be prepared by a conventional method, for example, based on the amino acid sequence of the complexed protein of the present invention. As a specific example, for the nucleic acid encoding the complexed protein of the present invention, for example, a base sequence encoding the amino acid sequence of each domain described above is obtained, and based on the base sequence, a molecular biology method and/or Alternatively, it can be prepared by a chemical synthesis method. The base sequence of the nucleic acid may be codon-optimized, for example, depending on the origin of cells expressing the complexed protein of the present invention.

The complexed protein contains the first binding domain and the second binding domain, as described above. Therefore, the nucleic acid of the present invention includes a nucleic acid sequence (polynucleotide) encoding the first binding domain and a nucleic acid sequence (polynucleotide) encoding the second binding domain.

Specific examples of the polynucleotide encoding the first binding domain include the following (sa) polynucleotides.
(Sa) Polynucleotide (nucleic acid) of the following (s-a1), (s-a2), (s-a3), (s-a4), (s-a5), or (s-a6)
(S-a1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:15 (s-a2) An amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:15, which is a human IgG Polynucleotide (s-a3) encoding a polypeptide that binds to human, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 15, Polynucleotide encoding a polypeptide that binds to (s-a4) Polynucleotide consisting of the nucleotide sequence of SEQ ID NO:21 (s-a5) Nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO:21 Consisting of the nucleotide sequence of polynucleotide (s-a6) SEQ ID NO: 21 encoding a polypeptide that binds to human IgG, in which one or several bases have been deleted, substituted, inserted and/or added And a polynucleotide encoding a polypeptide that binds to human IgG

A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 21
5'--3'

The polynucleotides (s-a1) to (s-a3) are nucleic acids encoding the polypeptides (S-A1) to (S-A3), respectively, and are (S-A1) to (S-A3). -The description of the polypeptide in A3) can be incorporated.

The polynucleotide (s-a4) is a polynucleotide encoding a polypeptide containing the amino acid sequences of SEQ ID NOs: 13 and 14 in the complexed protein of the present invention. In the polynucleotides (s-a5) and (s-a6), the polynucleotide encoding the amino acid sequence corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the nucleotide sequence of SEQ ID NO: 21 is, for example, , Saved. The nucleotide sequence encoding the amino acid sequence of each CDR in the nucleotide sequence of SEQ ID NO: 21 is, for example, respectively in the heavy chain variable region and the light chain variable region of the first binding domain in the complexed protein of the present invention. The amino acid sequence of CDR can be referred to.

In the base sequence (s-a5), the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, It is 97% or more, 98% or more, and 99% or more.

In the base acid sequence of (s-a6), “one or several” regarding substitution and the like is, for example, 1 to 216, 1 to 180, 1 to 144, 1 to 120, 1 to 90, 1-72, 1-60, 1-45, 1-30, 1-20, 1-9, 1-8, 1-7, 1- The number is 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, and 1.

Specific examples of the polynucleotide encoding the second binding domain include the following (sb) polynucleotides.
(Sb) Polynucleotide (nucleic acid) of (s-b1), (s-b2), (s-b3), (s-b4), (s-b5), or (s-b6) below
(S-b1) Polynucleotide encoding a polypeptide consisting of amino acid sequence of SEQ ID NO: 18 (s-b2) Comprising an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 18, and having CD3ε Polynucleotide encoding a binding polypeptide (s-b3) consists of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 18, and binds to CD3ε Polynucleotide encoding the polypeptide (s-b4) consisting of the nucleotide sequence of SEQ ID NO: 22 Polynucleotide (s-b5) consisting of the nucleotide sequence having 80% or more identity with the nucleotide sequence of SEQ ID NO: 22 , A polynucleotide encoding a polypeptide that binds to CD3ε (s-b6) consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted and/or added in the nucleotide sequence of SEQ ID NO: 22, Polynucleotide encoding a polypeptide that binds to CD3ε

A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 22
5'--3'

The polynucleotides of (s-b1) to (s-b3) are nucleic acids encoding the polypeptides of (S-B1) to (S-B3), respectively, and are (S-B1) to (S-B3). -The description of the polypeptide in B3) can be incorporated.

The polynucleotide (s-b4) is a polynucleotide encoding a polypeptide containing the amino acid sequences of SEQ ID NOs: 16 and 17 in the complexed protein of the present invention. In the polynucleotides (s-b5) and (s-b6), the polynucleotide encoding the amino acid sequence corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the nucleotide sequence of SEQ ID NO: 22 is, for example, , Saved. The nucleotide sequence encoding the amino acid sequence of each CDR in the nucleotide sequence of SEQ ID NO: 22 is, for example, respectively in the heavy chain variable region and the light chain variable region of the second binding domain in the complexed protein of the present invention. The amino acid sequence of CDR can be referred to.

In the base sequence of (s-b5), the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, respectively. It is 97% or more, 98% or more, and 99% or more.

In the base acid sequence of (s-b6), “one or several” regarding substitution and the like is, for example, 1 to 216, 1 to 180, 1 to 144, 1 to 120, 1 to 90, 1-72, 1-60, 1-45, 1-30, 1-20, 1-9, 1-8, 1-7, 1- The number is 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, and 1.

Specific examples of the polynucleotide encoding the complexed protein include the polynucleotide (b) below.
(B) Polynucleotide (nucleic acid) of the following (b1), (b2), (b3), (b4), (b5), or (b6)
(B1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 20 (b2) An amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 20, and comprising human IgG and/or CD3ε A polynucleotide (b3) encoding a polypeptide that binds to AA is composed of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 20, and human IgG and/or Alternatively, a polynucleotide encoding a polypeptide that binds to CD3ε (b4) consisting of the base sequence of SEQ ID NO:23 (b5) consisting of a base sequence having 80% or more identity with the base sequence of SEQ ID NO:23 (B6) a polynucleotide encoding a polypeptide that binds to human IgG and/or CD3ε, the nucleotide sequence of SEQ ID NO: 23 with one or several nucleotides deleted, substituted, inserted and/or added And a polynucleotide encoding a polypeptide that binds to human IgG and/or CD3ε

A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 23
5'--3'

The polynucleotides (b1) to (b3) are nucleic acids encoding the polypeptides (B1) to (B3), respectively, and the description of the polypeptides (B1) to (B3) can be incorporated.

The polynucleotide (b4) is a polynucleotide encoding a polypeptide containing the amino acid sequences of SEQ ID NOs: 15 and 18 in the complexed protein of the present invention. In the polynucleotides of (b5) and (b6), the polynucleotides encoding the amino acid sequences corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the nucleotide sequence of SEQ ID NO: 23 are conserved, for example. There is. The nucleotide sequence encoding the amino acid sequence of each CDR in the nucleotide sequence of SEQ ID NO: 23 has, for example, a heavy chain variable region and a light chain variable region of the first binding domain in the complexed protein of the present invention, respectively. The amino acid sequence of each CDR in the heavy chain variable region and the light chain variable region of the second binding domain can be referred to.

In the base sequence (b5), the “identity” is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97%, respectively. As described above, it is 98% or more and 99% or more.

In the nucleotide sequence of (b6), “one or several” regarding substitution and the like are, for example, 1 to 450, 1 to 400, 1 to 350, 1 to 300, 1 to 250, 1 to 216, 1 to 180, 1 to 144, 1 to 120, 1 to 72, 1 to 60, 1 to 45, 1 to 30, 1 to 20, 1 to 10, The numbers are 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, and 1.

<Treatment method>
In the method for treating a disease associated with the target antigen of the present invention, the complexed protein of the present invention and an antibody or an antigen-binding fragment thereof capable of binding to the target antigen are administered to a patient (administration step). The therapeutic method of the present invention is characterized by administering the complexed protein of the present invention to a patient, and other steps and conditions are not particularly limited. The description of the complexed protein, antibody complex, and pharmaceutical composition of the present invention can be applied to the therapeutic method of the present invention. According to the treatment method of the present invention, a disease associated with the target antigen can be treated.

In the administration step, the complexed protein and the antibody or the like may be administered separately or simultaneously. In the latter case, an antibody complex of the complexed protein and the antibody or the like may be administered in the administration step. In this case, the complexed protein binds to the antibody or the like by its first antigen-binding domain to form a complex. In the administration step, for example, a nucleic acid encoding the complexed protein may be administered as the complexed protein.

