Anti-vβ17 / Anti-CD123 bispecific antibodies

Abstract

To provide bispecific antibodies that may direct a cytotoxic T cell to an antigen-expressing cancer cell.SOLUTION: Anti-Vβ17 / anti-CD123 bispecific antibodies or antigen-binding fragments thereof are described. Also described are nucleic acids encoding the antibodies, compositions comprising the antibodies, methods of producing the antibodies, and methods of using the antibodies.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 816,464, filed March 11, 2019. No. 6,313,935, filed on Oct. 23, 2003, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to a monoclonal anti-Vβ17 / anti-CD123 bispecific antibody, Nucleic acids and expression vectors for encoding the antibodies, recombinant cells containing the vectors, and The present invention relates to compositions containing the antibodies, methods for producing the antibodies, and methods for using the antibodies to kill cancer cells. The law is also provided.

[0003] (Reference to electronically submitted sequence listing) This application is filed under the name "PRD4012WOPCT Sequence Listing g" and the creation date of March 11, 2020, as an ASCII sequence listing, It contains a sequence listing having a size of 85 kb, which has been submitted electronically via eb. The sequence listing submitted via FS-Web is incorporated herein by reference in its entirety. is incorporated herein. [Background technology]

[0004] Use of cytotoxic T cells (e.g., CD8+ T cells) to directly kill cancer cells Finding a way to target cytotoxic T cells to cancer cells could improve the killing of such cells. Cytotoxic T cells can result in the inhibition of tumor growth and the destruction of tumor cells. A bispecific antibody that binds to both the tumor and cancer cells is used to target the cytotoxic T cells to the cancer cells. By prolonged exposure, it can be activated against cancer cells expressing cancer-associated antigens. It has been demonstrated that the T cell and cancer cell antigens that mediate T cell activation can be selected. selecting a parent antibody with sufficient affinity to mediate binding in the context of the bispecific antibody; and to specifically act against cancer cells rather than inducing non-specific T cell activation. This approach to killing cancer cells involves selecting cancer cell antigens that activate T cells, such as There are a variety of potential complications for this approach. These complications are It consists solely of connections that attempt to activate T cells to destroy cancer cells in the host. Summary of the Invention [Means for solving the problem]

[0005] As used herein, Vβ17, an antigen associated with T cells, and Vβ18, an antigen associated with cancer cells, A bispecific antibody capable of binding to CD123 is provided. Cytotoxic T cells are capable of binding to Vβ17 and other They express T cell receptors consisting of α and β chains, which are involved in cancer-related Antigen-binding bispecific antibodies can target cytotoxic T cells to antigen-expressing cancer cells It is hypothesized that this type of bispecific antibody can be used to target cytotoxic T cells to antigens. By recruiting or redirecting T cells to current cancer cells, they are able to kill the cancer cells. do.

[0006] In one general aspect, the invention provides an isolated bispecific antibody that binds Vβ17 and CD123. The present invention relates to a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of the antibody or an antigen-binding fragment thereof.

[0007] Provided herein is an isolated Vβ17 bispecific antibody or antigen-binding fragment thereof. The isolated Vβ17 bispecific antibody or antigen-binding fragment thereof comprises: a. a first heavy chain (HC1); b. a second heavy chain (HC2); and c. the first light chain (LC1); d. a second light chain (LC2); HC1 associates with LC1, HC2 associates with LC2, HC1 is SEQ ID NO:1, SEQ ID NO: Heavy chain complementarity determining region 1 (HCDR) having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively 1), HCDR2, and HCDR3; LC1 comprises SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: Light chain complementarity determining region 1 (LCDR1), LCDR 2, and LCDR3, forming a binding site for a first antigen, and forms a binding site for a second antigen. In certain embodiments, the first antigen The binding site for binds to Vβ17 on the surface of CD8+ or CD4+ T cells. In certain embodiments, the binding site for the second antigen is a tumor antigen present on the surface of a cancer cell. Binds to antigens.

[0008] In certain embodiments, the targeting of Vβ17 present on the surface of CD8+ or CD4+ T cells The binding of the bispecific antibody and the binding of tumor antigens present on the surface of cancer cells results in the killing of the cancer cells. causes injury.

[0009] In certain embodiments, HC2 and LC2 bind to CD123.

[0010] In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is an antigen binding fragment, such as IgG4. It is an isotype.

[0011] In certain embodiments, the bispecific antibody or antigen-binding fragment thereof has a mAb concentration of less than about 0.2 pM. E-commerce50 In vitro CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells To induce.

[0012] Also provided is an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof. The anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof is a. a first heavy chain (HC1); b. a second heavy chain (HC2); and c. the first light chain (LC1); d. a second light chain (LC2); HC1 associates with LC1, HC2 associates with LC2, HC1 is SEQ ID NO:1, SEQ ID NO: Heavy chain complementarity determining region 1 (HCDR) having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively 1), HCDR2, and HCDR3; LC1 comprises SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: Light chain complementarity determining region 1 (LCDR1), LCDR a binding site for a first antigen that specifically binds to Vβ17, comprising LCDR3 and and HC2 has the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, LC2 has the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. A CD411 comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3) The antibody forms a dual site for a second antigen that specifically binds to 123. In one embodiment, HC1 comprises the amino acid sequence of SEQ ID NO:13, and LC1 comprises the amino acid sequence of SEQ ID NO:14. HC2 comprises the amino acid sequence of SEQ ID NO: 15, and LC2 comprises the amino acid sequence of SEQ ID NO: In certain embodiments, Vβ17 is a CD8+ or CD4 In certain embodiments, CD123 is on the surface of a cancer cell. In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is at a concentration of less than about 0.2 pM. CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells in vitro with an EC50 of Induce.

[0013] In certain embodiments, the anti-Vβ17 / anti-CD123 bispecific antibody or antigen binding thereof The fragments may be partially humanized or fully humanized chimeric.

[0014] Also provided is an isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof. The isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:28. The amino acid sequence may have at least 95% identity to the amino acid sequence of the specific In an embodiment, the isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof has the sequence Contains the amino acid sequence of sequence number 28.

[0015] The monoclonal antibodies or antigen-binding fragments thereof and bispecific antibodies or Also provided is an isolated nucleic acid encoding the antigen-binding fragment thereof.

[0016] The monoclonal antibodies or antigen-binding fragments thereof and bispecific antibodies or Also provided is a vector comprising the isolated nucleic acid encoding the antigen-binding fragment.

[0017] Also provided is a host cell comprising a vector that includes an isolated nucleic acid disclosed herein.

[0018] Methods for targeting Vβ17-expressing CD8+ or CD4+ T cells to cancer cells are also provided. The method comprises administering Vβ17-expressing CD8+ or CD4+ T cells to an anti-Vβ1 7 / Anti-CD123 bispecific antibody or antigen-binding fragment thereof. 7-expressing CD8+ or CD4+ T cells were treated with anti-Vβ17 / anti-CD123 bispecific antibody or By contacting Vβ17-expressing CD8+ or CD4+ T cells with an antigen-binding fragment of It can be directed to cells.

[0019] Also provided is a method for inhibiting the growth or proliferation of cancer cells. The method includes contacting cancer cells with the bispecific antibodies disclosed herein. Contacting the cancer cells may, for example, inhibit the growth or proliferation of the cancer cells or inhibit the T cells of the cancer cells. It can promote vector-mediated killing.

[0020] Methods for producing the bispecific antibodies or antigen-binding fragments thereof disclosed herein are also provided. The method includes culturing a cell containing nucleic acid encoding one heavy and light chain pair of a bispecific antibody. and culturing the antibody under conditions to produce heavy and light chains or antigen-binding fragments thereof; and recovering the heavy and light chains of the antibody or antigen-binding fragment thereof from the cell or culture. After collection of the heavy and light chains of both arms of a polyspecific antibody, the heavy and light chain pairs undergo self-assembly. The self-assembling bispecific antibodies are then collected under suitable conditions to allow for the formation of a self-assembled bispecific antibody. .

[0021] Bispecific antibodies or antigen-binding compositions disclosed herein, such as buffer compositions or purified compositions. Methods for producing compositions comprising the fragments are also provided. For example, the methods include producing bispecific antibodies or combining the antigen-binding fragment with an acceptable buffer for storage and use of the bispecific antibody; This may include:

[0022] Kits comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein, and Packaging for the product is also provided. [Brief description of the drawings]

