Anti-Vβ17 / anti-CD123 bispecific antibody

Abstract

The present invention describes an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof. The present invention also describes a nucleic acid encoding the antibody, a composition comprising the antibody, a method for producing the antibody, and a method for using the antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 816,464, filed on March 11, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a monoclonal anti-Vβ17 / anti-CD123 bispecific antibody, a nucleic acid encoding the antibody and an expression vector, a recombinant cell containing the vector, and a composition containing the antibody. A method for preparing the antibody and a method for killing cancer cells using the antibody are also provided.

[0004] Reference sequence listing submitted electronically

[0005] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing, with a file name of "PRD4012WOPCT Sequence Listing", a creation date of March 11, 2020, and a size of 85 kb. This sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0006] Cytotoxic T cells (e.g., CD8+ T cells) can be used to directly kill cancer cells. Finding a method for guiding cytotoxic T cells to cancer cells can lead to killing such cells and inhibiting cancer cell proliferation. It has been demonstrated that cytotoxic T cells against cancer cells expressing cancer-associated antigens can be activated, in a manner that uses a bispecific antibody that combines both cytotoxic T cells and cancer cells, so that the cytotoxic T cells and cancer cells are closely approached over an extended period of time. There are multiple potential complexities in this method of killing cancer cells, such as selecting T cell antigens and cancer cell antigens that mediate T cell activation, selecting parent antibodies that will have sufficient affinity to mediate binding in the case of bispecific antibodies, and selecting activated T cells to specifically play a role against cancer cells rather than triggering cancer cell antigens that activate nonspecific T cells. These complexities are only combined together in the case of attempting to activate T cells to destroy cancer cells in animal subjects. Summary of the invention

[0007] Provided herein are bispecific antibodies capable of binding to Vβ17 (antigens associated with T cells) and CD123 (antigens associated with cancer cells). Cytotoxic T cells express T cell receptors composed of α chains and β chains, such as Vβ17. It is assumed that bispecific antibodies can guide cytotoxic T cells to cancer cells expressing antigens in combination with Vβ17 and cancer-associated antigens (such as CD123). This bispecific antibody is used to recruit or redirect cytotoxic T cells to cancer cells expressing antigens, and T cells can be allowed to kill cancer cells.

[0008] In one general aspect, the disclosure relates to isolated bispecific antibodies or antigen-binding fragments thereof that bind to Vβ17 and CD123.

[0009] Provided herein is an isolated Vβ17 bispecific antibody or an antigen-binding fragment thereof. The isolated Vβ17 bispecific antibody or an antigen-binding fragment thereof comprises:

[0010] a. First heavy chain (HC1);

[0011] b. Second heavy chain (HC2);

[0012] c. the first light chain (LC1); and

[0013] d. Second light chain (LC2),

[0014] wherein HC1 associates with LC1 and HC2 associates with LC2, and wherein HC1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LC1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, to form a binding site for a first antigen, and wherein HC2 and LC2 form a binding site for a second antigen. In certain embodiments, the binding site for the first antigen binds to Vβ17 on the surface of a CD8+ T cell or a CD4+ T cell. In certain embodiments, the binding site for the second antigen binds to a tumor antigen present on the surface of a cancer cell.

[0015] In certain embodiments, binding of the bispecific antibody to Vβ17 present on the surface of a CD8+ T cell or a CD4+ T cell and to a tumor antigen present on the surface of a cancer cell results in killing of the cancer cell.

[0016] In certain embodiments, HC2 and LC2 bind to CD123.

[0017] In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is of the IgG isotype, such as IgG4.

[0018] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof are expressed in vitro at an EC of less than about 0.2 pM. 50 Induces CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells.

[0019] Also provided are isolated anti-Vβ17 / anti-CD123 bispecific antibodies or antigen-binding fragments thereof.

[0020] The anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment comprises:

[0021] a. First heavy chain (HC1);

[0022] b. Second heavy chain (HC2)

[0023] c. the first light chain (LC1); and

[0024] d. a second light chain (LC2), wherein HC1 associates with LC1 and HC2 associates with LC2, and wherein HC1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, the heavy chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LC1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, the light chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, to form a binding site that specifically binds to the first antigen of Vβ17, and wherein HC2 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, the heavy chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively, and LC2 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, the light chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:40, SEQ ID NO:40 NO:38 and SEQ ID NO:39 amino acid sequence to form a binding site for a second antigen that specifically binds CD123. In certain embodiments, HC1 comprises the amino acid sequence of SEQ ID NO:13 and LC1 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. In certain embodiments, Vβ17 is located on the surface of CD8+ T cells or CD4+ T cells. In certain embodiments, CD123 is located on the surface of cancer cells. In certain embodiments, the bispecific antibody or its antigen-binding fragment induces CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells in vitro with an EC50 of less than about 0.2pM.

[0025] In certain embodiments, the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof is chimeric, partially humanized, or fully humanized.

[0026] Also provided is an isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof. The isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof may comprise an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 28.

[0027] Also provided are isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof disclosed herein and the bispecific antibodies or antigen-binding fragments thereof disclosed herein.

[0028] Also provided are vectors comprising isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof disclosed herein and the bispecific antibodies or antigen-binding fragments thereof disclosed herein.

[0029] Also provided are host cells comprising a vector comprising an isolated nucleic acid disclosed herein.

[0030] Also provided is a method for directing CD8+ or CD4+ T cells expressing Vβ17 to cancer cells. The method comprises contacting CD8+ or CD4+ T cells expressing Vβ17 with an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof disclosed herein. Contacting CD8+ T cells or CD4+ T cells expressing Vβ17 with an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof can direct CD8+ T cells or CD4+ T cells expressing Vβ17 to cancer cells.

[0031] Also provided are methods for inhibiting the growth or proliferation of cancer cells. The methods include contacting cancer cells with a bispecific antibody disclosed herein. Contacting cancer cells with the antibody can, for example, inhibit the growth or proliferation of cancer cells, or promote T cell-mediated killing of cancer cells.

[0032] Also provided is a method for producing the bispecific antibodies or antigen-binding fragments thereof disclosed herein. The method includes culturing a cell comprising a nucleic acid encoding a heavy chain and light chain pair of a bispecific antibody under conditions that produce heavy chains and light chains or antigen-binding fragments thereof, and recovering the heavy and light chains of the bispecific antibody or antigen-binding fragments thereof from the cell or culture. After collecting the heavy and light chains of the two arms of the bispecific antibody, the heavy and light chain pairs are mixed under conditions suitable for allowing self-assembly, and then the self-assembled bispecific antibody is collected.

[0033] Also provided are methods of producing compositions (such as buffered compositions or purified compositions, etc.) comprising the bispecific antibodies or antigen-binding fragments disclosed herein. For example, the method can include combining the bispecific antibodies or antigen-binding fragments thereof with a buffer acceptable for storage and use of the bispecific antibodies.

[0034] Also provided are kits comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein and packaging thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The foregoing summary and the following detailed description of preferred embodiments of the present patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the precise embodiments shown in the drawings.