The disease related to the target antigen is not particularly limited, and examples thereof include tumors such as malignant lymphoma, leukemia, myeloma, lung cancer, breast cancer, kidney cancer, colon cancer, and ovarian cancer; allergic diseases or autoimmune diseases. And the like; genetic diseases such as hemophilia, idiopathic thrombocytopenic purpura, and von Willebrand disease; neurological diseases such as Alzheimer's disease, multiple sclerosis, Guillain-Barre syndrome, and the like.

The combination of the target antigen and the disease is not particularly limited. As a specific example, when the disease is cancer (tumor), the target antigen is a tumor antigen. In this case, the desired antigen in the complexed protein is, for example, an antigen expressed in the immune cells, preferably CD3. When the disease is B cell lymphoma, the target antigen is an antigen expressed on B cells such as CD19, CD20, CD38. In this case, the desired antigen in the complexed protein is, for example, an antigen expressed in the immune cells, preferably CD3. When the disease is an allergic disease, the target antigen is, for example, an allergen or cytokine. In this case, the desired antigen in the complexed protein is, for example, an allergen or cytokine, preferably an antigen different from the target antigen. When the disease is hemophilia, the target antigen is, for example, the aforementioned blood coagulation factor. In this case, the desired antigen in the complexed protein is, for example, the above-mentioned blood coagulation factor, preferably an antigen different from the target antigen.

<Use of complexed protein or antibody complex>
The present invention is the complexed protein or antibody complex of the present invention for use in a method for treating a disease associated with a target antigen. The present invention is also the use of the complexed protein or antibody complex of the present invention for the manufacture of a medicament for treating a disease associated with a target antigen. The use of the present invention can be incorporated by reference for the conjugated proteins, antibody conjugates, and pharmaceutical compositions of the present invention.

Next, an embodiment of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocol unless otherwise indicated.

[Example 1]
After preparing the complexed protein of the present invention, an antibody complex is prepared, and the antibody complex is provided with a binding property to a desired antigen, and the antibody complex is the same as the multispecific antibody. It was confirmed that the function of was exhibited.

(1) Preparation of B-BiTE First, a complexed protein having a first binding domain capable of binding to human IgG and a second binding domain capable of binding to CD3ε (hereinafter, also referred to as “B-BiTE”) ) Was prepared. Specifically, a polynucleotide encoding B-BiTE was synthesized using an artificial gene synthesis service (GeneArt (trademark), manufactured by Thermo Fisher Scientifics). The polynucleotide encoding B-BiTE was designed as follows. First, a heavy chain variable region and a light chain variable region derived from a mouse anti-human IgG mAb (clone: HP6017) were linked by a linker peptide (GGGGSGGGGSGGGGS: SEQ ID NO: 24), and a scFv capable of binding to human IgG (first binding) Domain) designed. The HP6017 antibody is known to be able to bind to all subclasses of human IgG.

Next, the heavy chain variable region and the light chain variable region derived from human CD3ε mAb (clone: OKT3) were linked by a linker peptide (GGGGSGGGGSGGGGS: SEQ ID NO: 24), and scFv (second binding domain) capable of binding to human CD3ε ) Was designed. Furthermore, the first binding domain and the second binding domain are linked in this order from the N terminus via a domain linker (SGSG: SEQ ID NO: 19), and the domain linker is further linked to the C terminus. B-BiTE was designed by connecting a His tag (His×6 tag) via Then, the base sequence of the polynucleotide (SEQ ID NO: 26) encoding B-BiTE was determined from the designed amino acid sequence of B-BiTE (SEQ ID NO: 25). Then, a polynucleotide encoding B-BiTE was synthesized.

Amino acid sequence of B-BiTE (SEQ ID NO:25)
MMRPIVLVLLFATSAAADIKMTQSPSSMYASVGERVTITCKASQDIKSYLTWYQKKPWKSPRTLIFYSTRLADGVPSRFSGSGSGQDFSLTISSLESDDTATYYCLQHDESPFTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTNYWINWVKQRPGQGLEWIGDIYPGGGITNYNEKFKTKATLTLDTSSSTVYMQLSSLTSEDSAVYYCSRSYGKYFDYWGQGTTLIVSSSGSGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINSGSGHHHHHH

Polynucleotide encoding B-BiTE (SEQ ID NO: 26, including stop codon)
5'--3'

The polynucleotide encoding B-BiTE was introduced into a pMX expression vector (B-BiTE expression vector). Next, a retrovirus was prepared by introducing the B-BiTE expression vector into Plat-A cells (provided by the Institute of Medical Science, University of Tokyo) using a transfection reagent (TransIT293, manufactured by Takara Bio). By infecting 293T cells with the retrovirus, a polynucleotide encoding B-BiTE was introduced into 293T cells to prepare B-BiTE-producing 293T cells (293T/B-BiTE cells). The Plat-A cells used were those maintained in DMEM medium containing 10% fetal calf serum (FCS), 1 μg/mL puromycin, and 10 μg/mL blasticidin. The obtained 293T/B-BiTE cells were cultured, and B-BiTE was produced from the obtained culture supernatant using a His-tag column (His GraviTrap column, manufactured by GE healthcare). The culture solution of 293T/B-BiTE cells was a DMEM medium supplemented with Ultra Low IgG FBS (manufactured by Thermo Fisher Scientific). The culture conditions of 293T cells and Plat-A cells were 37° C., 5% CO ₂ and a humid atmosphere. Hereinafter, unless otherwise stated, the cell culture conditions were the same.

(2) B-BiTE binding ability Next, it was confirmed that B-BiTE binds to human IgG and human CD3ε with the target antigen CD19. First, an antibody complex was formed between B-BiTE and an anti-CD19 antibody (human IgG1 chimeric antibody, clone FMC63) or a control antibody (human IgG1 chimeric antibody, clone MOPC21). The variable region of each antibody is a mouse antibody, and the constant region is a human antibody. Specifically, B-BiTE, an anti-CD19 antibody or a control antibody is added to a serum-free phosphate buffer (PBS) at a concentration of B-BiTE (B) and a concentration of the antibody (A). Was added so that the concentration ratio (B:A) was 1:3, and the mixture was incubated at room temperature (about 25° C.) for 1 hour. By setting the concentration (B) of B-BiTE and the concentration (A) of the antibody to 1:3, the ratio ( _MAb : _MCO ) becomes 1:about 1. Then, the obtained mixed solution was used as an antibody complex (CD19/B-BiTE or isotype/B-BiTE). The B-BiTE concentration was 10 μg/mL, and the antibody concentration was 30 μg/mL.

The cells that react with the antibody complex are KT1 cells (CD3 ^- CD19 ^- cells, chronic myelogenous leukemia-derived cell line (established in Ehime University School of Medicine, First University)), KT1/CD19 cells (CD3 ^- CD19 ⁺ cells). ), K562 cells (CD3 ⁻ CD19 ⁻ cells, obtained from ATCC), K562/CD19 cells (CD3 ⁻ CD19 ⁺ cells), Raji cells (CD3 ⁻ CD19 ⁺ cells), Jurkat76 cells (CD3 ⁻ CD19 ⁻ cells), and Jurkat. The cells (CD3 ⁺ CD19 ⁻ cells) were used. KT1/CD19 cells and K562/CD19 cells were prepared by introducing a polynucleotide encoding human CD19 using a retrovirus into KT1 cells and K562 cells, respectively. Specifically, a retrovirus was prepared in the same manner except that a pMX expression vector containing a polynucleotide (SEQ ID NO: 27) encoding human CD19 was introduced instead of the B-BiTE expression vector. KT1/CD19 cells and K562/CD19 cells were prepared by infecting KT1 cells and K562 cells with the obtained retrovirus. The culture solution of KT1 cells and KT1/CD19 cells was RPMI medium containing 10% FCS. The culture solution of K562 cells, K562/CD19 cells, Raji cells, Jurkat76 cells, and Jurkat cells, and K562/CD20 cells and K562/CD38 cells described later was 10% FCS-containing RPMI1640 medium.

Polynucleotide encoding human CD19 (SEQ ID NO:27)
5'--3'

KT1 cells, KT1/CD19 cells, K562 cells, K562/CD19 cells, Raji cells, Jurkat76 cells, and Jurkat cells are mixed with a mixed solution containing CD19/B-BiTE or isotype/B-BiTE, and the mixture is mixed for 30 minutes 4 Incubated at °C. After the incubation, each cell was washed and stained with a PE-labeled anti-His antibody (clone GG11-8F3.5.1). Then, for each cell after staining, binding between each cell and the antibody complex was examined using a flow cytometer (Gallios flow cytometer, manufactured by Beckman Coulter). The control was measured in the same manner except that the antibody complex was not added. The results are shown in FIG.