[0023] The above summary and the following detailed description of preferred embodiments of the present application should be taken in conjunction with the accompanying drawings. However, the present application is not intended to be a complete disclosure of the present invention, but is intended to be illustrative and not restrictive. It should be understood that there is no limitation to the precise embodiment. [Figure 1] FIG. 1 shows a schematic demonstrating anti-Vβ17 / anti-tumor antigen bispecific antibody binding to recruit T cells to cancer cells and induce cancer cell death. [Figure 2A] Figure 1 shows that Vβ17+CD8+ T cells are present in healthy subjects and that these cells can be expanded in vitro when cultured with M1 peptide. Figure 1 shows FACS histograms of gated peripheral blood mononuclear cells (PBMCs) for CD8+ T cells expressing Vβ17 on the cell surface (Vβ17+) from a healthy subject. [Figure 2B] 1 shows that Vβ17+CD8+ T cells are present in healthy subjects and that these cells can be expanded in vitro when cultured with M1 peptide. Shown are the HLA subtypes and presence of Vβ17+CD8+ T cells of different donors identified as day 0 and after 14 days of in vitro expansion with M1 peptide (day 14). [Diagram 3] Vβ17+CD8+ T cells have characteristics of killer cytotoxic cells. Bar graphs show expression of CD107a, CD69, Granzyme B (Gzb), and interferon-γ (IFNγ) on gated PBMCs for CD8+ T cells expressing Vβ17 on the cell surface (Vβ17+) on day 0 (without M1) and day 14 after stimulation with M1 peptide (+M1). [Figure 4]Figure 1 shows binding of VB11 [anti-Vβ17 / anti-CD123] bispecific and VB13 [Vβ17 null control bispecific] antibodies to CD8+ T cells. Data presented from CD8+ T cells isolated from PBMCs from three different donors (D203517, HPU09381, and HPU08694). The table below each graph shows the EC50 value of binding in nM. [Diagram 5] Figure 1 shows the binding of Vβ17 and CD123 bispecific (VB11), and Vβ17 null control bispecific (VB13) antibodies to AML cancer cell lines. The data presented shows the binding of the bispecific antibodies to the Kasumi3 AML cell line. The table below the graph shows the EC50 values ​​of binding in nM. [Figure 6] Figure 1 shows redirection of Vβ17+ T cells by bispecific antibodies, inducing efficient killing of AML cancer cells. Data in the left graph shows killing of Kasumi3 cancer cells at an effector to target (E:T) ratio of 0.5:1 and dose titration of bispecific antibodies. Data in the middle graph shows killing of Kasumi3 cancer cells at an E:T ratio of 1:1 and dose titration of bispecific antibodies. Data in the right graph shows killing of Kasumi3 cancer cells at an E:T ratio of 5:1 and dose titration of bispecific antibodies. The table below the graphs shows the EC50 values ​​calculated from the above graphs given in pM. [Figure 7A] Specific binding of anti-Vβ17 / anti-CD123 bispecific antibody (VB11) and Vβ17-specific bispecific antibody (VB13) to CD8+ T cells isolated from PBMCs. FACS histograms are shown from PBMCs gated on CD8+ T cells expressing Vβ17 on the cell surface (Vβ17+) from healthy subjects (left graph, Vβ17 non-depleted), and from PBMCs depleted of Vβ17+ T cells using negative selection (right graph, Vβ17 depleted). [Figure 7B]7A shows the specific binding of anti-Vβ17 / anti-CD123 bispecific antibody (VB11) and Vβ17 null bispecific antibody (VB13) to CD8+ T cells isolated from PBMC. 7B shows the specific binding of anti-Vβ17 / anti-CD123 bispecific antibody (VB11) and Vβ17 null bispecific antibody (VB13) to CD8+ T cells from FIG. 7A. The dose response of the bispecific antibodies is shown in the figure. The table below the graph shows the EC50 values ​​for binding calculated from the graph above given in nM. [Figure 8] Figure 1 shows the specific recruitment of Vβ17 T cells by Vβ17 bispecific antibodies for the killing of Kasumi3 cancer cells. The figure on the left shows the killing of Kasumi3 AML cell line when effector cells were isolated from PBMCs containing CD8α+ T cells (non-contacted CD8 T cells) expressing Vβ17 (Vβ17+) on the cell surface. The insert shows the presence of 10.1% Vβ17+ CD8 T cells in the effector cell population. The figure on the right shows the killing of Kasumi3 AML cell line when effector CD8+ T cells were isolated from PBMCs, but Vβ17+ T cells were depleted by negative selection. The insert shows the presence of a small population (0.086%) of Vβ17+ CD8+ T cells in the effector cell population. [Figure 9A] Figure 1 shows the absence of pan-activation of T cells when using Vβ17 bispecific antibodies. FACS plots of Vβ17+ and Vβ17- gated CD8+ T cells are shown. When T cells were activated with Vβ17 bispecific antibodies, there was a higher upregulation of CD69 on Vβ17+ (62.5%) compared to Vβ17-CD8+ T cells (1.80%). [Figure 9B] 1 shows the absence of pan-activation of T cells when using Vβ17 bispecific antibodies. FIG. 1 shows a bar graph of upregulation of CD69 on Vβ17+ and Vβ17- gated CD8+ T cells when activated using Vβ17 bispecific antibodies. [Figure 10]We show that Vβ17+ T cells from HLA A2-negative donors are also effector killer cells and do not require pre-stimulation of Vβ17+ cells. Efficient cytotoxicity mediated by Vβ17 bispecific antibodies of Kasumi3 cancer cells is shown from PBMCs containing Vβ17+ T cells from an HLA A2-negative donor (HPU09381). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Any discussion of documents, operations, materials, devices, articles and the like which has been included in the present specification is in accordance with the present invention. Such discussion is intended to provide a basis for the discussion of whether any or all of these things are: Admitted to constitute part of the prior art to any invention disclosed or claimed It is not something that can be achieved.

[0025] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used herein have the meanings set forth herein.

[0026] Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges, described herein are In all cases, the terms "about" should be understood to be modified. Thus, numerical values ​​typically include ±10% of the stated value. For example, A concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration of 1% to 10 The concentration range of % (w / v) includes 0.9% (w / v) to 11% (w / v). When used herein, the use of a numerical range shall include the range unless the context clearly indicates otherwise. All possible subranges, including integers and fractions of values, and all individual numbers within that range Explicitly included.

[0027] Unless otherwise noted, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the and recognize many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. is intended.

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and " It is important to note that "the" includes multiple referents unless the context makes clear otherwise. There is.

[0029] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having "," "contains," or "containing," or any of these terms Other variations of the invention are intended to include the stated integer or groups of integers, but are not limited to these. is not intended to exclude any other integer or group of integers other than , and is non-exclusive or non-limiting. It will be understood that the present invention is intended to be understood as including, for example, a composition, mixture, process, or the like that includes a series of elements. The methods, articles, or apparatus are not necessarily limited to those elements expressly set forth herein. Any composition, mixture, process, method, article, or apparatus not listed in the In addition, unless expressly stated to the contrary, "or" may be used without the words "or" or "without" as the term. " refers to an inclusive "or" and not an exclusive "or." For example, A condition A or B is true (or exists) and B is false (or does not exist). if A is false (or does not exist) and B is true (or exists), then If both A and B are true (or exist), then it is satisfied by either one of.

[0030] As used herein, the conjunctive term "and / or" between multiple listed elements means Both individual and combined options are understood to be encompassing. For example, When elements are connected by "and / or," the first option may be used without the second option. The first element is applicable. The second option is that the second element can be applied without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is within the meaning and therefore the As used in the specification, it is understood that the requirement of the term "and / or" is met. The simultaneous applicability of two or more of the following is also included within the meaning, and thus the term "and / or" It is understood that the above requirements are met.

[0031] The term "about" is used herein when referring to a size or characteristic of a component of the preferred invention. The terms "approximately," "generally," "substantially," and the like, as will be understood by those of ordinary skill in the art, are used to describe The dimensions / characteristics listed are not strict boundaries or parameters, but are functionally the same or similar. It should also be understood that the above does not exclude slight variations from the above. Such references involving numerical parameters, as used herein, are to be understood as being within the scope of the art. The mathematical and industrial principles used in the manufacture of products (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.) When using fractions such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3

[0032] The terms "identical" or percent "identity" refer to the degree to which two or more nucleic acid or polypeptide sequences (e.g. For example, anti-Vβ17 / anti-CD123 bispecific antibodies and polynucleotides encoding same. , Vβ17 polypeptide and Vβ17 polynucleotide encoding same, CD123 polypeptide and the CD123 polynucleotide encoding the same), the following sequence: Match when measured using one of the comparison algorithms or by visual inspection are the same when compared and aligned such that Two or more sequences or subsequences having the same amino acid residues or nucleotides at the same site Point to the column.

[0033] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer If necessary, subsequence coordinates are specified and the sequence algorithm program is executed. The sequence comparison algorithm is then run according to the designated program parameters. Based on the data, the percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0034] Optimal alignment of sequences for comparison can be performed using, for example, the method of Smith & Waterman. The local homology algorithm of Adv. Appl. Math. 2:482 (1981); Needleman & Wunsch, J. Mol. Biol. 48:443 (197 0) Homology Alignment Algorithm, Pearson & Lipman, Proc. Similarity search method in Nat'l Acad Sci USA 85:2444(1988) The method, computerized implementation of these algorithms (Wisconsin Gene tics Software Package,Genetics Computer GAP at Group, 575 Science Dr., Madison, WI; BESTFIT, FASTA, and TFASTA), or visual inspection (generally, Cur rent Protocols in Molecular Biology,FM Ausubel et al.,eds.,Current Protocols,a joint venture between Greene Publishing Associates, Inc. and John Wiley&Sons, Inc., (1995 Supplement) (Ausubel) Cut.

[0035] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is ltschul et al. (1990) J.Mol.Biol.215:403-4 10 and Altschul et al. (1997) Nucleic Acids R BLAST and BLAST The BLAST analysis software is provided by the National Center for Biotechnology Information. Publicly available through the National Center for Biotechnology Information. The algorithm first identifies words and alphanumerics of the same length in the database sequence. Either they match when interleaved, or they meet some positive threshold score T. By identifying short words of length W in the query sequence, The aim of this study is to identify high scoring sequence pairs (HSPs). These initial neighbors are referred to as the neighbor score threshold (Altschul et al., supra). A word hit is used to initiate searches to find longer HSPs containing it. It then acts as a seed. , and extend the word hits in both directions along each sequence.

[0036] For nucleotide sequences, the parameter M (the reward for a pair of matching residues) Core (always >0) and N (penalty score for mismatched residues, always <0) are used. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The cumulative alignment score is then reduced by an amount X from its maximum score. When the alignment is performed, the accumulation of one or more negative scoring residues results in When the cumulative score falls below zero or when the end of either sequence is reached, The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For the chido sequence, the default is word length (W) 11, expectation value 10, M=5, Use N=-4 and a comparison of both strands. For amino acid sequences, use the BLASTP program. Gram uses the default word length (W) of 3, expectation (E) of 10, and BLOSUM 62 Using the scoring matrix (Henikoff & Henikoff, Pr (see oc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0037] In addition to calculating percent sequence identity, the BLAST algorithm also Statistical analysis of similarities between columns is also performed (e.g., Karlin & Altschul, Proc. c. Nat'l. Acad. Sci. USA 90:5873-5787(1993) One measure of similarity provided by the BLAST algorithm is the minimum sum probability. The probability that a match between two nucleotide or amino acid sequences occurs by chance (P(N)). For example, the nucleic acid provides an indication of the probability of a minimum repeat occurrence in a comparison between a test nucleic acid and a reference nucleic acid. The small sum probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.0 If it is less than 01, it is considered similar to the reference sequence.

[0038] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. The objective of the present invention is to provide a method for the preparation of a polypeptide that is immunologically cross-reactive with the polypeptide to be administered. A polypeptide is typically substantially identical to a second polypeptide, e.g., two peptides Another indication that two nucleic acid sequences are substantially identical is It is when two molecules hybridize to each other under stringent conditions.

[0039] antibody The present specification provides an isolated anti-Vβ17 bispecific antibody or an antigen-binding fragment thereof, Nucleic acids and expression vectors encoding the same, recombinant cells containing the same, and The present invention further relates to compositions comprising isolated anti-Vβ17 / anti-CD123 antibodies. Polyspecific antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies and expression vectors, The present invention also relates to recombinant cells containing the vectors, as well as compositions comprising the bispecific antibodies. Methods of making the antibodies and methods of using the antibodies to treat diseases, including cancer, are also provided. The antibodies disclosed herein exhibit high affinity binding to Vβ17 and / or CD123, V High specificity for β17 and / or CD123, and either alone or in combination with other anti-cancer therapies including, but not limited to, the ability to treat or prevent cancer when administered together. The compound has one or more desirable functional properties.