[0036] Figure 1 A schematic diagram showing binding of anti-Vβ17 / anti-tumor antigen bispecific antibodies to recruit T cells to cancer cells and induce cancer cell death is shown.

[0037] FIG. 2A to FIG. 2B It was shown that Vβ17+CD8+ T cells are present in healthy subjects and that these cells can be expanded in vitro after culture with M1 peptide. Figure 2A Shown are FACS histograms of gated peripheral blood mononuclear cells (PBMCs) from healthy subjects for CD8+ T cells expressing Vβ17 on the cell surface (Vβ17+). Figure 2B Shown are the HLA subtypes of various donors, as well as the percentage of identified Vβ17+CD8+ T cells present at day 0 and after 14 days of in vitro expansion with M1 peptide (day 14).

[0038] Figure 3 It is shown that Vβ17+CD8+ T cells have markers for killing cytotoxic cells. The bar graph indicates the 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+) at day 0 (no M1) and day 14 (+M1) after stimulation with M1 peptide.

[0039] Figure 4 Binding of VB11 [anti-Vβ17 / anti-CD123] bispecific antibody and VB13 [Vβ17null control bispecific] antibody to CD8+ T cells is shown. Data presented for CD8+ T cells isolated from PBMCs from 3 different donors (D203517, HPU09381, and HPU08694). Each graph in the table below represents the EC value of binding in nM. 50 value.

[0040] Figure 5 Binding of the Vβ17 and CD123 bispecific (VB11) antibody and the Vβ17null control bispecific (VB13) antibody to AML cancer cell lines is shown. The data presented show binding of the bispecific antibodies to the Kasumi3 AML cell line. The table below the figure shows the EC values ​​of binding in nM. 50 value.

[0041] Figure 6The redirection of Vβ17+ T cells by bispecific antibodies that induce effective killing of AML cancer cells is shown. The data in the left figure show the killing of Kasumi3 cancer cells at an effector to target (E:T) ratio of 0.5:1 and the dose titration of the bispecific antibody. The data in the middle figure show the killing of Kasumi3 cancer cells at an E:T ratio of 1:1 and the dose titration of the bispecific antibody. The data in the right figure show the killing of Kasumi3 cancer cells at an E:T ratio of 5:1 and the dose titration of the bispecific antibody. The table below the figure shows the EC calculated from the above figure. 50 Values ​​(given in pM).

[0042] FIG. 7A to FIG. 7B Specific binding of the anti-Vβ17 / anti-CD123 bispecific antibody (VB11) and the Vβ17null bispecific antibody (VB13) to CD8+ T cells isolated from PBMCs is shown. Fig. 7A Shown are FACS histograms of gated PBMCs for CD8+ T cells expressing Vβ17 on the cell surface (Vβ17+) from healthy subjects (left panel, Vβ17 non-depleted) and from PBMCs depleted of Vβ17+ T cells using negative selection (right panel, Vβ17 depleted). Figure 7B The anti-Vβ17 / anti-CD123 bispecific antibody (VB11) and the Vβ17null bispecific antibody (VB13) are shown in Figure 1. Fig. 7A The dose response of the bispecific antibody is shown in the figure. The table below the figure shows the binding EC calculated from the above figure. 50 Values ​​(given in nM).

[0043] Figure 8 It is shown that the Vβ17 bispecific antibody specifically recruits Vβ17T cells to kill Kasumi3 cancer cells. The left figure shows the killing of the Kasumi3 AML cell line when effector cells are separated from PBMCs containing CD8+ T cells (Vβ17+) expressing Vβ17 on the cell surface (uncontacted CD8 T cells). The illustration shows that there are 10.1% Vβ17+CD8 T cells in the effector cell population. The right figure shows the killing of the Kasumi3 AML cell line when effector CD8+ T cells are separated from PBMCs, but Vβ17+ T cells are exhausted by negative selection. The illustration shows that there is a trace amount (0.086%) of Vβ17+CD8+ T cell population in the effector cell population.

[0044] FIG. 9A to FIG. 9B It was shown that there was no pan-activation of T cells when the Vβ17 bispecific antibody was used. Fig. 9AFACS plots of Vβ17+ gated CD8+ T cells and Vβ17- gated CD8+ T cells are shown. When T cells were activated with the Vβ17 bispecific antibody, there was a high level of upregulation of CD69 on Vβ17+CD8+ T cells (62.5%) compared to Vβ17-CD8+ T cells (1.80%). Fig. 9B A bar graph showing upregulation of CD69 on Vβ17+ gated CD8+ T cells and Vβ17- gated CD8+ T cells when activated using the Vβ17 bispecific antibody.

[0045] Fig.10 It was shown that Vβ17+ T cells from HLA A2-negative donors are also effector killer cells and that no prior stimulation of Vβ17+ cells is required.Efficient cytotoxicity mediated by Vβ17 bispecific antibody against Kasumi3 cancer cells was shown by PBMCs containing Vβ17+ T cells from an HLA A2-negative donor (HPU 09381). DETAILED DESCRIPTION

[0046] The discussion of documents, acts, materials, devices, articles and the like included in this specification is intended to provide context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification.

[0048] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include the value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0049] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0050] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, should be understood to mean including the specified integer or group of integers but not excluding any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to these compositions, mixtures, processes, methods, articles, or apparatuses. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0052] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0053] It should also be understood that when referring to dimensions or characteristics of components of the preferred invention, the use of "about," "approximately," "substantially," and similar terms herein indicates that the described dimensions / characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) without changing the least significant digits.

[0054] In the context of two or more nucleic acids or polypeptide sequences (e.g., anti-Vβ17 / anti-CD123 bispecific antibodies and polynucleotides encoding these antibodies, Vβ17 polypeptides and Vβ17 polynucleotides encoding these polypeptides, CD123 polypeptides and CD123 polynucleotides encoding these polypeptides), the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0055] For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and program parameters for the sequence algorithm are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage for the test sequence relative to the reference sequence based on the specified program parameters.

[0056] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John A joint venture of Wiley & Sons, Inc. (1995 supplemented edition) (Ausubel).

[0057] Examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in J. Mol. Biol. 215: 403-410, Altschul et al., 1990, and Nucleic Acids Res. 25: 3389-3402, Altschul et al., 1997, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0058] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction stops when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains by default. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix by default (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0059] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0060] Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0061] Antibody

[0062] Described herein are isolated anti-Vβ17 bispecific antibodies or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. The present invention further relates to isolated anti-Vβ17 / anti-CD123 bispecific antibodies or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the bispecific antibodies. Also provided are methods for preparing the antibodies, and methods for using the antibodies to treat diseases (including cancer). The antibodies disclosed herein have one or more desired functional properties, including but not limited to high affinity binding to Vβ17 and / or CD123, high specificity to Vβ17 and / or CD123, and the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies.