FIG. 2 is a histogram showing the binding between the antibody complex and cells. In FIG. 2, (A) shows KT1 cells, (B) shows KT1/CD19 cells, (C) shows K562 cells, (D) shows K562/CD19 cells, (E) shows Raji cells, (F). ) Shows the results of Jurkat76 cells, and (G) shows the results of Jurkat cells. 2A to 2G, the horizontal axis represents the fluorescence intensity of the anti-His antibody, and the vertical axis represents the relative value of the count number. As shown in FIGS. 2(A) to (G), no binding was observed in the control. Further, as shown in FIG. 2(G), the antibody complex of the complexed protein and the control antibody (isotype/B-BiTE) showed binding to CD3ε and therefore bound to Jurkat cells. .. Furthermore, as shown in FIGS. 2(B), (D), (E) and (G), the antibody complex (CD19/B-BiTE) of the complexed protein and the anti-CD19 antibody was added to CD19 and CD3ε. In order to show the binding property, it bound to KT1/CD19 cells, K562/CD19 cells, Raji cells and Jurkat cells. From these results, it was found that the complexed protein of the present invention can add a binding property to a desired antigen to the binding target antibody.

(3) Function of antibody complex The bispecific antibody can activate T cells by recruiting T cells to cells expressing the target antigen. Therefore, it was examined whether B-BiTE/CD19 has a similar function. Specifically, except that the concentration of B-BiTE was 2.5 μg/mL and the concentration of the antibody was 7.5 μg/mL, CD19/B-BiTE or Isotype/B-BiTE was prepared.

Next, CD4 ⁺ T cells and CD8 ⁺ T cells in peripheral blood were prepared. Specifically, human peripheral blood was collected from a healthy person. Next, after extracting human peripheral blood mononuclear cells from the peripheral blood, using the obtained human peripheral blood mononuclear cells and a T cell purification kit (Pan T cell isolation kit, manufactured by Miltenyi Biotec), CD3 ⁺ T cells were purified. Then, 2.5×10 ⁵ T cells and 5.0×10 ⁴ target cells were cultured for 2 hours in the presence of the above CD19/B-BiTE or isotype/B-BiTE (n= 3). The target cells were K562 cells, K562/CD19 cells and Raji cells. After the culture, Brefeldin A (BFA) was added so that the concentration was 5 μg/mL, and the culture was further continued for 18 hours. After recovering T cells, the cells were fixed with 1% paraformaldehyde (PFA) and then permeabilized to give PE-labeled anti-human TNF-α antibody (clone MAb11) and APC-labeled anti-human IL-2 antibody (clone MQ1). -17H12) and PC7-labeled anti-human IFN-γ antibody (clone B27), PC5-labeled anti-human CD8 antibody (clone B9.11), FITC-labeled anti-CD4 antibody (clone OKT4) and V450 anti-human CD3 antibody (clone UCHT1). Stained with. The stained T cells were measured by the flow cytometer, and the ratio of cells producing each cytokine was calculated. The results are shown in Figures 3 and 4.

3 and 4 are graphs showing the ratio of cytokine-producing cells. 3 and 4, the horizontal axis represents the type of antibody or antibody complex, and the vertical axis represents the proportion of each cytokine-producing cell. FIG. 3 shows the results when the target cells were K562 cells or K562/CD19 cells, and FIG. 4 shows the results when the target cells were Raji cells. As shown in FIG. 3, when K562 cells that did not express CD19 and CD3 were used as target cells, no cytokine production was observed in the CD19/B-BiTE and isotype/B-BiTE addition groups. On the other hand, when K562/CD19 cells expressing CD19 were used as target cells, the CD19/B-BiTE-added group had a significantly higher proportion of cytokine-producing cells than the isotype/B-BiTE-added group. Was increasing. Further, as shown in FIG. 4, when Raji cells expressing CD19 were used as target cells, the CD19/B-BiTE-added group showed significantly more cytokine-producing cells than the isotype/B-BiTE-added group. The proportion was increasing. From these results, it was found that the antibody complex of the present invention, like the bispecific antibody, can recruit T cells to cells expressing the target antigen and activate T cells.

From the above, the complexed protein of the present invention forms an antibody complex, the antibody complex is imparted with binding properties to a desired antigen, and the antibody complex has multispecificity. It was found that it exerts the same function as an antibody.

[Example 2]
It was found that an antibody complex of the present invention was prepared, that the antibody complex has a binding property to a desired antigen, and that the antibody complex exhibits a function similar to that of a multispecific antibody. It was

In place of the anti-CD19 antibody, an anti-CD20 antibody (Rituximab) or an anti-CD38 antibody (Daratumumab) was used, except that the antibody complex (CD20/B-BiTE or CD38) was used in the same manner as in Example 1(2). /B-BiTE) was prepared.

Cells that react with the antibody complex are K562 cells (CD3 ^- CD20 ^- CD38 ^- cells, obtained from ATCC), K562/CD20 cells (CD3 ^- CD20 ⁺ CD38 ^- cells), K562/CD38 cells (CD3 ^- CD20 ^- CD38 ⁺ cells). Cells), Raji cells (CD3 ⁻ CD20 ⁺ CD38 ⁺ cells), Jurkat76 cells (CD3 ⁻ CD20 ⁻ CD38 ⁻ cells), and Jurkat cells (CD3 ⁺ CD20 ⁻ CD38 ⁻ cells). K562/CD20 cells and K562/CD38 cells used retroviruses except that a polynucleotide encoding human CD20 and a polynucleotide encoding human CD38 were used in place of the polynucleotide encoding human CD19, respectively. Was prepared in the same manner as in Example 1(2) above.

Polynucleotide encoding human CD20 (SEQ ID NO: 28, including stop codon)
5'--3'

Polynucleotide encoding human CD38 (SEQ ID NO: 29, including stop codon)
5'--3'

Then, K562/CD20 cells and K562/CD38 cells were used instead of KT1 cells, KT1/CD19 cells, and K562/CD19 cells, and CD20/B-BiTE or CD38/B- was used instead of CD19/B-BiTE. The binding between each cell and the antibody complex was examined in the same manner as in Example 1(2) except that BiTE was used. The control was measured in the same manner except that the antibody complex was not added. These results are shown in FIG.

FIG. 5 is a histogram showing the binding between the antibody complex and cells. In FIG. 5, (A) is K562 cells, (B) is K562/CD20 cells, (C) is K562/CD38 cells, (D) is Raji cells, (E) is Jurkat cells, (F). ) Shows the results of Jurkat76 cells. 5A to 5F, the horizontal axis represents the fluorescence intensity of the anti-His antibody, and the vertical axis represents the relative value of the count number. As shown in FIGS. 5(A) to 5(F), no binding was observed in the control. Further, as shown in FIG. 5(E), the antibody complex of the complexed protein and the control antibody (isotype/B-BiTE) showed binding to CD3ε and therefore bound to Jurkat cells. .. Furthermore, as shown in FIGS. 5(B), (D), and (E), the antibody complex of the complexed protein and the anti-CD20 antibody (CD20/B-BiTE) binds to CD20 and CD3ε. To show sex, it bound to K562/CD20 cells, Raji cells and Jurkat cells. Then, as shown in FIGS. 5(C) to (E), the antibody complex of the complexed protein and the anti-CD38 antibody (CD38/B-BiTE) exhibits binding to CD38 and CD3ε, It bound to K562/CD38 cells, Raji cells and Jurkat cells. From these results, it was found that the complexed protein of the present invention can add a binding property to a desired antigen to the binding target antibody.

Next, we examined whether CD20/B-BiTE and CD38/B-BiTE exhibit the same function as the bispecific antibody. Specifically, instead of the anti-CD19 antibody, an anti-CD20 antibody (Rituximab) or an anti-CD38 antibody (Daratumumab) was used, the B-BiTE concentration was 2.5 μg/mL, and the antibody concentration was 7.5 μg/mL. CD20/B-BiTE or CD38/B-BiTE was prepared in the same manner as in Example 1(2) except that the amount was changed to mL.

Next, in place of CD19/B-BiTE, CD20/B-BiTE or CD38/B-BiTE was used, and instead of K562/CD19 cells, K562/CD20 cells or K562/CD38 cells were used, except that The proportion of cells producing cytokines was calculated in the same manner as in Example 1(3). The results are shown in FIGS. 6 and 7.