[0040] As used herein, the term "antibody" is used broadly and includes immunoglobulins, or monoclonal antibodies. Antibodies, including human, humanized, composite, and chimeric antibodies, which may be monoclonal or polyclonal. Generally, antibodies include antibodies that have binding specificity for a specific antigen. The structure of an antibody is well known. Immunoglobulins are proteins or peptide chains that There are five major classes of IgA, IgD, and IgE depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are classified as IgE, IgG, and IgM. Further classified as IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. The antibody light chains of vertebrate species are classified into two groups based on the amino acid sequence of their constant domains. can be assigned to one of two distinct types: κ and λ. Thus, the antibodies of the invention can contain either a kappa or a lambda light chain constant domain. According to embodiments, the antibodies disclosed herein comprise heavy chains and / or nucleotides derived from a mouse or human antibody. In addition to heavy and light constant domains, antibodies contain a light chain variable region and a heavy or light chain constant region. Each of these domains contains an antigen-binding region consisting of a variable chain region, each of which contains three domains (i.e. The light chain variable region contains complementarity determining regions 1 to 3 (CDR1, CDR2, and CDR3). The main heavy chain variable regions are alternatively referred to as LCDR1, LCDR2, and LCDR3. The domains are alternatively designated HCDR1, HCDR2, and HCDR3.

[0041] As used herein, the term "isolated antibody" refers to an antibody that is isolated from other antibodies having different antigen specificities. It refers to an antibody that is substantially free of antibodies (e.g., an isolated antibody that specifically binds Vβ17). The antibody is a monoclonal antibody that specifically binds to CD123, substantially free of antibodies that do not bind to Vβ17. The isolated antibody is substantially free of antibodies that do not bind to CD123. , substantially free of other cellular material and / or chemicals.

[0042] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies. This refers to antibodies obtained from a population of antibodies, i.e., the individual antibodies that make up the population may exist in minute quantities. The monoclonal antibodies disclosed herein are identical except for possible naturally occurring mutations. , hybridoma method, phage display technology, single lymphocyte gene cloning technology or by recombinant DNA methods. For example, monoclonal antibodies can be From a transgenic non-human animal, e.g., a transgenic mouse or rat. The hybridoma may be produced by a human B cell containing a human heavy chain transgene and a light chain transgene. It has a genome that contains genes.

[0043] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, a Fab, Fab', F(ab')2, Fv fragment, disulfide stabilized Fv fragment (dsFv), (d sFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diaFv Diabodies (dsDiabodies), Single Chain Antibody Molecules (scFv), Single Domain Antibodies (sdab ) scFv dimers (bivalent diabodies), portions of antibodies containing one or more CDRs Multispecific antibodies formed from single domain antibodies, camelized single domain antibodies, nanobodies, domain antibodies, Bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure An antigen-binding fragment refers to an antibody fragment that binds to the same antigen as the parent antibody or parent antibody fragment. According to certain embodiments, the antigen-binding fragment may comprise a light chain variable region, a light chain constant region, In another particular embodiment, the antigen-binding fragment comprises a Fa fragment of the heavy chain. b and F(ab').

[0044] As used herein, the term "humanized antibody" refers to an antibody that retains the antigen-binding properties of the antibody. However, the sequence homology is modified to that of a human antibody so that the antigenicity of the antigen in the human body is reduced. It refers to a non-human antibody raised against it.

[0045] As used herein, the term "multispecific antibody" refers to an antibody that is an antibody that contains multiple immunoglobulin variable domains. The term "antibody" refers to an antibody comprising an immunoglobulin variable domain sequence, the first of the plurality being a first immunoglobulin variable domain sequence. The antibody has binding specificity for one epitope and is a second one of a plurality of immunoglobulin variable domains. In one embodiment, the first and second epitopes have binding specificity for a second epitope. The first and second epitopes do not overlap or do not substantially overlap. The first and second epitopes may be located on different antigens, e.g., different proteins (or different multimers). In one embodiment, the multispecific antibody comprises a third, In one embodiment, the multispecific antibody comprises a first, second, third, fourth, or fifth immunoglobulin variable domain. is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0046] As used herein, the term "bispecific antibody" refers to an antibody that binds two or fewer epitopes or A bispecific antibody refers to a multispecific antibody that binds to the following antigens: A first immunoglobulin having binding specificity for an epitope on the Vβ17 antigen, for example. A heavy and light chain pair and a second epitope (e.g., an epitope on the CD123 antigen) The antibody is characterized by a second immunoglobulin heavy and light chain pair having binding specificity for In one embodiment, the first and second epitopes are located on different antigens, e.g., different proteins (or subunits of different multimeric proteins. In one embodiment, a bispecific antibody The invention relates to a method for producing a medicament comprising the steps of: a heavy chain variable domain sequence having binding specificity for a main sequence and a second epitope; and a light chain variable domain sequence. In one embodiment, the bispecific antibody comprises a first epitope A half antibody or fragment thereof having binding specificity for a second epitope and a second antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a first half antibody or fragment thereof having the same molecular weight as the first half antibody or fragment thereof. and a scFv or fragment thereof having binding specificity for a first epitope and a scFv or fragment thereof having binding specificity for a second epitope. In one embodiment, the first enzyme comprises an scFv or fragment thereof having binding specificity for The first epitope is located on Vβ17 and the second epitope is located on CD123. In an embodiment, the first epitope is located on Vβ17 and the second epitope is located on PD-1. , PD-L1, CTLA-4, EGFR, HER-2, CD19, CD20, CD3, and and / or other tumor-associated immunosuppressive factors or surface antigens.

[0047] As used herein, the term "half antibody" refers to one antibody associated with one immunoglobulin light chain. An exemplary half antibody is depicted in SEQ ID NO: 28. If so, the half antibody can include a fragment thereof, for example a single variable domain derived from Camelidae. It will be readily understood that the antigen-binding domain may also consist of a main

[0048] As used herein, the term "Vβ17" refers to a gene that mediates an immune response on cytotoxic T cells. Vβ17-expressing CD8+ T cells are typically expressed in a subject. It is produced in response to influenza A virus exposure in Vβ17-expressing CD8α+ T cells. The cells provide a greater recall in response to influenza exposure in subjects. β17" is a polypeptide that is naturally expressed by cells (including T cells) or that encodes a polypeptide. Any V that can be expressed in a cell transfected with a gene or cDNA encoding the V Includes Vβ17 variants, isoforms, and interspecies homologs. Unless otherwise specified, Vβ1 Preferably, Vβ7 is human Vβ17. The human Vβ17 amino acid sequence is k Accession number AAB49730.1.

[0049] The term "CD123" refers to an interleukin-123 (IC) receptor, a soluble cytokine important in the immune system. CD123 refers to a molecule found on cells that helps transmit the signal of kinin-3. The receptor is a type I cytokine receptor, which may also be referred to as the "interleukin-3 receptor." family and are paired with a common beta subunit (βc or CD131). The CD123 receptor is a heterodimer with the alpha chain of It can be used to induce tyrosine phosphorylation in cells and promote proliferation and differentiation in hematopoietic cell lines. CD123 can also promote the progression of acute myeloid leukemia. The term "CD123" can also be expressed in T-cell (AML, AML) subtypes. The genes or polypeptides that are naturally expressed by the host (including cells) or that encode those polypeptides or any CD123 variant that can be expressed on cells transfected with the cDNA. Unless otherwise specified, "CD123" refers to the human The amino acid sequence of human CD123 is preferably human CD123. This data is provided by accession number AY789109.1.

[0050] As used herein, an antibody that "specifically binds to Vβ17" is an antibody that is more than 1×10 -7 More than M Lower, preferably 1 x 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 Vβ17, with a KD of M or less Preferably, it refers to an antibody that binds to human Vβ17. The term "KD" refers to the ratio of Kd to Ka (i.e. The dissociation constant of an antibody is calculated from the ratio of its molecular weight (Kd) to its molecular weight (Ka) and expressed as a molar concentration (M). KD values ​​can be determined using methods known in the art in light of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, e.g., Biac or by using a biosensor system from the Ore® System; Using biolayer interferometry techniques such as the Octet RED96 system, It can be found by using

[0051] As used herein, an antibody that "specifically binds to CD123" refers to an antibody that specifically binds to CD123, Preferably, 1 × 10 for human CD123 -7 M or less, preferably 1×10 -8 M or less , more preferably 5 × 10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or is 1×10 -10 The term "KD" refers to an antibody that binds with a KD equal to or less than K. That is, it refers to the dissociation constant, which is obtained from the ratio of Kd / Ka and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art in light of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, e.g., By using the biosensor system of the iacore (registered trademark) system, or biolayer interferometry techniques such as the Octet RED96 system. It can be found by using techniques.

[0052] The smaller the KD value of an antibody, the higher the affinity with which the antibody binds to a target antigen.

[0053] In a particular aspect, the present invention provides an isolated Vβ17 bispecific antibody or an antigen-binding With respect to the fragment, (a) a first heavy chain (HC1), (b) a second heavy chain (HC2), and (c) a second heavy chain (HC3). (d) a second light chain (LC2), and (e) a first light chain (LC3), wherein HC3 associates with LC3. HC1 can be associated with LC2. HC1 can be associated with SEQ ID NO: 1, SEQ ID NO: heavy chain complementarity determining region 1 (HCD 2) containing the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively; R1), HCDR2, and HCDR3, wherein LC1 is SEQ ID NO: 4, SEQ ID NO: and light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, respectively. ), LCDR2, and LCDR3.

[0054] HC1 and LC1 form a binding site for the first antigen, and HC2 and LC2 form a binding site for the second antigen. In one example, the binding site for the first antigen is C It can bind to Vβ17 on CD8+ or CD4+ T cells and can bind to a second antigen. The binding site can, for example, bind to a tumor antigen present on the surface of a cancer cell. Binding of Vβ17 bispecific antibodies to Vβ17 present on the surface of CD8+ or CD4+ T cells and binding of tumor antigens present on the surface of cancer cells can result in, for example, killing of the cancer cells. do.

[0055] The present invention relates to a method for treating a cancer cell comprising administering to a patient an anti-Vβ17 antibody or an antigen-binding fragment thereof and an anti-CD123 antibody or an antigen-binding fragment thereof. Also provided herein are anti-Vβ17 / anti-CD123 bispecific antibodies or antigen-binding fragments thereof. In certain embodiments, the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen The binding fragment comprises: (a) a first heavy chain (HC1); (b) a second heavy chain (HC2); and (c) a third heavy chain (HC3). (d) a second light chain (LC2), and (e) a first light chain (LC3), wherein HC3 associates with LC3. and HC2 is associated with LC2. In certain embodiments, HC1 is associated with SEQ ID NO:1 , SEQ ID NO: 2, and SEQ ID NO: 3, respectively. CDR1), HCDR2, and HCDR3, and LC1 is SEQ ID NO: 4, SEQ ID NO: 5, and light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequences of SEQ ID NO:6, respectively; In certain embodiments, HC2 comprises SEQ ID NO: 34, Heavy chain complementarity determining region 1 (HCR1) containing the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively HCDR1), HCDR2, and HCDR3, LC2 is SEQ ID NO: 37, SEQ ID NO: 38, and light chain complementarity determining region 1 (LCDR) comprising the amino acid sequences of SEQ ID NO: 39, respectively. 1), LCDR2, and LCDR3.