[0063] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, composite antibodies and chimeric antibodies and monoclonal or polyclonal antibody fragments. Generally speaking, an antibody is a protein or peptide chain that exhibits binding specificity for a specific antigen. The structure of an antibody is well known. According to the heavy chain constant domain amino acid sequence, immunoglobulins can be designated as five major classes (i.e., IgA, IgD, IgE, IgG, and IgM). IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of a vertebrate species can be designated as one of two completely different types, i.e., κ and λ. Therefore, the antibody of the present invention may contain κ or λ light chain constant domains. According to a specific embodiment, the antibody disclosed herein includes a heavy chain constant region and / or a light chain constant region from a mouse antibody or a human antibody. In addition to the heavy and light chain constant domains, antibodies also comprise an antigen binding region consisting of a light chain variable region and a heavy chain variable region, each variable region comprising three domains (i.e., complementary determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0064] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to Vβ17 is substantially free of antibodies that do not bind to Vβ17; an isolated antibody that specifically binds to CD123 is substantially free of antibodies that do not bind to CD123). In addition, the isolated antibody may be substantially free of other cellular material and / or chemical substances.

[0065] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies disclosed herein can be prepared by hybridoma methods, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, a monoclonal antibody can be produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal such as a transgenic mouse or rat, the transgenic non-human animal having a genome comprising a human heavy chain transgene and a light chain transgene.

[0066] As used herein, the term "antigen binding fragment" refers to an antibody fragment, such as, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab), a scFv dimer (bivalent diabody), a multispecific antibody formed by a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen binding fragment is capable of binding to the same antigen as the antigen bound by the parent antibody or parent antibody fragment. According to specific embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain. According to other specific embodiments, the antigen binding fragment comprises Fab and F(ab').

[0067] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with a human antibody, such that the antigen-binding properties of the antibody are retained but its antigenicity in the human body is reduced.

[0068] As used herein, the term "multi-specific antibody" refers to an antibody comprising multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the multiple immunoglobulin variable domain sequences has binding specificity to the first epitope, and the second immunoglobulin variable domain sequence in the multiple immunoglobulin variable domain sequences has binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope do not overlap or do not overlap substantially. In one embodiment, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of multimeric proteins). In one embodiment, the multi-specific antibody comprises the third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multi-specific antibody is a bispecific antibody molecule, a tri-specific antibody molecule or a tetra-specific antibody molecule.

[0069] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. Bispecific antibodies are characterized by having a first immunoglobulin heavy chain and light chain pair with binding specificity to a first epitope (e.g., an epitope on a Vβ17 antigen), and a second immunoglobulin heavy chain and light chain pair with binding specificity to a second epitope (e.g., an epitope on a CD123 antigen). In one embodiment, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a half antibody or fragment thereof with binding specificity to the first epitope and a half antibody or fragment thereof with binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises an scFv or fragment thereof with binding specificity to the first epitope and an scFv or fragment thereof with binding specificity to the second epitope. In one embodiment, the first epitope is located on Vβ17, and the second epitope is located on CD123. In one 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 / or other tumor-associated immunosuppressive factors or surface antigens.

[0070] As used herein, the term "half antibody" refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain. An exemplary half antibody is shown in SEQ ID NO: 28. Those skilled in the art will readily appreciate that half antibodies may encompass fragments thereof and may also have an antigen binding domain consisting of a single variable domain, such as from Camelidae.

[0071] As used herein, the term "Vβ17" refers to a T cell receptor expressed in response to an immune response on cytotoxic T cells. CD8+ T cells expressing Vβ17 are generally produced in response to a subject being exposed to influenza A virus. CD8+ T cells expressing Vβ17 provide great recall in response to a subject being exposed to influenza. The term "Vβ17" includes any Vβ17 variants, isotypes, and species homologs that are naturally expressed by cells (including T cells) or can be expressed on cells transfected with a gene or cDNA encoding the polypeptide. Unless otherwise indicated, preferably, Vβ17 is human Vβ17. The human Vβ17 amino acid sequence is provided by GenBank Accession No. AAB49730.1.

[0072] The term "CD123" refers to a molecule present on a cell that helps transmit the signal of interleukin-3 (a soluble cytokine that is very important in the immune system). CD123 may also be referred to as "interleukin-3 receptor". The receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique α chain paired with a common β subunit (βc or CD131). The CD123 receptor may be present on multipotent progenitor cells, and may induce tyrosine phosphorylation in the cell and promote proliferation and differentiation in the hematopoietic cell line. CD123 may also be expressed in acute myeloid leukemia (AML) subtypes. Unless otherwise indicated, the term "CD123" includes any CD123 variants, isoforms, and species homologs that are naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides, preferably "CD123" is human CD123. Human CD123 amino acid sequences are provided by GenBank accession number AY789109.1.

[0073] As used herein, an antibody that "specifically binds to Vβ17" refers to an antibody that specifically binds to Vβ17 at a concentration of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 An antibody that binds to Vβ17, preferably human Vβ17, with a KD of M or less. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). According to the present disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD of an antibody can be determined using surface plasmon resonance, such as by using a biosensor system (e.g., system), or by using bio-layer interferometry techniques (such as the Octet RED96 system).

[0074] As used herein, an antibody that "specifically binds to CD123" refers to an antibody that specifically binds to CD123 at a concentration of 1×10 -7 M or less, preferably 1×10 - 8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller or 1×10 -10 M or less KD and CD123 binding, preferably with human CD123 binding antibody.Term " KD " refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e. Kd / Ka) and expressed as molar concentration (M).According to the present disclosure, the KD value of the antibody can be determined using methods in the art.For example, the KD of the antibody can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g. system), or by using bio-layer interferometry techniques (such as the Octet RED96 system).

[0075] The smaller the KD value of an antibody, the higher the affinity of the antibody for binding to the target antigen.

[0076] According to a specific aspect, the present invention relates to an isolated Vβ17 bispecific antibody or antigen-binding fragment thereof, comprising: (a) a first heavy chain (HC1); (b) a second heavy chain (HC2); (c) a first light chain (LC1); and (d) a second light chain (LC2). HC1 can associate with LC1, and HC2 can associate with LC2. HC1 can comprise a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, the heavy chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LC1 can comprise a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, the light chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0077] HC1 and LC1 form a binding site for a first antigen, and HC2 and LC2 form a binding site for a second antigen. By way of example, the binding site for the first antigen can bind to Vβ17 on a CD8+ T cell or a CD4+ T cell, and the binding site for the second antigen can, for example, bind to a tumor antigen present on the surface of a cancer cell. The binding of the Vβ17 bispecific antibody to Vβ17 present on the surface of a CD8+ T cell or a CD4+ T cell and to a tumor antigen present on the surface of a cancer cell can, for example, lead to killing of the cancer cell.

[0078] Also provided herein is an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, which comprises an anti-Vβ17 antibody or an antigen-binding fragment thereof and an anti-CD123 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof comprises: (a) a first heavy chain (HC1); (b) a second heavy chain (HC2); (c) a first light chain (LC1); and a second light chain (LC2). HC1 associates with LC1, and HC2 associates with LC2. In certain embodiments, HC1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and the heavy chain complementary determining region comprises the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LC1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, and the light chain complementary determining region comprises the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In certain embodiments, HC2 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and LC2 comprises a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively.