6 and 7 are graphs showing the ratio of cytokine-producing cells. 6 and 7, the horizontal axis represents the type of antibody or antibody complex, and the vertical axis represents the proportion of each cytokine-producing cell. FIG. 6 shows the results when the target cells were K562 cells, K562/CD20 cells or K562/CD38 cells, and FIG. 7 shows the results when the target cells were Raji cells. As shown in FIG. 6, when K562 cells that do not express CD20, CD38 and CD3 were used as target cells, cytokine production was not observed in the CD20/B-BiTE, CD38/B-BiTE and isotype/B-BiTE addition groups. I couldn't see it. On the other hand, when K562/CD20 cells expressing CD20 were used as target cells, the CD20/B-BiTE-added group had a significantly higher percentage of cytokine-producing cells than the isotype/B-BiTE-added group. Was increasing. In addition, when K562/CD38 cells expressing CD38 were used as target cells, the proportion of cytokine-producing cells was significantly increased in the CD38/B-BiTE-added group as compared with the isotype/B-BiTE-added group. It was Furthermore, as shown in FIG. 7, when Raji cells expressing CD20 and CD38 were used as target cells, the CD20/B-BiTE-added group and the CD38/B-BiTE-added group had The proportion of cytokine-producing cells was significantly increased as compared with. From these results, it was found that the antibody complex of the present invention, like the bispecific antibody, can recruit T cells to cells expressing the target antigen and activate T cells.

From the above, the complexed protein of the present invention forms an antibody complex, the antibody complex is imparted with binding properties to a desired antigen, and the antibody complex has multispecificity. It was found that the antibody functions similarly to the antibody.

[Example 3]
It was confirmed that the antibody complex of the present invention can induce the proliferation of other T cells while substantially suppressing the proliferation of regulatory T cells.

CD3 ⁺ T cells were prepared in the same manner as in Example 1(3) above. The T cells obtained were incubated in PBS containing 0.5 μmol/L CFSE (5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester) for 15 minutes at 37° C., and the T cells were labeled with CFSE.

Next, 2.0×10 ⁵ CFSE-labeled T cells and 2.0×10 ⁴ target cells were treated with the above CD20/B-BiTE, CD38/B-BiTE, or isotype/B-BiTE. In the presence, the cells were cultured for 5 days (n=3). The target cells were K562/CD19 cells, K562/CD20 cells, K562/CD38 cells and Raji cells. After the culture, the T cells were treated with Alexa647-labeled anti-human FoxP3 antibody (clone 206D), PE-labeled anti-human CD45RA antibody (clone HI100), the PC5-labeled anti-human CD8 antibody (clone B9.11), and APC-Cy7-labeled anti-human. The cells were stained with a CD4 antibody (clone OKT4) and a BV421 anti-human CD25 antibody (clone BC96). The stained T cells were measured by the flow cytometer, and the proportion of CFSE-attenuated cells and the proportion of regulatory T cells in the T cells were measured. These results are shown in FIGS. 8 and 9 (ns: not siginificant, *: p<0.05, **: p<0.01, ***: p<0.001, and so on).

FIG. 8 is a graph showing the percentage of cells in which CFSE is attenuated. In FIG. 8, the horizontal axis represents the type of antibody complex and the vertical axis represents the proportion of proliferated cells. As shown in the dot plot of FIG. 8, in isotype/B-BiTE, since T cell proliferation does not occur, CFSE strongly positive cells are obtained as indicated by arrows in the figure. On the other hand, in K562/CD38 cells and CD38/B-BiTE, the proliferation of T cells causes the attenuation of CFSE, so that the cells that proliferate are weakly positive for CFSE, as shown by the arrow in the figure. Become. When K562/CD19 cells expressing CD19 were used as the target cells, the CD19/B-BiTE-added group showed significantly expanded CD4 ⁺ T cells and CD8 ⁺ compared to the isotype/B-BiTE-added group. The proportion of T cells was increasing. Moreover, when K562/CD20 cells expressing CD20 were used as target cells, the CD20/B-BiTE-added group showed significantly proliferated CD4 ⁺ T cells and CD8 ⁺ compared to the isotype/B-BiTE-added group. The proportion of T cells was increasing. Furthermore, when K562/CD38 cells expressing CD38 were used as target cells, the CD38/B-BiTE-added group showed significantly increased CD4 ⁺ T and CD8 ⁺ T compared with the isotype/B-BiTE-added group. The proportion of cells was increasing. When Raji cells expressing CD19, CD20 and CD38 are used as target cells, the CD19/B-BiTE addition group, the CD20/B-BiTE addition group and the CD38/B-BiTE addition group are isotype/B- The proportion of significantly proliferated CD4 ⁺ T cells and CD8 ⁺ T cells was increased compared to the BiTE-added group.

FIG. 9 is a graph showing the ratio of activated CD4 ⁺ T cells (CD4 ⁺ CD25 ⁺ ) and regulatory T cells (CD4 ⁺ CD25 ⁺ CD45RA ⁺ FoxP3 ⁺ ). In FIG. 9, the horizontal axis represents the type of antibody complex or the subset of regulatory T cells, and the vertical axis represents the ratio of activated CD4 ⁺ T cells (CD4 ⁺ CD25 ⁺ ), or activated. The percentage of regulatory T cell subsets in CD4 ⁺ T cells (CD4 ⁺ CD25 ⁺ ) is shown. ^{Incidentally,} the CD4 + CD25 ⁺ cells, and developed with the expression of CD45RA and ^FoxP3, and CD45RA + FoxP3 ⁺ cells ^group, CD45RA - ^FoxP3 a cell population ⁺⁺⁺ was defined as regulatory T cells. In addition, the CD45RA ⁻ FoxP3 ⁺ cell group was determined to be activated T cells based on Reference Document 5 below. In this example, no CD45RA ⁻ FoxP3 ⁺⁺ cell group was found in the peripheral blood T cells after culture. When K562/CD19 cells expressing CD19 were used as target cells, the proportion of CD4 ⁺ T cells that were significantly activated in the CD19/B-BiTE addition group was significantly higher than that in the isotype/B-BiTE addition group. Although increased, the proportion of naive regulatory T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁺ ) and activated T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁻ ) remained unchanged. When K562/CD20 cells expressing CD20 were used as target cells, the CD20/B-BiTE-added group showed significantly activated CD4 ⁺ T cells as compared with the isotype/B-BiTE-added group. Although the proportion was increased, the proportion of naive regulatory T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁺ ) remained unchanged, and the proportion of activated T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁻ ) was slightly increased. did. Furthermore, when K562/CD38 cells expressing CD38 were used as target cells, the CD38/B-BiTE addition group showed significantly activated CD4 ⁺ T cells compared to the isotype/B-BiTE addition group. Although the proportion was increased, the proportion of naive regulatory T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁺ ) remained unchanged, and the proportion of activated T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁻ ) was slightly increased. did. When Raji cells expressing CD19, CD20 and CD38 are used as target cells, the CD19/B-BiTE addition group, the CD20/B-BiTE addition group and the CD38/B-BiTE addition group are isotype/B- Although the ratio of CD4 ⁺ T cells that were significantly activated was increased compared to the BiTE-added group, the ratio of naive regulatory T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁺ ) did not change, and the activity was unchanged. The ratio of activated T cells (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CD45RA ⁻ ) slightly increased in the CD19/B-BiTE-added group.
Reference 5: Makoto Miyara et.al., “Functional Delineation and Differentiation Dynamics of Human CD4 ⁺ T Cells Expressing the FoxP3 Transcription Factor”, Immunity, 2009, volume 30, pages 899-911.

From these results, it was found that the antibody complex of the present invention can substantially suppress the proliferation of regulatory T cells while inducing the proliferation of other T cells.

[Example 4]
It was confirmed that the antibody complex of the present invention can activate NK cells alone or NK cells and T cells.

(1) Activation of NK cells In place of the anti-CD19 antibody, an anti-CD20 antibody (Rituximab) or an anti-CD38 antibody (Daratumumab) was used, the concentration of B-BiTE was 2.5 μg/mL, and the concentration of the antibody was 7 An antibody complex (CD20/B-BiTE or CD38/B-BiTE) was prepared in the same manner as in Example 1(2) except that the concentration was 0.5 μg/mL.