[0056] In certain embodiments, HC1 comprises the amino acid sequence of, for example, SEQ ID NO: 13. For example, LC1 may include the amino acid sequence of SEQ ID NO: 14 and may specifically bind to Vβ17. HC2 can form a binding site for the first antigen to which it binds. 15, and LC2 can comprise, for example, the amino acid sequence of SEQ ID NO: 16. and forming a binding site for a second antigen that specifically binds to CD123. This can be done.

[0057] In certain embodiments, Vβ17 is on the surface of a CD8+ or CD4+ T cell. In certain embodiments, CD123 is on the surface of a cancer cell (e.g., a leukemia cell). .

[0058] In some embodiments, the bispecific antibodies disclosed herein comprise As such, diabodies, crosslinks, or bispecific antibodies obtained through controlled Fab arm exchange can be produced. This may take the form of a specific antibody.

[0059] In some embodiments, the bispecific antibody contains complementary CH3 domains that promote heterodimerization. IgG-like molecules with a small main chain, recombinant IgG-like dual targeting molecules (each of the two sides of the molecule has a small containing Fab fragments or portions of Fab fragments of at least two different antibodies), IgG fusions molecule (full-length IgG antibody fused to an extra Fab fragment or part of a Fab fragment), F c Fusion molecule (single chain Fv molecule or stabilized bidiabody) containing heavy chain constant domain, F c region or part thereof), Fab fusion molecules (different Fab fragments fused together ), ScFv and diabody-based antibodies and heavy chain antibodies (e.g., domain Antibodies, Nanobodies) (different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) can be fused to each other or to another protein or is fused to a carrier molecule).

[0060] In some embodiments, the IgG-like molecule having a complementary CH3 domain molecule includes a Trio mab / Quadroma(Trion Pharma / Fresenius Biot ech), Knobs-into-Holes (Genentech), CrossMA bs (Roche) and electrostatically modified (Amgen), LUZ-Y (Gene ntech), Strand Exchange Engineered Domain body(SEEDbody)(EMD Serono), Biclonic(Mer) us), and DuoBody (Genmab A / S).

[0061] In some embodiments, the recombinant IgG-like dual targeting molecule comprises a Dual Targeting g(DT)-Ig(GSK / Domantis), Two-in-one Antibo dy (Genentech), Cross-linked Mabs (Karmanos) Cancer Center), mAb2 (F-Star), and CovX-body (CovX / Pfizer) are examples.

[0062] In some embodiments, the IgG fusion molecule comprises a Dual Variable Domain. n(DVD)-Ig(Abbott), IgG-like Bispecific(In nClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec) , as well as TvAb (Roche).

[0063] In some embodiments, Fc fusion molecules include ScFv / Fc Fusions (Acad emic Institution), SCORPION (Emergent BioS olutions / Trubion,Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine-Medium countries) may be mentioned.

[0064] In some embodiments, the Fab fusion bispecific antibody includes F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalen t Bispecific (Biotecnol), and Fab-Fv (UCB-Cel ScFv antibodies, diabody-based antibodies, and domain antibodies. The body contains a bispecific T cell engager (BiTE) (M icromet), Tandem Diabody, Tandab (Affi med), Dual Affinity Retargeting Technology (DART) (MacroGenics), single chain diabody (Academic), TCR-like antibody (AIT, ReceptorLogics), human serum albumin ScFv fusion (Me rrimack), and COMBODY (Epigen Biotech), dual-targeting nanoparticles Nobodies (Ablynx), including but not limited to dual targeting heavy chain only domain antibodies Not done.

[0065] The full-length bispecific antibodies disclosed herein can be used, for example, in a cell-free environment in vitro. The synthesis of two antibody half molecules with different specificities, either in a single cell or using co-expression Introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation By using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies The Fab arm exchange reaction can be generated by disulfide bond isomerization and CH3 doping. The main dissociation-association events are the result of heavy chain disulfide bonds in the hinge region of monospecific parent antibodies. The resulting free cysteine ​​in one of the monospecific parent antibodies is Forming intra-heavy chain disulfide bonds with cysteine ​​residues of the second monospecific parent antibody molecule, and at the same time The CH3 domain of the parent antibody then opens and reforms by dissociation-association. The CH3 domain may be engineered to favor heterodimerization over homodimerization The resulting products each have a different epitope, i.e., an epitope on Vβ17 and Bispecific with two Fab arms or half molecules that bind to an epitope on a tumor antigen It is an antibody.

[0066] As used herein, "homodimerization" refers to formation of a homodimer having identical CH3 amino acid sequences. As used herein, "homodimer" refers to the interaction of two heavy chains in a homodimer. It refers to an antibody having two heavy chains with the same CH3 amino acid sequence.

[0067] As used herein, "heterodimerization" refers to formation of heterodimers having non-identical CH3 amino acid sequences. As used herein, a "heterodimer" refers to the interaction of two heavy chains having " refers to an antibody having two heavy chains that have non-identical CH3 amino acid sequences.

[0068] "Knob-in-hole" strategies (e.g., PCT International Publication No. 2006 / 02893 6) can be used to generate full length bispecific antibodies. Selected amino acids that form the interface of the CH3 domain in human IgG are To promote somatic formation, The amino acid with a small side chain (hole) is the site of the antibody that specifically binds to the first antigen. Amino acids with large side chains (knobs) are introduced into the heavy chain that specifically bind to a second antigen After co-expression of the two antibodies, the heterodimer is transformed into a "hole" It is formed as a result of preferential interaction between a heavy chain having a "knob" and a heavy chain having a "knob". Exemplary CH3 substitution pairs forming a bridge and a hole are T366Y / F405A, T366 W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y4 07A, T366W / T394S, F405W / T394S, and T366W / T366 S_L368A_Y407V (altered position in the first CH3 domain of the first heavy chain) position / modification position in the second CH3 domain of the second heavy chain).

[0069] Substituting a positively charged residue on one CH3 surface and a negatively charged residue on the second CH3 surface Other strategies, such as promoting heavy chain heterodimer formation using electrostatic interactions by incorporating , U.S. Patent Publication No. US2010 / 0015133, U.S. Patent Publication No. US2009 / 01 No. 82127, U.S. Patent Publication No. US2010 / 028637, or U.S. Patent Publication No. US In another strategy, Heterodimerization may be achieved by any of the methods described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. As described in US2013 / 0195849, L351Y_F405AY40 7V / T394W, T366I_K392M_T394W / F405A_Y407V, T 366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y40 7A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W substitutions (first CH3 of the first heavy chain As modified position in domain / modified position in the second CH3 domain of the second heavy chain This can be facilitated by

[0070] In addition to the above methods, the bispecific antibodies disclosed herein can be used in combination with other methods described in International Patent Publication No. W02 The two monospecific homodimeric antibodies were synthesized according to the method described in US Pat. No. 5,311,317,6. Asymmetric mutations were introduced into the CH3 region, and the disulfide bonds were isomerized under reducing conditions. By forming a bispecific heterodimeric antibody from two monospecific homodimeric parent antibodies, In this method, the first One monospecific bivalent antibody (e.g., an anti-CD33 antibody) and a second monospecific bivalent antibody (e.g., For example, anti-CD3 antibodies contain specific substitutions in the CH3 domain that promote heterodimer stability. These antibodies are engineered to have a disulfide bond between the cysteines in the hinge region. Incubate together under reducing conditions sufficient to allow the isomerization of the The antibodies are then transferred to the Fab arm of the target antibody, thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions may optionally be returned to non-reducing conditions. Exemplary reducing agents that may be used are , 2-mercaptoethylamine (2-MEA), dithiothreate dithiothreitol (DTT), dithioerythritol (DTE) , glutathione, tris(2-carboxyethyl)phosphine osphine, TCEP), L-cysteine, and β-mercaptoethanol, among which preferred For example, 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxylate) For example, the reducing agent is selected from the group consisting of ethyl phosphine. At a temperature of In the presence of dithiothreitol, at pH 5 to 8, for example, pH 7.0 or pH 7.4 For this purpose, an incubation period of at least 90 minutes may be used.

[0071] In certain embodiments, the anti-Vβ17 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region Region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, which are a. having the polypeptide sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; The anti-CD123 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), H CDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCD R3, which are 1. The polypeptide sequences of SEQ ID NOs: 34, 35, 36, 37, 38, and 39, respectively. has.

[0072] According to another particular aspect, the present invention relates to a method for the treatment of antibody-dependent cell-mediated cytotoxicity. The isolated anti-Vβ17 / anti-Vβ17 antibody induces dent cell-mediated cytotoxicity (ADCC). The present invention relates to a CD123 bispecific antibody or an antigen-binding fragment thereof. The binding fragment can, for example, induce ADCC in vitro. The antigen-binding fragment has an EC value of less than about 1 pM. 50 ADCC can be induced by In certain embodiments, EC 50 is less than about 1 pM, less than about 0.9 pM, less than about 0.8 pM less than about 0.7 pM, less than about 0.6 pM, less than about 0.5 pM, less than about 0.4 pM, Less than 300 pM, less than about 0.2 pM, less than about 0.19 pM, less than about 0.18 pM, less than about 0 Less than 0.17 pM, less than about 0.16 pM, less than about 0.15 pM, less than about 0.14 pM, less than about 0 Less than .13 pM, less than about 0.12 pM, less than about 0.11 pM, less than about 0.1 pM, less than about 0. Less than about 0.09 pM, less than about 0.08 pM, less than about 0.07 pM, less than about 0.06 pM, less than about 0. less than about 0.05 pM, less than about 0.04 pM, less than about 0.03 pM, less than about 0.02 pM, or In certain embodiments, the anti-Vβ17 / anti-CD123 bispecific The antibody or antigen-binding fragment thereof comprises an IgG1, IgG2, IgG3, or IgG4 backbone. In one such embodiment, the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding The fragment has an antibody backbone of the IgG4 isotype.