[0079] In certain embodiments, HC1 may, for example, comprise the amino acid sequence of SEQ ID NO: 13 and LC1 may, for example, comprise the amino acid sequence of SEQ ID NO: 14, to form a binding site for a first antigen that specifically binds to Vβ17. HC2 may, for example, comprise the amino acid sequence of SEQ ID NO: 15 and LC2 may, for example, comprise the amino acid sequence of SEQ ID NO: 16, to form a binding site for a second antigen that specifically binds to CD123.

[0080] In certain embodiments, Vβ17 is located on the surface of CD8+ T cells or CD4+ T cells. In certain embodiments, CD123 is located on the surface of cancer cells (eg, leukemia cells).

[0081] In some embodiments, the bispecific antibodies disclosed herein may take the form of diabodies, crossover antibodies, or bispecific antibodies obtained via controlled Fab arm exchange as described herein.

[0082] In some embodiments, the bispecific antibodies include IgG-like molecules with complementary CH3 domains that promote heterodimerization; recombinant IgG-like dual-targeting molecules, wherein each side of the molecule contains a portion of the Fab fragment or Fab fragment of at least two different antibodies; IgG fusion molecules, in which the full-length IgG antibody is fused with an additional Fab fragment or a portion of the Fab fragment; Fc fusion molecules, in which a single-chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; Fab fusion molecules, in which different Fab fragments are fused together; heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and diabodies, in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0083] In some embodiments, IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes antibodies (Genentech), CrossMAbs (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A / S).

[0084] In some embodiments, recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK / Domantis), two-in-one antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX bodies (CovX / Pfizer).

[0085] In some embodiments, IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), IgG-like bispecific antibodies (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

[0086] In some embodiments, Fc fusion molecules may include ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART) (MacroGenics), and Dual (ScFv)2-Fab (National Research Center for Antibody Medicine--China).

[0087] In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-based, bivalent antibody-based domain antibodies include but are not limited to bispecific T cell engagers (BiTE) (Micromet), tandem bivalent antibodies (Tandab) (Affimed), dual affinity retargeting technology (DART) (MacroGenics), single-chain bivalent antibodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual-targeted nanobodies (Ablynx), dual-targeted heavy chain domain antibodies only.

[0088] The full-length bispecific antibodies disclosed herein can be produced, for example, using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 junction in each half molecule to promote the formation of heterodimers of two antibody half molecules with different specificities in a cell-free environment in vitro or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of a second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and re-formed by dissociation-association. The CH3 domain of the Fab arm can be engineered to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each of which binds to a different epitope, i.e., an epitope on Vβ17 and an epitope on a tumor antigen.

[0089] As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains having the same CH3 amino acid sequence.

[0090] As used herein, "heterodimerization" refers to the interaction of two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody with two heavy chains containing different CH3 amino acid sequences.

[0091] The "knob-in-hole" strategy (see, e.g., PCT International Publication WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the interface of the CH3 domains in human IgG can be mutated at positions that affect the interaction of the CH3 domains, thereby promoting heterodimer formation. Amino acids with small side chains (holes) are introduced into the heavy chains of antibodies that specifically bind to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chains of antibodies that specifically bind to a second antigen. Upon co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chains with the "hole" with the heavy chains with the "knob". Exemplary CH3 substitution pairs that form a knob-in-hole structure (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0092] Other strategies may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in U.S. Patent Publication No. US2010 / 0015133, U.S. Patent Publication No. US2009 / 0182127, U.S. Patent Publication No. US2010 / 028637, or U.S. Patent Publication No. US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (indicated as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W, as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849.

[0093] In addition to the above methods, the bispecific antibodies disclosed herein can be produced in a cell-free environment in vitro by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization according to the method described in International Patent Publication No. WO2011 / 131746. In the method, a first monospecific bivalent antibody (e.g., an anti-CD33 antibody) and a second monospecific bivalent antibody (e.g., an anti-CD33 antibody) are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of cysteine ​​in the hinge region; thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions can optionally be restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine ​​and β-mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions can be used: in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5-8, such as pH 7.0 or pH 7.4, at a temperature of at least 20° C., incubated for at least 90 minutes.

[0094] In certain embodiments, the anti-Vβ17 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0095] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0096] And the anti-CD123 antibody or its antigen-binding fragment comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0097] 1. SEQ ID NO: 34, 35, 36, 37, 38 and 39, respectively .

[0098] According to another specific aspect, the present invention relates to an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof that induces antibody-dependent cell-mediated cytotoxicity (ADCC). The bispecific antibody or antigen-binding fragment thereof is capable of inducing ADCC, for example, in vitro. The bispecific antibody or antigen-binding fragment thereof is capable of inducing ADCC with an EC of less than about 1 pM. 50 Inducing ADCC. In certain embodiments, EC 50 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 about 0.300 pM, less than about 0.2 pM, less than about 0.19 pM, less than about 0.18 pM, less than about 0.17 pM, less than about 0.16 pM, less than about 0.15 pM, less than about 0.14 pM, less than about 0.13 pM, less than about 0.12 pM, less than about 0.11 pM, less than about 0.1 pM, 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 0.05 pM, less than about 0.04 pM, less than about 0.03 pM, less than about 0.02 pM, or less than about 0.01 pM. In certain embodiments, the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment comprises an IgG1, IgG2, IgG3 or IgG4 main chain. In one such embodiment, the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment has an antibody main chain of an IgG4 isotype.

[0099] In some embodiments described herein, the immune effector properties of anti-Vβ17 / anti-CD123 bispecific antibodies can be enhanced or silenced via Fc modification by techniques known to those skilled in the art. For example, Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), cell surface receptors (e.g., B cell receptors; BCR) down-regulation, etc., can be provided and / or controlled by modifying the residues in the Fc that contribute to these activities.

[0100] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0101] The ability of antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Most of the glycans in human IgG1 or IgG3 are N-glycosylated at Asn297 in the form of well-known bibranched G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unengineered CHO cells typically have a glycan fucose content of about at least 85%. Removing core fucose from bibranched complex-type oligosaccharides attached to the Fc region enhances the ADCC of antibodies via improved FcγRIIIa binding without changing antigen binding or CDC activity. Such antibodies can be achieved using different methods reported for the successful expression of relatively highly defucosylated antibodies with bibranched complex-type Fc oligosaccharides, such as controlling the culture osmotic pressure (Konno et al., Cytotechnology 64:249-65, 2012), using the variant CHO cell line Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), using the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID: 20562582), using the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), introducing small interfering RNA specific for the α-1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or a potent α-mannosidase I inhibitor, kifnosine (Ferrara et al., J Biol Chem 281:5032-5036, 2006; Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng, 99:652-65, 2008).

[0102] In some embodiments described herein, ADCC caused by anti-Vβ17 / anti-CD123 bispecific antibodies can also be enhanced by some replacements in antibody Fc. Exemplary replacements include, for example, replacements at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residues are numbered according to the EU index), as described in U.S. Patent No. 6,737,056.

[0103] According to another specific aspect, the present invention relates to an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof, which is capable of inducing T cell-dependent cytotoxicity in cells expressing Vβ17 and / or cells expressing CD123. The bispecific antibody or antigen-binding fragment thereof can, for example, be used in vitro in cells expressing Vβ17 and / or cells expressing CD123 with an EC of less than about 2 nM. 50 In some embodiments, EC 50 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 about 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, 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 nM, less than about 0.2 nM and less than about 0.1 nM.