Next, NK cells were prepared from peripheral blood. Specifically, human peripheral blood was collected from a healthy person. Next, after extracting human peripheral blood mononuclear cells from the peripheral blood, the obtained human peripheral blood mononuclear cells and NK cell purification kit (NK cell isolation kit, manufactured by Miltenyi Biotec) were used to obtain NK cells. Was purified. Then, 1×10 ⁵ NK cells and 1.0×10 ⁵ target cells were treated with the anti-CD20 antibody, anti-CD38 antibody, control antibody, CD20/B-BiTE, CD38/B-BiTE, or isotype. /B-BiTE, and 100 U/mL IL-2 and 10 ng/mL IL-15 were incubated for 1 hour (n=3). The concentration of the antibody was 7.5 μg/mL when the antibody was used alone. The target cells were K562/CD20 cells, K562/CD38 cells and Raji cells. After the culture, BFA was added so that the concentration was 5 μmol/L, and the culture was further continued for 4 hours. After fixing NK cells with 1% PFA, permeabilization of the cell membrane was performed, and further, APC-labeled anti-human CD107a (clone H4A3), the PC7-labeled anti-human IFN-γ antibody, and FITC-labeled anti-human CD16 antibody (clone). 3G8) and PE-labeled anti-human CD56 antibody (clone HCD56). The stained NK cells were measured with the flow cytometer. The control was measured in the same manner except that the target cells were not added. Then, based on the expression of CD107a in the control, the rate of increase in CD107a expression was calculated. The results are shown in Figures 10 and 11.

10 and 11 are graphs showing the ratio of IFN-γ producing cells and the ratio of increased CD107a expression. 10 and 11, the horizontal axis represents the type of antibody or antibody complex, and the vertical axis represents the proportion of IFN-γ producing cells or the proportion of increased CD107a expression. As shown in FIGS. 10 and 11, when there are target cells that express the target antigen CD20 or CD38, and when there is an antibody that binds to the target antigen, when there is a target that does not express the target antigen, and the target It was found that NK cells were activated due to an increase in the percentage of IFN-γ producing cells and an increase in CD107a expression, as compared with the case where an antibody that does not bind to the antigen was present. Similarly, when there is a target cell that expresses the target antigen CD20 or CD38, and when there is an antibody complex that binds to the target antigen, when there is a target that does not express the target antigen, and when there is a target antigen It was found that NK cells were activated due to an increase in the ratio of IFN-γ producing cells and an increase in the expression of CD107a, as compared with the case where there was an antibody complex that did not bind. In addition, there was no difference in the increase in the ratio of IFN-γ producing cells and the increase in the expression of CD107a between the anti-CD20 antibody-added group and the CD20/B-BiTE added group. There was no difference between the anti-CD38 antibody-added group and the CD38/B-BiTE-added group in the increase in the ratio of IFN-γ producing cells and the increase in the expression of CD107a. From these, it was found that the antibody complex of the present invention can induce the activation of NK cells similarly to the antibody.

(2) Activation of NK cells and T cells In addition to the anti-CD19 antibody, an anti-CD20 antibody (Rituximab) or an anti-CD38 antibody (Daratumumab) was used to adjust the concentration of B-BiTE to 2.5 μg/mL, An antibody complex (CD19/B-BiTE, CD20/B-BiTE or CD38/B-BiTE) was prepared in the same manner as in Example 1(2) except that the concentration was 7.5 μg/mL.

Human peripheral blood was collected from a healthy person. Next, human peripheral blood mononuclear cells were extracted from the peripheral blood, and then the obtained human peripheral blood mononuclear cells and B cell removal kit (CD19 microbeads, manufactured by Miltenyi Biotec) were used to remove B cells. By doing so, a cell group containing NK cells, T cells and monocytes was purified. After the purification, 2×10 ⁵ cell groups and 2.0×10 ⁵ target cells were treated with the anti-CD19 antibody, anti-CD20 antibody, anti-CD38 antibody, control antibody, CD19/B-BiTE, CD20/ The cells were cultured for 5 hours in the presence of B-BiTE, CD38/B-BiTE, or isotype/B-BiTE (n=3). The concentration of the antibody was 7.5 μg/mL when the antibody was used alone. The target cells were K562/CD19 cells, K562/CD20 cells and K562/CD38 cells. After the culture, BFA was added so that the concentration became 5 μmol/L, and the culture was further continued for 5 hours. Then, after culturing, the cells were fixed with 1% PFA, and then the cell membrane was permeabilized. Furthermore, APC-Cy7 anti-human CD4 antibody (clone RPA-T4), Alexa700-labeled anti-human CD56 antibody (clone 5.1H11), V450 anti-human CD107a antibody (clone H4A3), PE-labeled anti-human TNF-α antibody (clone MAb11). , APC-labeled anti-human IL-2 antibody (clone MQ1-17H12), PC7-labeled anti-human IFN-γ antibody (clone B27), PC5-labeled anti-human CD8 antibody (clone B9.11), and FITC-labeled anti-human CD16 antibody ( It was stained with clone 3G8). The cells after the staining were measured with the flow cytometer. Then, the rate of increase in CD107a expression was calculated based on the cytokine expression and CD107a expression in the target cells as controls. The results are shown in FIGS.

12 to 14 are graphs showing activation of NK cells and T cells. 12 to 14, the horizontal axis represents the type of antibody or antibody complex, and the vertical axis represents the proportion of cells producing each cytokine or the proportion of increased expression of CD107a. As shown in FIGS. 12 to 14, when target cells that express the target antigen CD19, CD20 or CD38 exist, and when an antibody that binds to the target antigen exists, and when a target that does not express the target antigen exists It was found that the NK cells were activated by the increase in the ratio of IFN-γ producing cells in the NK cells and the increase in the expression of CD107a, as compared with the case where the antibody that does not bind to the target antigen was present. Similarly, when there is a target cell that expresses the target antigen, CD19, CD20, or CD38, and when there is an antibody complex that binds to the target antigen, there is a target that does not express the target antigen, and Increased proportion of IL-2, IFN-γ and TNF-α producing cells in CD4 ⁺ T cells and CD8 ⁺ T cells and increased expression of CD107a compared to the presence of antibody complexes that do not bind antigen. Occurred, resulting in an increase in the percentage of IFN-γ producing cells in NK cells and an increase in the expression of CD107a. That is, it was found that CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells were activated. From these, it was found that the antibody complex of the present invention can induce the activation of NK cells and T cells similarly to the antibody.

(3) Induction of NK cell and T cell damaging activity In place of the anti-CD19 antibody, an anti-CD20 antibody (Rituximab) or an anti-CD38 antibody (Daratumumab) was used, and the concentration of B-BiTE was set to 1 μg/mL. An antibody complex (CD19/B-BiTE, CD20/B-BiTE or CD38/B-BiTE) was prepared in the same manner as in Example 1(2) except that the concentration was 3 μg/mL.

Next, 5.0×10 ³ target cells of K562 cells, K562/CD20 cells, K562/CD38 cells and Raji cells were cultured in the presence of chromium 51 ( ⁵¹ Cr) for 1.5 hours. , Labeled with chrome 51. Next, regarding the cell group (E) purified in Example 4(1) and the target cells (T) labeled with the chromium 51, E:T was 20:1, 10:1, 5:1 or The purified cell group was added to the well in which the target cells were cultured so that the ratio was 2.5:1. Further, the above-mentioned anti-CD20 antibody, anti-CD38 antibody, control antibody, isotpye/B-BiTE, CD20/B-BiTE or CD38/B-BiTE was added. The concentration of the antibody was 3 μg/mL. Then, after culturing for 18 hours, the supernatant was collected, and the counting rate (CPM: count per minute) of radiation per minute was measured using AccuFLEXg7010 (manufactured by HITACHI). The control was measured in the same manner except that the purified cell group was not added. Then, the cytotoxicity (specific melting rate) was measured based on the following formula (1). The results are shown in FIG.