[0073] In some embodiments described herein, the anti-Vβ17 / anti-CD123 bispecific antibody The immune effector properties of the antibody can be activated or inhibited via Fc modification using techniques known to those skilled in the art. For example, Fc effector functions, such as C1q binding, complement-dependent cytotoxicity, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (CDC); ADCC), antibody-dependent cell-mediated phagocytosis sis, ADCP), cell surface receptors (e.g., B cell receptor, BCR) Downregulation of Fc and other functions can be achieved by modifying the residues in Fc responsible for these activities. may be provided and / or controlled.

[0074] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a type of immunotherapy that uses Fc receptors (Fc receptors, Nonspecific cytotoxic cells expressing FcR (e.g., Natural Killer NK cells, neutrophils, and macrophages recognize the antibodies bound to the target cells, It subsequently refers to a cell-mediated reaction that results in the lysis of target cells.

[0075] The ability of an antibody to induce ADCC can be enhanced by engineering its oligosaccharide moiety. Human IgG1 or IgG3 is N-glycosylated at Asn297. The majority of glycans are of the well-known biantennary G0, G0F, G1, G1F, G2, or G2F Antibodies produced by non-genetically engineered CHO cells are typically in the form of The glycan fucose content is at least about 85%. Removal of core fucose from various types of oligosaccharides does not alter antigen binding or CDC activity. Enhanced ADCC of antibodies through improved FcγRIIIa binding without Such Abs are relatively highly defucosylated, with biantennary and complex type Fc oligosaccharides. Different methods have been reported to result in successful expression of mutant antibodies, e.g., changing the culture osmolarity. Control (Konno et al., Cytotechnology 64:249-65 , 2012), and application of the mutant CHO line Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002) , application of the mutant CHO line EB66 as a host cell line (Olivier et al., MAbs;2(4),2010, Electronic publication ahead of print, PMID:20562582) , Application of rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), Small molecule interferon specific for the α-1,6-fucosyltransferase (FUT8) gene Introduction of interfering RNA (Mori et al., Biotechnol Bioeng 88 :901-908,2004), or β-1,4-N-acetylglucosaminyl trans Ferrase III and Golgi α-mannosidase II or potent α-mannosidase Co-expression of kifunensine, an inhibitor of I (Ferrara et al., J Biol Chem281:5032-5036,2006, Ferrara et al.,B iotechnol Bioeng93:851-861,2006, Xhou et al., Biotechnol Bioeng 99;652-65,2008) was used. This can be achieved by:

[0076] In some embodiments described herein, the anti-Vβ17 / anti-CD123 bispecific antibody ADCC elicited by IgG1 is also activated by certain substitutions in the antibody Fc. Exemplary substitutions include, for example, those described in U.S. Pat. No. 6,737,056: Amino acids 256, 290, 298, 312, 356, 330, 333, 334, 360, Substitutions at positions 378 or 430 (residue numbering according to the EU index) be.

[0077] In another particular embodiment, the present invention relates to a method for the detection of Vβ17-expressing cells and CD123-expressing cells. Isolated anti-Vβ17 / anti-CD123 antibodies capable of inducing T cell-dependent cytotoxicity The present invention relates to a bispecific antibody or an antigen-binding fragment thereof. The bispecific antibody or an antigen-binding fragment thereof comprises: For example, an EC of less than about 2 nM 50 Values ​​in vitro in Vβ17-expressing cells / CD123 In certain embodiments, the expressing cells can induce T cell dependent cytotoxicity. 0 is less than about 2.0 nM, less than about 1.9 nM, less than about 1.8 nM, less than about 1.7 nM, Less than 1.6 nM, less than about 1.5 nM, less than about 1.4 nM, less than about 1.3 nM, less than about 1.2 nM M or less, less than about 1.1 nM, less than about 1.0 nM, less than about 0.9 nM, less than about 0.8 nM, Less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 less than about 0.1 nM, less than about 0.2 nM, and less than about 0.1 nM.

[0078] According to another particular aspect, the present invention provides an isolated anti-Vβ17 / anti-CD123 bispecific Regarding the antibody or antigen-binding fragment thereof, an anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment The original binding fragment is a chimera.

[0079] According to another particular aspect, the present invention provides an isolated anti-Vβ17 / anti-CD123 bispecific Regarding the antibody or antigen-binding fragment thereof, an anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment The original binding fragments are human or humanized.

[0080] In another general aspect, the invention provides an isolated humanized Vβ17 monoclonal antibody or The present invention relates to an isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof. The binding fragment has at least 85%, preferably 90%, more preferably 10% identical to the amino acid sequence of SEQ ID NO:28. Preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identity. In certain embodiments, the humanized Vβ17 monoclonal antibody comprises an amino acid sequence having the following structure: The antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:28.

[0081] In another general aspect, the invention provides a monoclonal antibody or a method thereof as disclosed herein. In another general aspect, the invention relates to an isolated nucleic acid encoding an antigen-binding fragment. An isolated antibody encoding the bispecific antibody or antigen-binding fragment thereof disclosed herein. It relates to nucleic acids. It is possible to modify the coding sequence of a protein without changing the amino acid sequence of the protein. It will be understood by those skilled in the art that the sequences may be altered (e.g., substituted, deleted, inserted, etc.) in any manner. It will be understood that the amino acid sequence of the protein may be modified as disclosed herein without altering the amino acid sequence. By modifying the nucleic acid sequences encoding the monoclonal and / or bispecific antibodies to be It will be understood by those skilled in the art that

[0082] In another general aspect, the invention provides a vector comprising an isolated nucleic acid disclosed herein. The present invention relates to a vector, such as a plasmid, a cosmid, a phage vector, or a viral vector. Any vector known to one of skill in the art in light of the present disclosure may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. , promoters, ribosome binding elements, terminators, enhancers, selection markers, and an origin of replication, which are optional elements for establishing the conventional functions of an expression vector. The promoter may be a constitutive, inducible, or reconfigurable promoter. Numerous expression vectors capable of delivering nucleic acids to cells are available in the art. are known and are used herein to produce antibodies or antigen-binding fragments thereof intracellularly. The present invention can be produced using conventional cloning techniques or artificial gene synthesis. Recombinant expression vectors can be produced according to the embodiments shown. will be well known to those of skill in the art in view of the present disclosure.

[0083] In another general aspect, the invention provides the monoclonal antibodies and / or or a host cell comprising an isolated nucleic acid encoding the antibody, or a bispecific antibody, or an antigen-binding fragment thereof. In view of the present disclosure, any host cell known to one of skill in the art is referred to herein as a host cell. The method can be used for recombinant expression of the antibodies or antigen-binding fragments disclosed in some embodiments. In some embodiments, the host cell is E. coli TG1 or BL21 cells (e.g., scFv or Fab For antibody expression, CHO-DG44 or CHO-K1 cells, or HEK293 cells In certain embodiments, the recombinant Expression vectors are designed to stably integrate into the host cell genome so that recombinant nucleic acids can be efficiently expressed. These include chemical transfection, heat shock, or electroporation. Transformation into host cells is by conventional methods.

[0084] In another general aspect, the invention provides a bispecific antibody or an antigen thereof as disclosed herein. The present invention relates to a method for producing a binding fragment of a bispecific antibody or a a method for producing a bispecific antibody or an antigen-binding fragment thereof, comprising: and extracting the antibody from the cell or cell culture (e.g., from the supernatant). and recovering the expressed antibody or antigen-binding fragment thereof. The cells are harvested according to conventional techniques known in the art and as described herein. It can be purified as described.

[0085] How to use In another general aspect, the invention relates to a method of targeting CD123 on the surface of a cancer cell. The method comprises treating cancer cells with an anti-Vβ1717 / anti-CD123 bispecific antibody or an antigen thereof. This involves exposing the antibody to a binding fragment.

[0086] Functions of bispecific antibodies and antigen-binding fragments thereof that bind to Vβ17 and / or CD123 Activity is characterized by methods known in the art and as described herein. Antibodies and antigen-binding fragments thereof that bind to Vβ17 and / or CD123. Methods for characterization include Biacore, ELISA, and OctetRed. Affinity and specificity assays, including FACS, of antibodies to CD123 on cancer cells Binding assays to detect binding of antibodies to Vβ17 on CD8+ or CD4+ T cells These include, but are not limited to, binding assays in which the binding is detected. According to the present invention, antibodies and antigen-binding fragments thereof that bind to Vβ17 and / or CD123 are characterized. Methods for doing so include those described below.

[0087] In another general aspect, the invention relates to a method for the treatment of cancer cells by isolating Vβ17-expressing CD8+ or CD4+ T cells. The method includes a method for directing Vβ17-expressing CD8+ or CD4+ T cells to contacting the subject with an anti-Vβ1717 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof; The antibody or antibody fragment inhibits Vβ17-expressing CD8+ or CD4+ T cells by inhibiting CD123. It is directed to cancer cells that have it on their surface.

[0088] In another general aspect, the invention relates to a method for inhibiting the growth or proliferation of cancer cells. This method involves the cloning of Vβ17-expressing CD8+ T cells with anti-Vβ1717 / anti-CD123 bispecific antibodies. contacting the cancer cells with a specific antibody or antigen-binding fragment thereof; Inducing the activation of the IL-1 receptor inhibits the growth or proliferation of cancer cells.

[0089] According to an embodiment of the invention, the described anti-Vβ1717 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof may be provided in a buffered composition for storage or use. A preferred method for preserving an anti-Vβ1771 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof A suitable buffer will minimize degradation during storage and will prevent aggregation of the antibody or antibody fragment. or minimize adhesion to storage containers, thereby preventing the antibody or antibody fragment from This helps maintain the stability of the

[0090] Embodiment The present invention provides the following non-limiting embodiments.

[0091] Embodiment 1 is an isolated Vβ17 bispecific antibody or antigen-binding fragment thereof, The resulting Vβ17 bispecific antibody or antigen-binding fragment thereof is a. a first heavy chain (HC1); b. a second heavy chain (HC2); and c. the first light chain (LC1); d. a second light chain (LC2); HC1 associates with LC1, HC2 associates with LC2, HC1 is SEQ ID NO:1, SEQ ID NO: Heavy chain complementarity determining region 1 (HCDR) having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively 1), HCDR2, and HCDR3; LC1 comprises SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: Light chain complementarity determining region 1 (LCDR1), LCDR 2, and LCDR3, forming a binding site for a first antigen, and forms a binding site for a second antigen.

[0092] Embodiment 2 is a Vβ17 bispecific antibody or an antigen-binding fragment thereof according to embodiment 1. The binding site for the first antigen is bound to Vβ17 on CD8+ or CD4+ T cells. To combine.

[0093] Embodiment 3 is a Vβ17 bispecific antibody or an antigen-binding fragment thereof according to embodiment 1 or 2. and the binding site for the second antigen is a fragment that binds to a tumor antigen present on the surface of a cancer cell. do.