[0104] According to another specific aspect, the present invention relates to an isolated anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, wherein the anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof is chimeric.

[0105] According to another specific aspect, the present invention relates to an isolated anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, wherein the anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof is human or humanized.

[0106] In another general aspect, the present invention relates to an isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof. The isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof comprises an amino acid sequence having at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 28.

[0107] In another general aspect, the present invention relates to isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof disclosed herein. In another general aspect, the present invention relates to isolated nucleic acids encoding bispecific antibodies or antigen-binding fragments thereof disclosed herein. It will be appreciated by those skilled in the art that the coding sequence of a protein may be altered (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, it will be appreciated by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibodies and / or bispecific antibodies disclosed herein may be altered without changing the amino acid sequence of the protein.

[0108] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid disclosed herein. According to the present disclosure, any vector known to those skilled in the art, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element of the conventional function of establishing an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce antibodies or their antigen-binding fragments in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to the embodiments disclosed herein. According to the present disclosure, such techniques are well known to those skilled in the art.

[0109] In another general aspect, the present invention relates to a host cell comprising a nucleic acid encoding a monoclonal antibody and / or a bispecific antibody disclosed herein or an antigen-binding fragment thereof. In view of the present disclosure, any host cell known to those skilled in the art can be used for recombinant expression of an antibody disclosed herein or an antigen-binding fragment thereof. In some embodiments, the host cell is an Escherichia coli TG1 or BL21 cell (for expressing, for example, scFv or Fab antibodies), a CHO-DG44 or CHO-K1 cell or a HEK293 cell (for expressing, for example, a full-length IgG antibody). According to a specific embodiment, a recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein the recombinant expression vector is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0110] In another general aspect, the present invention relates to a method for producing a bispecific antibody or antigen-binding fragment thereof disclosed herein. The method includes culturing a cell comprising a nucleic acid encoding a bispecific antibody or antigen-binding fragment thereof under conditions that produce a bispecific antibody or antigen-binding fragment thereof disclosed herein, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art and as described herein.

[0111] How to use

[0112] In another general aspect, the present invention relates to a method of targeting CD123 on the surface of a cancer cell, the method comprising exposing the cancer cell to an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof.

[0113] The functional activity of bispecific antibodies and antigen-binding fragments thereof bound to Vβ17 and / or CD123 can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies and antigen-binding fragments thereof bound to Vβ17 and / or CD123 include, but are not limited to, affinity and specificity determinations, including Biacore, ELISA, and OctetRed analysis; binding assays for detecting the binding of antibodies to CD123 on cancer cells by FACS; binding assays for detecting the binding of antibodies to Vβ17 on CD8+ T cells or CD4+ T cells. According to a specific embodiment, methods for characterizing antibodies and antigen-binding fragments thereof bound to Vβ17 and / or CD123 include those described below.

[0114] In another general aspect, the present invention relates to a method for directing CD8+ T cells or CD4+ T cells expressing Vβ17 to cancer cells. The method comprises contacting CD8+ T cells or CD4+ T cells expressing Vβ17 with an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, wherein the antibody or antibody fragment directs CD8+ T cells or CD4+ T cells expressing Vβ17 to cancer cells having CD123 on their surface.

[0115] In another general aspect, the present invention relates to a method for inhibiting the growth or proliferation of cancer cells. The method comprises contacting CD8+ T cells expressing Vβ17 with an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, wherein contacting the cancer cells with the antibody or antibody fragment inhibits the growth or proliferation of the cancer cells.

[0116] According to an embodiment of the present invention, the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment can be provided in a buffer composition for storage or use. Suitable buffer for storing the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment will play a role in maintaining the stability of the antibody or antibody fragment by minimizing the deterioration during storage, not promoting the aggregation of the antibody or antibody fragment, or minimizing the adhesion to the storage container.

[0117] Implementation

[0118] The present invention provides the following non-limiting embodiments.

[0119] Embodiment 1 is an isolated Vβ17 bispecific antibody or an antigen-binding fragment thereof, wherein the isolated Vβ17 bispecific antibody or an antigen-binding fragment thereof comprises:

[0120] a. First heavy chain (HC1);

[0121] b. Second heavy chain (HC2);

[0122] c. the first light chain (LC1); and

[0123] d. a second light chain (LC2), wherein HC1 associates with LC1 and HC2 associates with LC2, and wherein HC1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, wherein the heavy chain complementary determining regions comprise the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LC1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the light chain complementary determining regions comprise the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, to form a binding site for a first antigen, and wherein HC2 and LC2 form a binding site for a second antigen.

[0124] Embodiment 2 is the Vβ17 bispecific antibody or antigen-binding fragment thereof according to embodiment 1, wherein the binding site of the first antigen binds to Vβ17 on CD8+ T cells or CD4+ T cells.

[0125] Embodiment 3 is the Vβ17 bispecific antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the binding site of the second antigen binds to a tumor antigen present on the surface of a cancer cell.

[0126] Embodiment 4 is the Vβ17 bispecific antibody or antigen-binding fragment according to any one of embodiments 1 to 3, wherein HC1 and LC1 are humanized.

[0127] Embodiment 5 is the Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein HC2 and LC2 bind to CD123.

[0128] Embodiment 6 is the Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the bispecific antibody or antigen-binding fragment thereof is of IgG isotype.

[0129] Embodiment 7 is the Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the bispecific antibody or antigen-binding fragment thereof is of the IgG4 isotype.

[0130] Embodiment 8 is a Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 7, wherein the bispecific antibody or antigen-binding fragment thereof has an EC of less than about 0.2 pM in vitro. 50 Induces CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells.

[0131] Embodiment 9 is an isolated nucleic acid encoding HC1 and LC1 of the Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8.

[0132] Embodiment 10 is an isolated nucleic acid encoding HC2 and LC2 of the Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8.

[0133] Embodiment 11 is a vector comprising the isolated nucleic acid according to embodiment 9 or embodiment 10.

[0134] Embodiment 12 is a host cell comprising the vector according to embodiment 11.

[0135] Embodiment 13 is a buffer composition comprising the isolated Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 8, and a buffer solution.

[0136] Embodiment 14 is an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof, comprising:

[0137] a. First heavy chain (HC1);

[0138] b. Second heavy chain (HC2)

[0139] c. the first light chain (LC1); and

[0140] d. a second light chain (LC2), wherein HC1 associates with LC1 and HC2 associates with LC2, and wherein HC1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, the heavy chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LC1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, the light chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, to form a binding site that specifically binds to the first antigen of Vβ17, and wherein HC2 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, the heavy chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively, and LC2 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, the light chain complementary determining regions comprising the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:40, SEQ ID NO:40 NO:38 and the amino acid sequence of SEQ ID NO:39 to form a binding site for a second antigen that specifically binds to CD123.