K=(L _E −L _S )/(L _Max −L _S )×100(%) (1)
K: Cytotoxicity L _E: measurement of CPM L _S: measured value of CPM in the case of target cells only L _Max: measurement of CPM in the case of adding 0.2% Triton-X in PBS to wells of target cells only

FIG. 15 is a graph showing cytotoxicity. In FIG. 15, the horizontal axis represents the E/T ratio and the vertical axis represents the cytotoxicity. As shown in FIG. 15, when K562, which does not express the target antigens CD20 and CD38, was used as the target cells, specific cytotoxicity did not occur, and the cytotoxicity in each group was similar. On the other hand, when K562 and Raji cells expressing the target antigen CD20 or CD38 are used as target cells, the presence of an antibody or antibody complex that binds to the target antigen causes cytotoxicity in a dose ratio-dependent manner on the effector. Increased. In addition, when the antibody-added group and the antibody-complex-added group were compared, the antibody-complex-added group had increased cytotoxicity as compared with the antibody-added group. From these results, it was found that the antibody complex of the present invention can induce the cytotoxic activity and its inducing ability is enhanced as compared with the antibody alone.

[Example 5]
It was confirmed that the antibody complex of the present invention induces cytokine production of T cells in a concentration-dependent manner.

In addition to the anti-CD19 antibody, an anti-CD20 antibody (Rituximab) or an anti-CD38 antibody (Daratumumab) was used, except that the B-BiTE concentration was 2.5 μg/mL and the antibody concentration was 7.5 μg/mL. An antibody complex (CD19/B-BiTE, CD20/B-BiTE or CD38/B-BiTE) was prepared in the same manner as in Example 1(2) above.

CD3 ⁺ T cells were purified in the same manner as in Example 1(3) above. 2.5×10 ⁵ CD3 ⁺ T cells and 5.0×10 ⁴ target cells were used at a predetermined concentration (total concentration of B-BiTE and antibody; 10 μg/mL) of CD19/B-BiTE, CD20. Culture was performed for 2 hours in the presence of /B-BiTE, CD38/B-BiTE, or isotype/B-BiTE (n=3). The target cells were K562/CD19 cells, K562/CD20 cells or K562/CD38 cells. After the culture, BFA was added so that the concentration became 5 μmol/L, and the culture was further continued for 18 hours. Then, after culturing, the cells were fixed with 1% PFA, and then the cell membrane was permeabilized. Further, the ratio of cells producing each cytokine was calculated in the same manner as in Example 1 (3) above. Then, in each of the cytokines of CD4 ⁺ T cells and CD8 ⁺ T cells, the ratio of the cells producing the cytokine when the antibody complex was added at 10 μg/mL was set to 100%, and the relative ratio of the producing cells was calculated. .. The results are shown in FIG.

FIG. 16 is a graph showing the relative proportion of producer cells. In FIG. 16, the upper row shows the results of CD8 ⁺ T cells, and the lower row shows the results of CD4 ⁺ T cells. In FIG. 16, the horizontal axis represents the total concentration of B-BiTE and the antibody, and the vertical axis represents the relative proportion of producing cells. Moreover, the numerical value described adjacent to the name of the antibody complex in each graph shows EC50. As shown in FIG. 16, CD8 ⁺ T cells and CD4 ⁺ T cells increased the proportion of cells producing each cytokine, depending on the concentration of the antibody complex. From these results, it was found that the antibody complex of the present invention induces T cell cytokine production in a concentration-dependent manner.

[Example 6]
It was confirmed that the antibody complex of the present invention exhibits antitumor activity in vivo.

Raji cells expressing SLR (Raji/SLR cells) were the same as those of the above except that Raji cells were used instead of K562 cells and a polynucleotide encoding SLR was used instead of the polynucleotide encoding human CD19. Prepared as in Example 1(2). Since SLR is an intracellular protein, a polynucleotide encoding a truncated NGFR gene (ΔNGFR) can be used as a polynucleotide encoding SLR by using a furin cleavage site (RAKR: SEQ ID NO: 32) and a spacer. Ligation was performed via a polynucleotide encoding the sequence (SGSG (same sequence as the domain linker)) and the codon-optimized P2A sequence (ATNFSLLKQAGDVEENPGP: SEQ ID NO: 33).

SLR protein (SLR/furin-sgsg-p2a/dNGFR, SEQ ID NO: 30)
MEEENIVNGDRPRDLVFPGTAGLQLYQSLYKYSYITDGIIDAHTNEVISYAQIFETSCRLAVSLEKYGLDHNNVVAICSENNIHFFGPLIAALYQGIPMATSNDMYTEREMIGHLNISKPCLMFCSKKSLPFILKVQKHLDFLKKVIVIDSMYDINGVECVFSFVSRYTDHAFDPVKFNPKEFDPLERTALIMTSSGTTGLPKGVVISHRSITIRFVHSSDPIYGTRIAPDTSILAIAPFHHAFGLFTALAYFPVGLKIVMVKKFEGEFFLKTIQNYKIASIVVPPPIMVYLAKSPLVDEYNLSSLTEIACGGSPLGRDIADKVAKRLKVHGILQGYGLTETCSALILSPNDRELKKGAIGTPMPYVQVKVIDINTGKALGPREKGEICFKSQMLMKGYHNNPQATRDALDKDGWLHTGDLGYYDEDRFIYVVDRLKELIKYKGYQVAPAELENLLLQHPNISDAGVIGIPDEFAGQLPSACVVLEPGKTMTEKEVQDYIAELVTTTKHLRGGVVFIDSIPKGPTGKLMRNELRAIFAREQAKSKLRAKRSGSGATNFSLLKQAGDVEENPGPMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKRWNS

Polynucleotide encoding SLR (SEQ ID NO: 31)
5'--3'

A 5-week-old NOG mouse (NOD/Shi-scid, IL-2RγKO.Jic mouse, purchased from In-Vivo Science) was irradiated with 1.5 Gy of γ-ray. Next, as shown in FIG. 17(A), 5×10 ⁵ Raji/SLR cells were intravenously administered to the NOG mouse and transplanted. On

day

6 and 11 after the transplantation, activated effector cells containing 5×10 ⁶ T cells and NK cells and 30 μg/150 μL of the anti-CD20 antibody or 40 μg/150 μL of the effector cells. CD20/B-BiTE was administered intravenously. For effector cells, 1.0×10 ⁶ peripheral blood mononuclear cells were cultured for about 7 to 10 days in the presence of IL-2 100 U/mL and anti-CD3 antibody (clone OKT3) 50 ng/mL. Prepared. CD20/B-BiTE was prepared by mixing 30 μg of anti-CD20 antibody and 10 μg of B-BiTE at room temperature for 1 hour and then diluting with PBS to 40 μg/150 μL. Then, the size of the tumor on the 5th, 8th and 12th days after the transplantation was measured by an image analyzer (AEQUORIA-2D/8600 bioluminescence imaging assays, manufactured by Hamamatsu Photonics). The control was measured in the same manner except that the antibody and antibody complex were not administered. These results are shown in FIG.

FIG. 17 is a diagram relating to antitumor activity in a living body. In FIG. 17, (A) shows a protocol, (B) is a photograph measured by an image analyzer, and (C) is a graph showing the size of a tumor. In FIG. 17B, a region surrounded by a white line is a region where a tumor exists. As shown in FIGS. 17(B) and (C), the tumor size was reduced in the anti-CD20 antibody or antibody complex administration group as compared with the control. In addition, the tumor in the antibody-conjugated group was significantly reduced as compared with the anti-CD20 antibody-administered group. From these results, it was found that the antibody complex of the present invention exhibits antitumor activity in vivo, and that it has higher antitumor activity than the antibody alone. Rituximab used as the anti-CD20 antibody is known as an antibody having high ADCC activity by NK cells. It is speculated that when an antibody preparation having a low ADCC activity and a weak therapeutic effect is combined with a complexed protein, the antitumor effect can be more clearly enhanced in vivo as compared with the antibody alone.

From the above, it was found that the antibody complex of the present invention exhibits antitumor activity in vivo.

Although the present invention has been described above with reference to the exemplary embodiments, the present invention is not limited to the above exemplary embodiments. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims the priority right based on Japanese Patent Application No. 2019-024976 filed on Feb. 15, 2019, and incorporates all the disclosure thereof.