[0094] Embodiment 4 is a method for producing a Vβ17 bispecific antibody or an anti-Vβ17 antibody according to any one of embodiments 1 to 3. The original binding fragments, HC1 and LC1, have been humanized.

[0095] Embodiment 5 is a method for producing a Vβ17 bispecific antibody or a Vβ17 bispecific antibody thereof according to any one of embodiments 1 to 4. and HC2 and LC2 bind to CD123.

[0096] Embodiment 6 is a Vβ17 bispecific antibody or a combination thereof according to any one of embodiments 1 to 5. and the bispecific antibody or antigen-binding fragment thereof is an IgG isotype. be.

[0097] Embodiment 7 is a method for producing a Vβ17 bispecific antibody or a Vβ17 antibody thereof according to any one of embodiments 1 to 6. and the bispecific antibody or antigen-binding fragment thereof is an IgG4 isotype. It is.

[0098] Embodiment 8 is a method for producing a Vβ17 bispecific antibody or a Vβ17 antibody according to any one of embodiments 1 to 7. and the bispecific antibody or antigen-binding fragment thereof has an E of less than about 0.2 pM. C 50 Induce CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells in vitro do.

[0099] Embodiment 9 is a method for producing a Vβ17 bispecific antibody or a Vβ17 antibody thereof according to any one of embodiments 1 to 8. and an isolated nucleic acid encoding the antigen-binding fragments HC1 and LC1 of said antibody.

[0100] Embodiment 10 is a Vβ17 bispecific antibody according to any one of embodiments 1 to 8, or and an isolated nucleic acid encoding the antigen-binding fragments HC2 and LC2.

[0101] Embodiment 11 relates to a vector comprising the isolated nucleic acid of embodiment 9 or embodiment 10. -It is.

[0102] Embodiment 12 is a host cell comprising the vector of embodiment 11.

[0103] Embodiment 13 is an isolated Vβ17 bispecific antibody according to any one of embodiments 1 to 8. The buffer composition comprises a reactive antibody or antigen-binding fragment thereof and a buffer solution.

[0104] Embodiment 14 is an isolated anti-Vβ17 / anti-CD123 bispecific antibody or its antigen binding site. An isolated anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof One side is a. a first heavy chain (HC1); b. a second heavy chain (HC2); and c. the first light chain (LC1); d. a second light chain (LC2); HC1 associates with LC1, HC2 associates with LC2, HC1 is SEQ ID NO:1, SEQ ID NO: Heavy chain complementarity determining region 1 (HCDR) having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively 1), HCDR2, and HCDR3; LC1 comprises SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: Light chain complementarity determining region 1 (LCDR1), LCDR a binding site for a first antigen that specifically binds to Vβ17, comprising LCDR3 and and HC2 has the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36. heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, LC2 has the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. A CD411 comprising a light chain complementarity determining region 1 (LCDR1), a light chain complementarity determining region 2 (LCDR2), and a light chain complementarity determining region 3 (LCDR3) The second antigen specifically binds to 123.

[0105] Embodiment 15 is an isolated anti-Vβ17 / anti-CD123 bispecific antibody according to embodiment 14. HC1 comprises the amino acid sequence of SEQ ID NO: 13; and L C1 comprises the amino acid sequence of SEQ ID NO:14, and HC2 comprises the amino acid sequence of SEQ ID NO:15. and LC2 comprises the amino acid sequence of SEQ ID NO:16.

[0106] Embodiment 16 is an isolated anti-Vβ17 / anti-CD123 antibody according to embodiment 14 or 15. A bispecific antibody or antigen-binding fragment thereof, wherein Vβ17 binds to CD8+ or CD4+ T cells. on the surface.

[0107] Embodiment 17 is an isolated anti-Vβ17 / antibody according to any one of embodiments 14 to 16. An anti-CD123 bispecific antibody or an antigen-binding fragment thereof, It's on top.

[0108] Embodiment 18 is an anti-Vβ17 / anti-CD12 antibody according to any one of embodiments 14 to 17. 3. A bispecific antibody or an antigen-binding fragment thereof, , EC<0.2 pM 50 In vitro, CD8+ or CD4+ T cell dependent Induces cell damage.

[0109] Embodiment 19 is an anti-Vβ17 / anti-CD12 antibody according to any one of embodiments 14 to 18. 3. An isolated nucleic acid encoding HC1 and LC1 of a bispecific antibody or an antigen-binding fragment thereof It is an acid.

[0110] Embodiment 20 is an anti-Vβ17 / anti-CD12 antibody according to any one of embodiments 14 to 18. 3. An isolated nucleic acid encoding HC2 and LC2 of a bispecific antibody or an antigen-binding fragment thereof It is an acid.

[0111] Embodiment 21 relates to a vector comprising the isolated nucleic acid of embodiment 19 or embodiment 20. It is Tar.

[0112] Embodiment 22 is a host cell comprising the vector of embodiment 21.

[0113] Embodiment 23 is an anti-Vβ17 / anti-CD12 antibody according to any one of embodiments 14 to 18. 3. A buffer composition comprising a bispecific antibody or an antigen-binding fragment thereof and a buffer solution.

[0114] Embodiment 24 is a method for directing Vβ17-expressing CD8+ or CD4+ T cells to cancer cells. The method further comprises administering to the subject a Vβ17-expressing CD8+ or CD4+ T cell according to any one of the embodiments 1 to 8 or 19. The anti-Vβ17 / anti-CD123 bispecific antibody according to any one of 14 to 18, or an antibody thereof contacting Vβ17-expressing CD8+ or CD4+ T cells with an anti-Vβ17 binding fragment; Contacting with the Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof Targeting CD8+ or CD4+ T cells expressing CD123 to cancer cells bearing CD123 on their surface .

[0115] Embodiment 24(a) is the method of embodiment 24, wherein the Vβ17-expressing CD8+ or The CD4+ T cells are anti-Vβ17 / Contact with an anti-CD123 bispecific antibody.

[0116] Embodiment 24(b) is the method of embodiment 24, wherein the Vβ17-expressing CD8+ or The CD4+ T cells are anti-Vβ17 / Contacted with an anti-CD123 bispecific antibody fragment.

[0117] Embodiment 25 inhibits the growth or proliferation of cancer cells expressing CD123 on their surface. The method comprises: and contacting the antibody with an anti-Vβ17 / anti-CD123 bispecific antibody or fragment thereof, Contacting cancer cells with the antibody or antibody fragment inhibits the growth or proliferation of the cancer cells.

[0118] Embodiment 25(a) is the method of embodiment 25, wherein the CD123-expressing cancer cells are: While in the presence of Vβ17-expressing CD8+ T cells, anti-Vβ17 / anti-CD123 bispecific The antibody or fragment thereof is in contact with the antibody.

[0119] Embodiment 25(b) is the method according to embodiment 25 or 25(a), further comprising administering to said patient CD123 The expressing cancer cells are the anti-Vβ17 / anti-C- D123 bispecific antibody.

[0120]

[0036] Embodiment 25(c) is the method according to embodiment 25 or 25(a), further comprising administering to said patient CD123 The expressing cancer cells are the anti-Vβ17 / anti-C- D123 bispecific antibody fragment.

[0121] Embodiment 26 is a Vβ17 bispecific antibody according to any one of embodiments 1 to 8, or A kit comprising the antigen-binding fragment and packaging therefor.

[0122] Embodiment 27 is an anti-Vβ17 / anti-CD12 antibody according to any one of embodiments 14 to 18. 3. A kit comprising a bispecific antibody or an antigen-binding fragment thereof and packaging therefor.

[0123] Embodiment 28 is a method for producing a Vβ17 bispecific antibody or an antigen-binding fragment thereof. The method comprises injecting a host cell according to embodiment 12 with a Vβ17 bispecific antibody or an antigen thereof. and culturing under conditions that produce a binding fragment thereof. and recovering the fragment from the cell or culture.

[0124] Embodiment 29 is an anti-Vβ17 / anti-CD12 antibody according to any one of embodiments 14 to 18. 3. A method for producing a bispecific antibody or an antigen-binding fragment thereof, the method comprising the steps of: 2. The host cell according to claim 2, further comprising an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof. and culturing the anti-Vβ17 / anti-CD123 bispecific antibody or its and recovering the antigen-binding fragment from the cell or culture.

[0125] Embodiment 30 is an isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof. and the Vβ17 monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:28. It includes an amino acid sequence having at least 95% identity to the sequence.

[0126] Embodiment 31 is an isolated humanized Vβ17 monoclonal antibody according to embodiment 30. or an antigen-binding fragment thereof, and the Vβ17 monoclonal antibody or the antigen-binding fragment thereof is Contains the amino acid sequence of sequence number 28.

[0127] Embodiment 32 is a humanized Vβ17 monoclonal antibody according to embodiment 30 or embodiment 31. The present invention is an isolated nucleic acid encoding a human antibody or antigen-binding fragment thereof.

[0128] Embodiment 33 is a vector comprising the isolated nucleic acid of embodiment 32.

[0129] Embodiment 34 is a host cell comprising the vector of embodiment 33.

[0130] Embodiment 35 relates to an isolated humanized Vβ17 mutant of embodiment 30 or embodiment 31. The present invention relates to a buffered composition comprising a monoclonal antibody or an antigen-binding fragment thereof. EXAMPLES

[0131] The following examples show that influenza virus-derived peptide M1 induces a select set of T cells. These cells are TCR haplotype V The majority of these cells express β17 and exhibit efficient cytotoxicity of tumor target cells. This ability is due to one arm binding to the Vβ17 structure and the other arm being targeted by cancer cells. The method is utilized using bispecific antibodies constructed to bind to antigens expressed on the target cell. Thus, bispecific antibodies can bridge effector and target cells together and mediate cancer cell killing. This mechanism of action is depicted in the schematic outlined in FIG.

[0132] The following example shows (1) the V of the T-cell receptor (TCR) on the CTL. Generation of bispecific antibodies capable of binding to the β17 arm (Examples 1 and 2), and (2) Bispecific antibody-enabled target cells by in vitro expanded CTLs Evidence of lethality (Example 3) can be divided into the categories of

[0133] Example 1: Human framework adaptation of anti-Vβ17 mAb E17.5F Mouse IgG1 anti-human T cell receptor Vβ17 clone E17.5F was used as a Beckman Purified water was obtained from Altair, Inc. (Brea, CA). Sample preparation and LC / MSMS analysis were performed. , Protea Bioscience Inc. (Morgantown, WV) The sample was reduced, alkylated, divided into seven aliquots, and purified using trypsin / LysC. , chymotrypsin, LysC, pepsin, and AspN, elastase, and proteinase The resulting peptides were digested with the enzyme ELISA using a ZipTip C18 pipette tip. The fractions were desalted using a HPLC digest and separated on-line using reversed-phase chromatography. Mass spectrometry on a Thermo Q-Exactive spectrometer using fragmentation The MS dataset was analyzed by integrating de novo sequence tags with the IMGT-based antibody sequence database. The sequences were analyzed using the PEAKS software by matching the GAPs are assigned using Contig sequence assembly of de novo identified peptides All CDRs and hypermutations were confirmed by examining the MS / MS spectra. Ta.