[0141] Embodiment 15 is an isolated anti-Vb17 / anti-CD123 bispecific antibody or antigen-binding fragment according to embodiment 14, wherein HC1 comprises the amino acid sequence of SEQ ID NO: 13 and LC1 comprises the amino acid sequence of SEQ ID NO: 14, and wherein HC2 comprises the amino acid sequence of SEQ ID NO: 15 and LC2 comprises the amino acid sequence of SEQ ID NO: 16.

[0142] Embodiment 16 is an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to embodiment 14 or embodiment 15, wherein the Vβ17 is located on the surface of CD8+ T cells or CD4+ T cells.

[0143] Embodiment 17 is an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 14 to 16, wherein the CD123 is located on the surface of a cancer cell.

[0144] Embodiment 18 is an isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 14 to 17, wherein the bispecific antibody or antigen-binding fragment thereof has an EC of less than about 0.2 pM in vitro.50 Induces CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells.

[0145] Embodiment 19 is an isolated nucleic acid encoding the HC1 and the LC1 of the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 14 to 18.

[0146] Embodiment 20 is an isolated nucleic acid encoding the HC2 and the LC2 of the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 14 to 18.

[0147] Embodiment 21 is a vector comprising the isolated nucleic acid according to embodiment 19 or embodiment 20.

[0148] Embodiment 22 is a host cell comprising the vector according to embodiment 21.

[0149] Embodiment 23 is a buffer composition comprising the isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 14 to 18, and a buffer solution.

[0150] Embodiment 24 is a method for guiding CD8+ T cells or CD4+ T cells expressing Vβ17 to cancer cells, the method comprising contacting CD8+ T cells or CD4+ T cells expressing Vβ17 with the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment according to any one of Embodiments 1 to 8 or 14 to 18, wherein contacting the CD8+ T cells or CD4+ T cells expressing Vβ17 with the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment guides the CD8+ T cells or CD4+ T cells expressing Vβ17 to cancer cells having CD123 on their surface.

[0151] Embodiment 24(a) is a method according to Embodiment 24, wherein the CD8+ T cells or CD4+ T cells expressing Vβ17 are contacted with the anti-Vβ17 / anti-CD123 bispecific antibody according to any one of Embodiments 1 to 8 or 14 to 18.

[0152] Embodiment 24(b) is a method according to Embodiment 24, wherein the CD8+ T cells or CD4+ T cells expressing Vβ17 are contacted with the anti-Vβ17 / anti-CD123 bispecific antibody fragment according to any one of Embodiments 1 to 8 or 14 to 18.

[0153] Embodiment 25 is a method for inhibiting the growth or proliferation of cancer cells expressing CD123 on their surface, the method comprising contacting the cancer cells with the anti-Vβ17 / anti-CD123 bispecific antibody or fragment thereof according to any one of Embodiments 1 to 8 or 14 to 18, wherein contacting the cancer cells with the antibody or antibody fragment inhibits the growth or proliferation of the cancer cells.

[0154] Embodiment 25(a) is a method according to Embodiment 25, wherein the cancer cells expressing CD123 are contacted with an anti-Vβ17 / anti-CD123 bispecific antibody or a fragment thereof simultaneously in the presence of CD8+ T cells expressing Vβ17.

[0155] Embodiment 25(b) is a method according to Embodiment 25 or 25(a), wherein the CD123-expressing cancer cells are contacted with the anti-Vβ17 / anti-CD123 bispecific antibody according to any one of Embodiments 1 to 8 or 14 to 18.

[0156] Embodiment 25(c) is a method according to Embodiment 25 or 25(a), wherein the CD123-expressing cancer cells are contacted with the anti-Vβ17 / anti-CD123 bispecific antibody fragment according to any one of Embodiments 1 to 8 or 14 to 18.

[0157] Embodiment 26 is a kit comprising the Vβ17 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 8 and packaging thereof.

[0158] Embodiment 27 is a kit comprising the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 14 to 18 and packaging thereof.

[0159] Embodiment 28 is a method for producing a Vβ17 bispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to embodiment 12 under conditions where the Vβ17 bispecific antibody or an antigen-binding fragment thereof is produced, and recovering the Vβ17 bispecific antibody or an antigen-binding fragment thereof from the cell or culture.

[0160] Embodiment 29 is a method for producing the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment according to any one of embodiments 14 to 18, the method comprising culturing the host cell according to embodiment 22 under conditions where the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment is produced, and recovering the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment from the cell or culture.

[0161] Embodiment 30 is an isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof, wherein the Vβ17 monoclonal antibody or the antigen-binding fragment thereof comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:28.

[0162] Embodiment 31 is an isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof according to embodiment 30, wherein the Vβ17 monoclonal antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:28.

[0163] Embodiment 32 is an isolated nucleic acid encoding the humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof according to embodiment 30 or embodiment 31.

[0164] Embodiment 33 is a vector comprising the isolated nucleic acid according to embodiment 32.

[0165] Embodiment 34 is a host cell comprising the vector according to embodiment 33.

[0166] Embodiment 35 is a buffer composition comprising the isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof according to embodiment 30 or embodiment 31.

[0167] Example

[0168] The following examples are based on the premise that the peptide M1 derived from influenza virus is able to expand a group of selected T cells. These cells express the TCR-haplotype-Vβ17, and the majority of these cells show potent cytotoxicity against tumor target cells. This ability is then exploited using a bispecific antibody constructed such that one arm binds to the Vβ17 structure and the other arm binds to an antigen expressed by cancer cells. Thus, the bispecific antibody bridges effector and target cells together, resulting in cancer cell killing. This mechanism of action is Figure 1 The schematic diagram outlined in FIG.

[0169] The examples that follow can be divided into the following categories: (1) generation of bispecific antibodies capable of binding to the Vβ17 arm of the T cell receptor (TCR) on CTLs (Examples 1 and 2); and (2) evidence of bispecific antibody-initiated target cell killing by in vitro expanded CTLs (Example 3).

[0170] Example 1: Human framework adaptation of anti-Vβ17 mAb E17.5F

[0171] Mouse IgG1 anti-human T cell receptor Vβ17 clone E17.5F was obtained from BeckmanCoulter, Inc. (Brea, CA). Sample preparation and LC / MSMS analysis were performed at Protea Bioscience Inc. (Morgantown, WV). The sample was reduced and alkylated, divided into seven aliquots, and proteolytic digestion was performed with trypsin / LysC, chymotrypsin, LysC, pepsin and AspN, elastase and proteinase K enzymes. The resulting peptides were desalted using the ZipTip C18 pipette tip and separated online using reverse phase chromatography. Mass spectrometry was performed using HCD fragmentation on a Thermo Q-Exactive mass spectrometer. PEAKS software was used to analyze the MS data set by matching the de novo sequence tags with the IMGT-based antibody sequence database. The gaps in the sequence were specified using the overlapping group sequence assembly of the peptides identified de novo. All CDRs and hypermutations were confirmed by checking the MS / MS spectra.

[0172] The obtained sequences are shown in Tables 1 and 2.