<Appendix>
The whole or part of the exemplary embodiments and examples described above can be described as, but not limited to, the following supplementary notes.
(Appendix 1)
Including a first antigen-binding domain and a second antigen-binding domain,
The first antigen-binding domain is capable of binding an immunoglobulin,
The second antigen-binding domain is a complexed protein capable of binding a desired antigen.
(Appendix 2)
The complexed protein of Appendix 1, wherein the first antigen binding domain is capable of binding to an immunoglobulin constant (Fc) region.
(Appendix 3)
3. The complexed protein according to Appendix 2, wherein the first antigen-binding domain has an epitope within the region of positions 237 to 447 in the site represented by EU numbering of the Fc region.
(Appendix 4)
4. The complexed protein according to any one of appendices 1 to 3, wherein the first antigen-binding domain comprises a first heavy chain variable region and a first light chain variable region.
(Appendix 5)
The first heavy chain variable region comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3,
The first light chain variable region comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3,
CDRH1 is a polypeptide containing the following amino acid sequence (H1-A),
CDRH2 is a polypeptide containing the following amino acid sequence (H2-A),
CDRH3 is a polypeptide containing the following amino acid sequence (H3-A),
CDRL1 is a polypeptide containing the following amino acid sequence (L1-A),
CDRL2 is a polypeptide containing the amino acid sequence of (L2-A) below,
CDRL3 is a polypeptide containing the amino acid sequence of (L3-A) below, The complex protein according to note 4:
(H1-A) The following (H1-A1), (H1-A2) or (H1-A3) amino acid sequence (H1-A1) SEQ ID NO: 1 amino acid sequence (H1-A2) SEQ ID NO: 1 amino acid sequence And an amino acid sequence (H1-A3) having 80% or more identity, in which one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 1 (H2-A3). A) The following (H2-A1), (H2-A2) or (H2-A3) amino acid sequence (H2-A1) SEQ ID NO: 2 amino acid sequence (H2-A2) SEQ ID NO: 2 amino acid sequence is 80 Amino acid sequence having% or more identity (H2-A3) Amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, inserted and/or added (H3-A) (H3-A1), (H3-A2) or (H3-A3) amino acid sequence (H3-A1) SEQ ID NO:3 amino acid sequence (H3-A2) 80% or more of the amino acid sequence of SEQ ID NO:3 Amino acid sequence having identity (H3-A3) In the amino acid sequence of SEQ ID NO: 3, one or several amino acids have been deleted, substituted, inserted and/or added (L1-A) the following (L1- A1), (L1-A2) or (L1-A3) amino acid sequence (L1-A1) SEQ ID NO: 4 amino acid sequence (L1-A2) SEQ ID NO: 4 amino acid sequence with 80% or more identity Amino acid sequence (L1-A3) which has the amino acid sequence of SEQ ID NO: 4 in which one or several amino acids have been deleted, substituted, inserted and/or added (L2-A) the following (L2-A1), (L2-A2) or (L2-A3) amino acid sequence (L2-A1) SEQ ID NO: 5 amino acid sequence (L2-A2) SEQ ID NO: 5 amino acid sequence having at least 80% identity to the amino acid sequence (L2-A3) The amino acid sequence of SEQ ID NO: 5 in which one or several amino acids are deleted, substituted, inserted and/or added (L3-A) The following (L3-A1), (L3- A2) or (L3-A3) amino acid sequence (L3-A1) SEQ ID NO: 6 amino acid sequence (L3-A2) SEQ ID NO: 6 amino acid sequence having an identity of 80% or more (L3- A3) An amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 6.
(Appendix 6)
The complexed protein according to any one of appendices 1 to 5, wherein the first antigen-binding domain comprises a single chain antibody (scFv).
(Appendix 7)
7. The complexed protein according to any one of appendices 1 to 6, wherein the desired antigen is an antigen expressed on T cells.
(Appendix 8)
7. The complexed protein according to appendix 7, wherein the antigen expressed on the T cell is CD3.
(Appendix 9)
9. The complexed protein according to any one of appendices 1 to 8, wherein the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region.
(Appendix 10)
The second heavy chain variable region comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3,
The second light chain variable region comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3,
CDRH1 is a polypeptide containing the following amino acid sequence (H1-B),
CDRH2 is a polypeptide containing the following amino acid sequence (H2-B),
CDRH3 is a polypeptide containing the following amino acid sequence (H3-B),
CDRL1 is a polypeptide containing the following amino acid sequence (L1-B),
CDRL2 is a polypeptide containing the amino acid sequence of (L2-B) below,
CDRL3 is a polypeptide containing the following amino acid sequence (L3-B), wherein the complexed protein according to note 9:
(H1-B) The following (H1-B1), (H1-B2) or (H1-B3) amino acid sequence (H1-B1) SEQ ID NO: 7 amino acid sequence (H1-B2) SEQ ID NO: 7 amino acid sequence And an amino acid sequence (H1-B3) having 80% or more identity, in which one or several amino acids have been deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 7 (H2-B3). B) The following (H2-B1), (H2-B2) or (H2-B3) amino acid sequence (H2-B1) SEQ ID NO:8 amino acid sequence (H2-B2) SEQ ID NO:8 relative to the amino acid sequence 80 Amino acid sequence having% or more identity (H2-B3) Amino acid sequence of SEQ ID NO: 8 in which one or several amino acids are deleted, substituted, inserted and/or added (H3-B) (H3-B1), (H3-B2) or (H3-B3) amino acid sequence (H3-B1) SEQ ID NO: 9 amino acid sequence (H3-B2) 80% or more of the amino acid sequence of SEQ ID NO: 9 Amino acid sequence having identity (H3-B3) In the amino acid sequence of SEQ ID NO: 9, one or several amino acids have been deleted, substituted, inserted and/or added (L1-B) below (L1- B1), (L1-B2) or (L1-B3) amino acid sequence (L1-B1) SEQ ID NO: 10 amino acid sequence (L1-B2) SEQ ID NO: 10 amino acid sequence with 80% or more identity Amino acid sequence (L1-B3) having, in the amino acid sequence of SEQ ID NO: 10, one or several amino acids have been deleted, substituted, inserted and/or added (L2-B) the following (L2-B1), (L2-B2) or (L2-B3) amino acid sequence (L2-B1) SEQ ID NO: 11 amino acid sequence (L2-B2) SEQ ID NO: 11 amino acid sequence having an amino acid sequence having 80% or more identity (L2-B3) Amino acid sequence of SEQ ID NO: 11 in which one or several amino acids are deleted, substituted, inserted and/or added (L3-B) The following (L3-B1), (L3- B2) or (L3-B3) amino acid sequence (L3-B1) SEQ ID NO: 12 amino acid sequence (L3-B2) SEQ ID NO: 12 amino acid sequence having an identity of 80% or more (L3- B3) An amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 12.
(Appendix 11)
11. The complexed protein according to any one of appendices 1 to 10, wherein the second antigen binding domain comprises a single chain antibody (scFv).
(Appendix 12)
12. The complexed protein according to any one of appendices 1 to 11, comprising a photoreactive crosslinkable portion capable of crosslinking with the immunoglobulin.
(Appendix 13)
13. The complexed protein according to appendix 12, wherein the amino acid forming the first antigen-binding domain has a photoactive reactive functional group as the photoreactive cross-linking portion.
(Appendix 14)
An antibody or an antigen-binding fragment thereof capable of binding to a target antigen, and a complexed protein,
The complexed protein is the complexed protein according to any one of appendices 1 to 13,
The antibody complex, wherein the complexed protein binds to the antibody or an antigen-binding fragment thereof through a first antigen-binding domain thereof to form a complex.
(Appendix 15)
15. The antibody complex according to appendix 14, wherein the antibody or antigen-binding fragment thereof and the complexed protein are crosslinked.
(Appendix 16)
16. The antibody complex according to appendix 14 or 15, wherein the target antigen is an antigen associated with a disease.
(Appendix 17)
The antibody complex according to any one of appendices 14 to 16, wherein the target antigen is a tumor antigen.
(Appendix 18)
A pharmaceutical composition comprising the complexed protein according to any one of appendices 1 to 13.
(Appendix 19)
19. The pharmaceutical composition according to appendix 18, comprising an antibody capable of binding to a target antigen or an antigen-binding fragment thereof.
(Appendix 20)
20. The pharmaceutical composition according to appendix 19, wherein the complexed protein binds to the antibody or the antigen-binding fragment thereof by the first antigen-binding domain thereof to form a complex.
(Appendix 21)
A nucleic acid encoding the complexed protein according to any one of appendices 1 to 13.
(Appendix 22)
A method for treating a disease associated with a target antigen, which comprises administering the complexed protein according to any one of appendices 1 to 13 and an antibody capable of binding to the target antigen or an antigen-binding fragment thereof to a patient.
(Appendix 23)
23. The therapeutic method according to appendix 22, wherein the complexed protein binds to the antibody or an antigen-binding fragment thereof through a first antigen-binding domain thereof to form a complex.
(Appendix 24)
The target antigen is a tumor antigen,
24. The treatment method according to

appendix

22 or 23, wherein the disease associated with the target antigen is cancer.