[0134] The sequences obtained are shown in Tables 1 and 2.

[0135] [Table 1]

[0136] [Table 2]

[0137] Changes were made in the sequence for the preparation of bispecific antibodies (Table 3). These changes included: 1) The framework mutation Asn1 in the heavy chain is not conserved, so the sequence is (2) another mutation identified in Fc, K337Y, was specifically modified to have the QLW sequence. (3) a heavy chain construct that does not have this mutation is synthesized. Potential secondary glycosylation sites above were observed, and therefore the N-linked site (Choth Two versions of this mAb were developed, with and without N82a (based on ia numbering). This includes synthesis.

[0138] [Table 3]

[0139] The two antibodies (B17B1 and B17B2) were expressed in HEK293Expi cells. The supernatant was tested for Vβ17 binding (B17B1 and B17B2), and only B17B1 bound. Therefore, expression of B17B1 with an IgG4 constant region with Fc substitution was observed. He made him do so.

[0140] The anti-human TCR Vβ17 mouse mAb B17B1 was synthesized using human framework adapted (HF A) Law (Fransson J,et al.J.Mol.Biol.2010;398 The best combination of humanized heavy and light chains was found. To identify the V-region sequences, several human V-region sequences were selected for testing (Table 4). The selection of the cell line was based on overall sequence similarity with mouse antibodies in the framework (FR) regions. Neither the CDR sequences nor their length or canonical structure were taken into consideration in this selection. Ta.

[0141] The CDR definitions used in HFA are (Fransson J, et al. J. Mol. .Biol.2010;398:214-231) and Martin's definition ( Abhinandan KR and Martin AC.Mol.Immunol. 2008;45:3832-3839). The CDRs (Table 1) are defined as follows: The Chothia numbering scheme [Chothia C, and Lesk AJ Mol. Biol. 1987;196:901-917.] HCDR1 (SEQ ID NO: 1) 26-35 HCDR2 (SEQ ID NO: 2) 50 to 58 HCDR3 (SEQ ID NO: 3) 95-102 LCDR1 (SEQ ID NO: 4) 24-34 LCDR2 (SEQ ID NO: 5) 50-56 LCDR3 (SEQ ID NO: 6) 89-97

[0142] Selected human germline sequences are provided in Table 4 (in IMGT notation).

[0143] [Table 4] CDRs 1 to 3 are underlined.

[0144] The "back mutations" in some variants overlap with the VL / VH pairing and CDR structure. The selected human germline sequences are shown in Table 5 ( The IMGT designation is provided in the present specification and the back mutations are described.

[0145] [Table 5]

[0146] The amino acid sequences of all nine pairs of three heavy and three light chains were reverse translated into DNA. and gene synthesis technology (U.S. Patent Nos. 6,670,127 and 6,521,427). cDNA was prepared using an in-house expression vector containing a CMV promoter. Using standard molecular biology techniques, the heavy chain (HC) variable region was cloned onto the human IgG4 constant region. The expression vector, which contains the CMV promoter, was developed in-house and subcloned into the standard Using advanced molecular biology techniques, the light chain (LC) variable region was cloned onto the human lambda (λ) constant region. The resulting plasmid was subcloned into HEK EXPI cells (LifeTech). The mAb was transfected into chromosomes 10 (Human Immunology, Carlsbad, CA) and expressed on 100 ng / mL IgG. The protein A column (hiTrap MAbSelect SuRe column) was used to express the After elution, the pool was purified by standard methods using PBS, pH 7. The patient was dialyzed into 2.

[0147] [Table 6]

[0148] The humanized antibody was synthesized as follows: TCRVβ17 (SEQ ID NO: 27) / Va10.2-Fc (SEQ ID NO: 44 ) fusion protein was screened by ELISA. TCR Vβ17 / Va10.2-Fc fusion protein was coated with streptavidin. Unbound proteins were washed away and the mAb was added at a concentration range The plate was washed and anti-kappa:HRP detection antibody was added. The plate was washed, chemiluminescent detection reagent was added, and luminescence was detected by Perkinesis. Plates were read on an Elmer EnVision plate reader. 20 and B17B21 showed positive binding to the TCR Vβ17 protein. B22 showed weak binding to this protein. These were then selected for further study. The antibodies were purified as described above. B17B21 is a recombinant TCR Vβ17 protein and These showed the best binding to M1- and M2-stimulated T cells and therefore warrant further functional studies, specifically As a bispecific antibody, it was selected as a molecule for redirecting T cells to kill cancer cells.

[0149] Therefore, B17B21 (anti-Vβ17) and I3RB217 (anti-CD123) antibodies Using variable region sequences, we have investigated T cell redirected killing of acute myeloid leukemia (AML) cells. The present inventors have generated bispecific antibodies which are then tested using the same antibody as described above.

[0150] Example 2. Preparation of anti-Vβ17 / anti-CD123 bispecific antibodies VB11 (anti-Vβ17 / anti-CD123) and VB13 (Vβ17×null) bispecific antibodies The antibody was prepared as human IgG4 in a knob-into-hole format, as previously described. The antibody was generated as a marker for the chromatin-binding protein 1 (Agc121.1). Nucleic acid sequences encoding the variable regions were amplified using standard PCR restriction enzyme-based PCR. Using the cloning technique described above, we developed a custom mammalian host containing the constant region of an IgG4 expression cassette. The bispecific antibodies were expressed in Chinese hamster ovary cells and subcloned into a mammalian expression vector. The antibodies were expressed in Mab Sel ect SuRe Protein A column (GE healthcare, Pisc ataway,New Jersey)(Brown,Bottomley et al The column was first purified using a phosphate buffered saline (PBS) buffer at pH 7.2 (P The column was equilibrated with PBS ( The column was washed with 1000 sucrose (4 CV) and then eluted with 30 mM sodium acetate, pH 3.5. a) Absorbance at 280 nm on Explorer (GE healthcare) The fractions containing the protein peak monitored by the method were pooled together and diluted with 1% pH 9.0 water. The pH was neutralized to 5.0 by adding 3M sodium acetate. Preliminary size determination was performed using a Perdex 200 column (GE Healthcare). The antibodies were purified by size exclusion chromatography (SEC). Sample integrity was verified by endotoxin assay and SDS polyacrylamide gel electrophoresis. The final protein concentrations were evaluated under reducing and non-reducing conditions. The results were 0.48 mg / mL for Vβ17×null and 0.24 mg / mL for Vβ17×null. Final EU of anti-Vβ17 / anti-CD123 and Vβ17×null based on these protein concentrations The concentrations were 2.053 EU / mg and 4.219 EU / mg, respectively.

[0151] [Table 7-1]

[0152] [Table 7-2]

[0153] [Table 7-3]

[0154] [Table 7-4]

[0155] [Table 8]

[0156] Example 3. Kasumi-3 cells and human CD8 + Anti-Vβ17 / anti-C using T cells Evaluation of the binding and cytotoxic properties of the D123 bispecific antibody Vβ17 from total PBMCs + CD8 + T cell stimulation and proliferation Vβ17 + CD8 + To expand T cells, we used an HLA A2 donor (HPU-086 Total PBMCs from 94) were incubated with 1 μg / mL FLU MP58 peptide (in DMSO). Stimulated. Total CD8 + Vβ17 between T cells + The cell frequency was measured on days 8 and 14 of the culture period. The total CD8 + Vβ17 among T cells + To count the frequency of cells, total viable PB MCs were first gated, doublets were excluded, and total CD8 + Gate on T cells and then Vβ 17 + Cells were gated (Figure 3A). Day 0 total CD8 + Vβ17 between T cells + Cell frequency Compared to the control, a significant proliferation of these cells was observed on day 8 of the culture period (Figure 3B). Day 8 CD8 + A larger fraction of T cells was Vβ17 in this donor + Cells (Figure 3 B).

[0157] Anti-Vβ17 / anti-CD123 bispecific antibody binding assay On Kasumi-3 cells To understand the binding kinetics of anti-Vβ17 / anti-CD123 bispecific antibodies, Kasumi -3 cells were treated with anti-Vβ17 / anti-CD123 bispecific antibody and Vβ17 × null arm control. Both were incubated at various concentrations (range: 5 μg / mL to 0 μg / mL). The combined bispecific antibodies were detected with a mouse anti-human IgG4 Fc-PE secondary antibody. Positive Kas for PE (secondary antibody) when treated with different concentrations of bispecific antibody The frequency of umi-3 cells is shown. EC5 of anti-Vβ17 / anti-CD123 and null × CD123 0 were determined to be 6 and 42.7 nM, respectively (Table 9).

[0158] [Table 9]

[0159] Bispecific antibody binding affinity to Kasumi-3 cells was determined by flow cytometry. The half-maximal binding was determined as the bispecific concentration that yielded 50% of the maximum binding (PE-positive cells). Dose-effect concentration (EC 50 ) values ​​were calculated. ND: Not determined.

[0160] On enriched CD8+ T cells Enriched FLU MP58 peptide stimulated CD8 + T (from day 14 of culture) was measured using various Along with various concentrations of anti-Vβ17 / anti-CD123 bispecific and Vβ17× null arm control antibodies A mouse anti-human IgG4 Fc-PE secondary antibody was used to perform bispecific Table 10 shows the effect of treatment with different concentrations of bispecific antibodies on PE (bispecific antibody). CD8 positive for primary antibody + The frequency of T cells is shown. Anti-Vβ17 / anti-CD123, Vβ 17x Null EC 50 were determined to be 9.0 nm and 18.7 nm, respectively (Table 10).