[0173] Table 1: CDR sequences of TCR Vβ17 clone E17.5F

[0174]

[0175] Table 2: Heavy and light chain sequences of TCR Vb17 clone E17.5F

[0176]

[0177] Changes were made to the sequence to prepare the bispecific antibody (Table 3). The changes included the following: (1) the framework mutation Asn1 of the heavy chain was not conserved, so the sequence was modified to have a DVQLW sequence; (2) another mutation K337Y identified in the Fc was considered uncharacteristic, so a construct without this mutation was synthesized; and (3) a potential secondary glycosylation site was observed on the heavy chain, so two versions of this mAb were synthesized with and without the N-linked site (N82a, based on Chothia numbering).

[0178] Table 3: Heavy and light chain sequences of Vβ17 clone E17.5F antibody variant

[0179]

[0180] The two antibodies (B17B1 and B17B2) were expressed in HEK293Expi cells. The supernatants were tested for Vβ17 binding (B17B1 and B17B2) and only B17B1 showed binding. Therefore, B17B1 was expressed with an IgG4 constant region with an Fc substitution.

[0181] The anti-human TCR Vβ17 mouse mAb B17B1 was humanized using the human framework adaptation (HFA) method (Fransson J et al., J. Mol. Biol. 2010; 398: 214-231). In order to find the best combination of humanized heavy and light chains, several human V region sequences were selected for testing (Table 4). The selection of human germline was based only on the overall sequence similarity with mouse antibodies in the framework (FR) region. Neither CDR sequences nor their length or canonical structure were considered in this selection.

[0182] The CDR definitions used in HFA are described in (Fransson J et al., J. Mol. Biol. 2010; 398: 214-231) and correspond to the definition of Martin (Abhinandan KR and Martin AC., Mol. Immunol. 2008; 45: 3832-3839). The CDRs (Table 1) are defined as follows (using the Chothia numbering scheme [Chothia C and Lesk A. J. Mol. Biol. 1987; 196: 901-917]):

[0183] HCDR1 (SEQ ID NO: 1) 26-35

[0184] HCDR2 (SEQ ID NO:2) 50-58

[0185] HCDR3 (SEQ ID NO:3) 95-102

[0186] LCDR1 (SEQ ID NO:4) 24-34

[0187] LCDR2 (SEQ ID NO: 5) 50-56

[0188] LCDR3 (SEQ ID NO:6) 89-97

[0189] Selected human germlines are provided in Table 4 (in IMGT notation).

[0190] Table 4: VH variants and VL variants

[0191]

[0192]

[0193] CDRs 1 to 3 are underlined

[0194] "Back mutations" in several variants were introduced at FR positions known to be important for VL / VH pairing and CDR conformation. The selected human germlines are provided in Table 5 (in IMGT notation), where the back mutations are noted.

[0195] Table 5: Selected J regions

[0196]

[0197] The amino acid sequences of all nine paired combinations of three heavy chains and three light chains are reverse translated into DNA, and cDNA is prepared using gene synthesis technology (U.S. Patent No. 6,670,127, U.S. Patent No. 6,521,427). Using standard molecular biological techniques, the heavy chain (HC) variable region is subcloned onto the human IgG4 constant region using an internal expression vector with a CMV promoter. Using standard molecular biological techniques, the light chain (LC) variable region is subcloned onto the human lambda constant region using an internal expression vector with a CMV promoter. The resulting plasmid is transfected into HEK EXPI cells (Life Technologies; Carlsbad, CA), and mAb is expressed. Purification is performed by the standard method using a protein A column (hiTrap MAb Select SuRe column). After elution, the harvest is dialyzed into D-PBS (pH 7.2).

[0198] Table 6: Heavy and light chains of nine humanized Vβ17 antibodies

[0199]

[0200] The binding of humanized antibodies to TCRVβ17 (SEQID NO: 27) / Va10.2-Fc (SEQ ID NO: 44) fusion protein was screened by ELISA. The biotinylated TCRVβ17 / Va10.2-Fc fusion protein was added to a streptavidin-coated ELISA plate. Unbound protein was washed away, and mAb was added at a range of concentrations (0.01 μg / mL to 10 μg / mL). The plate was washed and anti-κ: HRP detection antibody was added. The plate was washed, chemiluminescent detection reagent was added, and the luminescence of the plate was read on a Perkin Elmer EnVision plate reader. B17B20 and B17B21 showed positive binding to the TCR-Vβ17 protein. B17B22 showed weak binding to the protein. These antibodies were then purified as described above for further study. B17B21 demonstrated the best binding to recombinant TCR-Vβ17 protein and M1-stimulated T cells and was therefore selected as the molecule for further functional studies, especially as a bispecific antibody for T cell redirected killing of cancer cells.

[0201] Therefore, the variable region sequences of B17B21 (anti-Vβ17) and I3RB217 (anti-CD123 antibody) were used to generate a bispecific antibody that was tested against T cell redirected killing of acute myeloid leukemia (AML) cells.

[0202] Example 2. Preparation of anti-Vβ17 / anti-CD123 bispecific antibodies

[0203] VB11 (anti-Vβ17 / anti-CD123) and VB13 (Vβ17×Null) bispecific antibodies were produced as full-length antibodies in the form of a knob-in-hole structure to obtain human IgG4, as previously described (Atwell et al., J. Mol. Biol. 270:26-35, 1997). The nucleic acid sequence encoding the variable region was subcloned into a custom mammalian expression vector containing the constant region of the IgG4 expression cassette using standard PCR restriction enzymes based on cloning technology. The bispecific antibodies were expressed by transient transfection in a Chinese hamster ovary cell line. The antibodies were initially purified by a Mab Select SuRe protein A column (GE healthcare, Piscataway, New Jersey) (Brown, Bottomley et al., 1998). The column was balanced with phosphate buffered saline (PBS) at pH 7.2, and the fermentation supernatant was loaded at a flow rate of 2 mL / min. After loading, the column was washed with PBS (4CV) and then eluted in 30 mM sodium acetate at pH 3.5. The fractions containing the protein peak were combined and neutralized to pH 5.0 by adding 1% 3M sodium acetate (pH 9.0), and the protein peak was monitored by the absorbance at 280nm in Akta Explorer (GE healthcare). As a refining step, the antibody was purified using a Superdex 200 column (GE healthcare) on a preparative size exclusion chromatography (SEC). The integrity of the sample was assessed under reducing and non-reducing conditions by endotoxin measurement and SDS polyacrylamide gel electrophoresis. The final protein concentration of anti-Vβ17 / anti-CD123 was 0.48mg / ml, and the final protein concentration of Vβ17×Null was 0.24mg / mL. Based on these protein concentrations, the final EU levels of anti-Vβ17 / anti-CD123 and Vβ17×Null were 2.053EU / mg and 4.219EU / mg, respectively.