As described above, according to the complexed protein of the present invention, it is possible to add the binding property for a desired antigen to the antibody or the antigen-binding fragment thereof. Therefore, the present invention can be said to be extremely useful in the field of medicine and the like.

Claims

Including a first antigen-binding domain and a second antigen-binding domain,
The first antigen-binding domain is capable of binding an immunoglobulin,
The second antigen-binding domain is a complexed protein capable of binding a desired antigen.
2. The complexed protein of claim 1, wherein the first antigen binding domain is capable of binding an immunoglobulin constant (Fc) region.
The complexed protein according to claim 2, wherein the first antigen-binding domain has an epitope within a region of positions 237 to 447 in the site represented by EU numbering of the Fc region.
4. The complexed protein of any one of claims 1-3, wherein the first antigen binding domain comprises a first heavy chain variable region and a first light chain variable region.
The first heavy chain variable region comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3,
The first light chain variable region comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3,
CDRH1 is a polypeptide containing the following amino acid sequence (H1-A),
CDRH2 is a polypeptide containing the following amino acid sequence (H2-A),
CDRH3 is a polypeptide containing the following amino acid sequence (H3-A),
CDRL1 is a polypeptide containing the following amino acid sequence (L1-A),
CDRL2 is a polypeptide containing the amino acid sequence of (L2-A) below,
The complexed protein according to claim 4, wherein CDRL3 is a polypeptide containing the amino acid sequence of (L3-A) below:
(H1-A) The following (H1-A1), (H1-A2) or (H1-A3) amino acid sequence (H1-A1) SEQ ID NO: 1 amino acid sequence (H1-A2) SEQ ID NO: 1 amino acid sequence And an amino acid sequence having 80% or more identity (H1-A3), the amino acid sequence of SEQ ID NO: 1 with one or several amino acids deleted, substituted, inserted and/or added;
(H2-A) The following (H2-A1), (H2-A2) or (H2-A3) amino acid sequence (H2-A1) SEQ ID NO: 2 amino acid sequence (H2-A2) SEQ ID NO: 2 amino acid sequence And an amino acid sequence having an amino acid sequence of 80% or more (H2-A3) SEQ ID NO: 2 in which one or several amino acids are deleted, substituted, inserted and/or added;
(H3-A) The following (H3-A1), (H3-A2) or (H3-A3) amino acid sequence (H3-A1) SEQ ID NO:3 amino acid sequence (H3-A2) SEQ ID NO:3 amino acid sequence And an amino acid sequence (H3-A3) having an identity of 80% or more (H3-A3), wherein one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 3.
(L1-A) The following (L1-A1), (L1-A2) or (L1-A3) amino acid sequence (L1-A1) SEQ ID NO: 4 amino acid sequence (L1-A2) SEQ ID NO: 4 relative to the amino acid sequence And an amino acid sequence (L1-A3) having an identity of 80% or more (L1-A3), in which one or several amino acids are deleted, substituted, inserted and/or added;
(L2-A) The following (L2-A1), (L2-A2) or (L2-A3) amino acid sequence (L2-A1) SEQ ID NO:5 amino acid sequence (L2-A2) SEQ ID NO:5 amino acid sequence And an amino acid sequence (L2-A3) having an identity of 80% or more (L2-A3), wherein one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 5.
(L3-A) The following (L3-A1), (L3-A2) or (L3-A3) amino acid sequence (L3-A1) SEQ ID NO: 6 amino acid sequence (L3-A2) SEQ ID NO: 6 amino acid sequence An amino acid sequence having an identity of 80% or more (L3-A3), wherein the amino acid sequence of SEQ ID NO: 6 has one or several amino acids deleted, substituted, inserted and/or added.
6. The complexed protein of any one of claims 1-5, wherein the first antigen binding domain comprises a single chain antibody (scFv).
7. The complexed protein according to any one of claims 1 to 6, wherein the desired antigen is an antigen expressed on T cells.
The complexed protein according to claim 7, wherein the antigen expressed on the T cell is CD3.
9. The complexed protein of any one of claims 1-8, wherein the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region.
The second heavy chain variable region comprises heavy chain complementarity determining region (CDRH) 1, CDRH2, and CDRH3,
The second light chain variable region comprises light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3,
CDRH1 is a polypeptide containing the following amino acid sequence (H1-B),
CDRH2 is a polypeptide containing the following amino acid sequence (H2-B),
CDRH3 is a polypeptide containing the following amino acid sequence (H3-B),
CDRL1 is a polypeptide containing the following amino acid sequence (L1-B),
CDRL2 is a polypeptide containing the amino acid sequence of (L2-B) below,
The complexed protein according to claim 9, wherein CDRL3 is a polypeptide containing the amino acid sequence of (L3-B) below:
(H1-B) The following (H1-B1), (H1-B2) or (H1-B3) amino acid sequence (H1-B1) SEQ ID NO: 7 amino acid sequence (H1-B2) SEQ ID NO: 7 amino acid sequence And an amino acid sequence (H1-B3) having 80% or more identity, in which one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO:7;
(H2-B) The following (H2-B1), (H2-B2) or (H2-B3) amino acid sequence (H2-B1) SEQ ID NO:8 amino acid sequence (H2-B2) SEQ ID NO:8 amino acid sequence And an amino acid sequence having 80% or more identity (H2-B3) SEQ ID NO: 8 with one or several amino acids deleted, substituted, inserted and/or added;
(H3-B) The following (H3-B1), (H3-B2) or (H3-B3) amino acid sequence (H3-B1) SEQ ID NO: 9 amino acid sequence (H3-B2) SEQ ID NO: 9 relative to the amino acid sequence An amino acid sequence (H3-B3) having an identity of 80% or more (H3-B3), wherein one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 9;
(L1-B) The following (L1-B1), (L1-B2) or (L1-B3) amino acid sequence (L1-B1) SEQ ID NO: 10 amino acid sequence (L1-B2) SEQ ID NO: 10 amino acid sequence And an amino acid sequence (L1-B3) having an identity of 80% or more (L1-B3), wherein one or several amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of SEQ ID NO: 10.
(L2-B) The following (L2-B1), (L2-B2) or (L2-B3) amino acid sequence (L2-B1) SEQ ID NO: 11 amino acid sequence (L2-B2) SEQ ID NO: 11 relative to the amino acid sequence And an amino acid sequence (L2-B3) having an identity of 80% or more (L2-B3), in which one or several amino acids are deleted, substituted, inserted and/or added;
(L3-B) The following (L3-B1), (L3-B2) or (L3-B3) amino acid sequence (L3-B1) SEQ ID NO: 12 amino acid sequence (L3-B2) SEQ ID NO: 12 amino acid sequence And an amino acid sequence (L3-B3) having an identity of 80% or more (L3-B3), in which one or several amino acids are deleted, substituted, inserted and/or added.
11. The complexed protein of any one of claims 1-10, wherein the second antigen binding domain comprises a single chain antibody (scFv).
The complexed protein according to any one of claims 1 to 11, which comprises a photoreactive cross-linking portion capable of cross-linking with the immunoglobulin.
13. The complexed protein according to claim 12, wherein the amino acid forming the first antigen-binding domain has a photoactive reactive functional group as the photoreactive cross-linking portion.
An antibody or an antigen-binding fragment thereof capable of binding to a target antigen, and a complexed protein,
The complexed protein is the complexed protein according to any one of claims 1 to 13,
The antibody complex, wherein the complexed protein binds to the antibody or an antigen-binding fragment thereof through a first antigen-binding domain thereof to form a complex.
The antibody complex according to claim 14, wherein the antibody or the antigen-binding fragment thereof and the complexed protein are cross-linked.
The antibody complex according to claim 14 or 15, wherein the target antigen is an antigen associated with a disease.
The antibody complex according to any one of claims 14 to 16, wherein the target antigen is a tumor antigen.
A pharmaceutical composition comprising the complexed protein according to any one of claims 1 to 13.
The pharmaceutical composition according to claim 18, comprising an antibody or an antigen-binding fragment thereof capable of binding to a target antigen.
20. The pharmaceutical composition according to claim 19, wherein the complexed protein binds to the antibody or the antigen-binding fragment thereof by the first antigen-binding domain thereof to form a complex.
A nucleic acid encoding the complexed protein according to any one of claims 1 to 13.