[0161] [Table 10]

[0162] CD8 + Bispecific antibody binding affinity to T cells was determined by flow cytometry. The half-maximal effective concentration (HAEC) was determined as the antibody concentration that produced 50% of the maximum binding (PE-positive cells). EC 50 ) values ​​were calculated. ND: Not measured

[0163] Bispecific-mediated cytotoxicity assay To analyze the efficacy of anti-Vβ17 / anti-CD123 bispecific antibody-mediated cytotoxicity, CFSE-labeled target (Kasumi-3) cells were incubated with various concentrations of anti-Vβ17 / anti-CD 123 bispecific and Vβ17× null arm control antibodies were used at 0.5:1, 1:1, 5 Stimulated CD8 cells at an effector-to-target (ET) ratio of 1:1 for 14 and 24 h from day 14 of culture. + T cells were co-cultured with the target Kasumi-3 cells using a commercially available anti-CD123 antibody. We investigated CD123 expression in target cells (Kasumi-3) by labeling with CFSE. CFSE during raw cytometry analysis + After the co-culture period, 7-AAD was added. 7-AAD was then used as an indicator of cytotoxicity. + CFSE + The percentage of cells was analyzed. The basal cytotoxicity observed in the absence of antibody was reduced, resulting in specific cell proliferation in response to the bispecific antibody. The assay was performed once on a single donor (HPU-08694). At time points between, anti-Vβ17 / anti-CD123 dual immunotherapy was performed at ET ratios of 0.5:1, 1:1, and 5:1. Polyspecific antibody EC 50 were 3.7, 0.1, and 0.133 pM, respectively (Table 11).

[0164] [Table 11] UD: Undetectable, activity too low for proper curve fitting.

[0165] At 24 hours, anti-Vβ17 / anti-CD at ET ratios of 0.5:1, 1:1, and 5:1 123 bispecific EC 50 were 0.4, 0.2, and 1.0 pM, respectively (Table 12).

[0166] [Table 12] UD: Undetectable

[0167] Similarly, anti-Vβ17 / anti-CD123 bispecific mediated non-stimulatory CD8 + T cell toxicity, 1 ET ratios of 0.5:1, 1:1, and 5:1 were tested for 4 (Table 13) and 24 (Table 14) hours. At an anti-Vβ17 / anti-CD123 bispecific concentration of 5 ng / ml and at the 14 hour time point, Unstimulated CD8 at ET ratios of 1:1 and 1:1 + T cells stimulate CD8 + 77% by T cells and 73% cytotoxicity, respectively, compared with 2.8% and 9.8% target cell damage. At an ET ratio of 5:1, unstimulated CD8 + T cells stimulate CD8 + T cell The results were similar for the 24-hour group, which showed 31.65% target cell death compared to 70.9% for the 24-hour group. (Tables 12, 15, 16, and 17). Anti-Vβ17 / anti-CD12 tested At the highest concentration of the three bispecifics (5ng / ml), unstimulated CD8 + T cells are more The ET ratio showed higher cytotoxicity against target cells.

[0168] [Table 13]

[0169] [Table 14]

[0170] [Table 15]

[0171] Maximum cytotoxicity (CFSE + 7AAD + ) The antibody concentration that produces 50% of the cells is the half-maximum Effective concentration (EC 50 ) values ​​were calculated. ND: Not determined.

[0172] [Table 16]

[0173] Maximum cytotoxicity (CFSE + 7AAD + ) The antibody concentration that produces 50% of the cells is the half-maximum Effective concentration (EC 50 ) values ​​were calculated. ND: Not measured

[0174] [Table 17]

[0175] Maximum cytotoxicity (CFSE + 7AAD + ) The antibody concentration that produces 50% of the cells is the half-maximum Effective concentration (EC 50 ) values ​​were calculated. UD: Not detectable.

[0176] Those skilled in the art may make changes to the above-described embodiments without departing from the broad inventive concept. It will be understood that the invention is therefore not limited to the particular embodiments disclosed. without limitation, and is intended to encompass modifications within the spirit and scope of the invention as defined by this description. is understood to be intended.

[0177]

Table 18-1

[0178]

Table 18-2

[0179]

Table 18-3

[0180]

Table 18-4

[0181]

Table 18-5

[0182]

Table 18-6

[0183]

Table 18-7

[0184]

Table 18-8

[0185]

Table 18-9

[0186]

Table 18-10

Claims

1. 1. An isolated Vβ17 bispecific antibody, or antigen-binding fragment thereof, comprising: a. a first heavy chain (HC1); and b. a second heavy chain (HC2); and c. the first light chain (LC1); d. a second light chain (LC2); HC1 associates with LC1, HC2 associates with LC2, and HC1 is SEQ ID NO: 1, SEQ ID NO: heavy chain complementarity determining region 1 (HCDR) having the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3, 1), HCDR2, and HCDR3, and LC1 is selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: Light chain complementarity determining region 1 (LCDR1), LCDR each containing the amino acid sequence of sequence number 6 2, and LCDR3 to form a binding site for a first antigen, and forms a binding site for a second antigen; The isolated Vβ17 bispecific antibody or antigen-binding fragment thereof.

2. The binding site for the first antigen is Vβ1 on a CD8+ or CD4+ T cell.

7. The Vβ17 bispecific antibody or antigen-binding fragment thereof of claim 1 .

3. The binding site for the second antigen binds to a tumor antigen present on the surface of a cancer cell. The Vβ17 bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The Vβ1 according to any one of claims 1 to 3, wherein HC1 and LC1 are humanized.

7. Bispecific antibodies or antigen-binding fragments.

5. The V antigen of any one of claims 1 to 4, wherein HC2 and LC2 bind to CD123. β17 bispecific antibody or antigen-binding fragment thereof.

6. The bispecific antibody or antigen-binding fragment thereof is of the IgG isotype.

6. A Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of claims 5 to 5.

7. 2. The method of claim 1, wherein the bispecific antibody or antigen-binding fragment thereof is of the IgG4 isotype.

7. A Vβ17 bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The bispecific antibody, or antigen-binding fragment thereof, has an EC ５０ Invitation In the step (b), the antibody induces CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells.

8. A Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of claims 7.

9. The Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 An isolated nucleic acid encoding said HC1 and said LC1.

10. The Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 An isolated nucleic acid encoding said HC2 and said LC2.

11. A vector comprising the isolated nucleic acid of claim 9 or 10.

12. A host cell comprising the vector of claim 11.

13. 1. An isolated anti-Vβ17 / anti-CD123 bispecific antibody, or antigen-binding fragment thereof, comprising: a. a first heavy chain (HC1); and b. a second heavy chain (HC2); and c. the first light chain (LC1); d. a second light chain (LC2); HC1 associates with LC1, HC2 associates with LC2, and HC1 is SEQ ID NO: 1, SEQ ID NO: heavy chain complementarity determining region 1 (HCDR) having the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3, 1), HCDR2, and HCDR3, and LC1 is selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: Light chain complementarity determining region 1 (LCDR1), LCDR each containing the amino acid sequence of sequence number 6 a binding site for a first antigen that specifically binds to Vβ17, comprising LCDR2, and LCDR3; and HC2 comprises the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:

36. heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, LC2 has the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. A light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, forming a binding site for a second antigen that specifically binds to 123; The isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof.

14. HC1 comprises the amino acid sequence of SEQ ID NO:13, and LC1 comprises the amino acid sequence of SEQ ID NO:

14. HC2 comprises the amino acid sequence of SEQ ID NO:15, and LC2 comprises the amino acid sequence of SEQ ID NO:

16. The isolated anti-Vβ17 / anti-CD123 bispecific antibody of claim 13, comprising the amino acid sequence An antibody or an antigen-binding fragment thereof.

15. 15. The method of claim 13 or 14, wherein the Vβ17 is on the surface of a CD8+ or CD4+ T cell.

2. An isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof as described above.

16. The CD123 is on the surface of a cancer cell or a CD34+ stem cell according to claims 13 to 15. The isolated anti-Vβ17 / anti-CD123 bispecific antibody or its antigen according to any one of claims 1 to 4. Combined fragment.

17. The bispecific antibody or antigen-binding fragment thereof has an EC ５０ In vitro Inducing CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells in the method according to claims 13 to 1.

7. An isolated anti-Vβ17 / anti-CD123 bispecific antibody according to any one of claims 6 to 61, or Antigen-binding fragment.

18. The anti-Vβ17 / anti-CD123 bispecific antibody according to any one of claims 13 to 17, An isolated nucleic acid encoding said HC1 and LC1 of said antigen-binding fragments.

19. The anti-Vβ17 / anti-CD123 bispecific antibody according to any one of claims 13 to 18, An isolated nucleic acid encoding said HC2 and LC2 of said antigen-binding fragments.

20. A vector comprising the isolated nucleic acid of claim 18 or 19.

21. A host cell comprising the vector of claim 20.

22. The isolated anti-Vβ17 / anti-CD123 bispecific antibody according to any one of claims 13 to 17. A buffered composition comprising an isomeric antibody or antigen-binding fragment thereof.

23. A method for targeting Vβ17-expressing CD8+ or CD4+ T cells to cancer cells, comprising:

14. The method of claim 13, further comprising administering to said patient an anti-Vβ17 / anti-CD123 antibody capable of expressing Vβ17 or CD4 T cells. contacting said Vβ17-expressing CD8+ or treating CD4+ T cells with said anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment with contacting the fragment with said Vβ17-expressing CD8+ or CD4+ T cells directed to cancer cells The method according to claim 1,

24. 14. A method for inhibiting the growth or proliferation of cancer cells, comprising: and contacting the subject with the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof. and treating said cancer cells with said anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof. wherein contacting with inhibits the growth or proliferation of the cancer cells.

25. The method of claim 23 or 24, wherein the cancer cells are CD123-expressing cancer cells.

26. The Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. , and packaging therefor, a kit.

27. The anti-Vβ17 / anti-CD123 bispecific antibody according to any one of claims 13 to 17, A kit comprising the antigen-binding fragment and packaging therefor.

28. A method for producing a Vβ17 bispecific antibody or antigen-binding fragment thereof, comprising the steps of: Culturing the host cell described herein to produce the Vβ17 bispecific antibody or antigen-binding fragment thereof. and extracting said Vβ17 bispecific antibody or antigen-binding fragment thereof from said cell or culture. and recovering.

29. A method for producing an anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof.

22. Culturing the host cell of claim 21 to produce the anti-Vβ17 / anti-CD123 bispecific Producing an antibody or antigen-binding fragment thereof and said anti-Vβ17 / anti-CD123 bispecific and recovering the antibody or antigen-binding fragment thereof from the cell or culture.

30. An isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof, comprising: The humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:

28. The isolated humanized Vβ1 comprises an amino acid sequence having at least 95% identity with 7. A monoclonal antibody or an antigen-binding fragment thereof.

31. The humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:

28.

31. The isolated humanized Vβ17 monoclonal antibody of claim 30, comprising the amino acid sequence An antigen-binding fragment thereof.

32. The isolated humanized Vβ17 monoclonal antibody or its antibody according to claim 30 or 31. An isolated nucleic acid encoding an original binding fragment.

33. A vector comprising the isolated nucleic acid of claim 32.

34. A host cell comprising the vector of claim 33.

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