[0204] Table 7: Sequences of half antibodies expressed in CHO cells

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] Table 8: Heavy chain sequences and light chain sequences of Vβ17 bispecific antibodies

[0212]

[0213]

[0214] Example 3. Use of Kasumi-3 cells and human CD8 + T cell evaluation of anti-Vβ17 / anti-CD123 bispecific antibody Binding and cytotoxic properties

[0215] Stimulation and expansion of Vβ17 from total PBMCs + CD8 + T cells

[0216] To amplify Vβ17 + CD8 + T cells, whole PBMCs from an HLA-A2 donor (HPU-08694) were stimulated with 1 μg / mL influenza MP 58 peptide in DMSO. Total CD8 + Vβ17 in T cells + In order to determine the frequency of CD8 + Vβ17 in T cells + The frequency of cells was counted by first gating the total live PBMCs to exclude doublets and then gating the total CD8 + T cells were gated and then Vβ17 + Cells were gated ( Figure 3 A). Total CD8 + Vβ17 in T cells + Compared with the frequency of cells, a significant expansion of these cells was observed on day 8 of the culture period ( Figure 3 B) On day 8, CD8 + The larger fraction of T cells is Vβ17 in this donor + cell( Figure 3 B).

[0217] Anti-Vβ17 / anti-CD123 bispecific antibody binding assay

[0218] About Kasumi-3 Cells

[0219] In order to understand the binding kinetics of anti-Vβ17 / anti-CD123 bispecific antibodies, Kasumi-3 cells were incubated with various concentrations (ranging from 5 μg / mL to 0 μg / mL) of anti-Vβ17 / anti-CD123 bispecific antibodies and Vβ17XNULL arm controls. The bispecific antibodies bound to the cells were detected with mouse anti-human IgG4 Fc-PE secondary antibodies. Table 9 shows the frequency of Kasumi-3 cells positive for PE (secondary antibody) when treated with different concentrations of bispecific antibodies. EC values ​​of anti-Vβ17 / anti-CD123 and NULLXCD123 50They were determined to be 6 nM and 42.7 nM, respectively (Table 9).

[0220] Table 9: Binding affinity of bispecific antibodies to Kasumi-3 cells

[0221]

[0222] The binding affinity of the bispecific antibody to Kasumi-3 cells was determined by flow cytometry. The half-maximal effective concentration (EC 50 ) values ​​were calculated as the bispecific concentration that yielded 50% maximal binding (PE-positive cells). ND: Not determined.

[0223] About enriched CD8 + T cells

[0224] The enriched CD8 + T cells (from day 14 culture) were incubated with various concentrations of anti-Vβ17 / anti-CD123 bispecific antibodies and Vβ17XNULL arm control antibodies. Mouse anti-human IgG4 Fc-PE secondary antibodies were used to detect the bispecific antibodies. Table 10 shows the CD8 positive for PE (secondary antibody) when treated with different concentrations of bispecific antibodies. + Frequency of T cells. ECs with anti-Vβ17 / anti-CD123 and Vβ17XNULL 50 They were determined to be 9.0 nM and 18.7 nM, respectively (Table 10).

[0225] Table 10: Bispecific antibodies against CD8 + T cell binding activity

[0226]

[0227] Flow cytometry analysis of the bispecific antibodies against CD8 + Binding affinity of T cells. Half maximal effective concentration (EC 50 ) values ​​were calculated as the antibody concentration that produced 50% maximal binding (PE-positive cells). ND: Not determined

[0228] Bispecific-mediated cytotoxicity assay

[0229] To analyze the efficacy of anti-Vβ17 / anti-CD123 bispecific antibody-mediated cytotoxicity, CFSE-labeled target (Kasumi-3) cells were incubated with stimulated CD8 +T cells (effectors) were co-cultured with various concentrations of anti-Vβ17 / anti-CD123 bispecific antibodies and Vβ17XNULL arm control antibodies at effector to target (E:T) ratios of 0.5:1, 1:1, and 5:1 for 14 and 24 hours. CD123 expression on target Kasumi-3 cells was examined by using commercially available anti-CD123 antibodies. Target cells (Kasumi-3) were labeled with CFSE so that they could be identified as CFSE during flow cytometry analysis. + After the co-culture period, 7-AAD was added to analyze 7-AAD + CFSE + The percentage of cells that were expressed as a measure of cytotoxicity was subtracted from the basal cytotoxicity observed in the absence of the bispecific antibody to obtain the specific cytotoxicity in response to the bispecific antibody. One assay was performed with a single donor (HPU-08694). The EC values ​​of the anti-Vβ17 / anti-CD123 bispecific antibody at the 14-hour time point were 0.5:1, 1:1, and 5:1 ET ratios. 50 They were 3.7 pM, 0.1 pM and 0.133 pM respectively (Table 11).

[0230] Table 11: CD8 stimulated by influenza MP 58 peptide + T cells and Kasumi-3 cells were mixed at a ratio of 0.5:1, 1:1, and 5:1 After 14 hours of co-culture, the EC of various bispecific antibodies 50 Aggregation of values .

[0231]

[0232]

[0233] UD: Not detectable because activity was too low for correct curve fitting.

[0234] EC values ​​of the anti-Vβ17 / anti-CD123 bispecific antibody at 24 h time point were calculated using ET ratios of 0.5:1, 1:1, and 5:1. 50 They were 0.4 pM, 0.2 pM and 1.0 pM respectively (Table 12).

[0235] Table 12: CD8 stimulated by influenza MP 58 peptide + T cells and Kasumi-3 cells were mixed at a ratio of 0.5:1, 1:1, and 5:1 After 24 hours of co-culture, the EC of various bispecific antibodies 50 Aggregation of values .

[0236]

[0237]

[0238]

[0239] UD: Undetectable

[0240] Similarly, the anti-Vβ17 / anti-CD123 bispecific mediated activation of unstimulated CD8 + T cell cytotoxicity. Unstimulated CD8 T cells were isolated at 5 ng / ml of anti-Vβ17 / anti-CD123 bispecific concentration and 14 hours time point using ET ratios of 0.5:1 and 1:1. + T cells showed 2.8% and 9.8% target cell cytotoxicity, respectively (Table 13). In contrast, stimulated CD8 + T cells showed 77% and 73% cytotoxicity. + T cells showed 31.65% target cell cytotoxicity, compared to stimulated CD8 + T cells showed 70.9% target cell toxicity. Similar results were obtained from the 24 hour time point (Tables 12, 15, 16 and 17). At the highest concentration tested (5 ng / ml) of anti-Vβ17 / anti-CD123 bispecific, unstimulated CD8 + T cells showed higher cytotoxicity against target cells when using higher ET ratios.

[0241] Table 13: Unstimulated CD8 + T cells were cultured for 14 hours for cytotoxicity assay. The frequencies of CFSE-positive cells and 7-AAD-positive cells when treated with different concentrations of bispecific antibodies .

[0242]

[0243] Table 14: Unstimulated CD8 + T cells were cultured for 24 hours to determine cytotoxicity. The frequencies of CFSE-positive cells and 7-AAD-positive cells when treated with different concentrations of bispecific antibodies .

[0244]

[0245] Table 15: In the case of 0.5:1 ET ratio (stimulated CD8 + T cells: Kasumi-3 cells) were incubated for 14 hours. When treated with different concentrations of bispecific antibodies at a 0.5:1 ET ratio for 14 h, CFSE-positive The frequency of cells and 7-AAD positive cells .

[0246]

[0247] The half-maximal effective concentration (EC 50 ) value was calculated as the value that produced 50% maximal cytotoxicity (CFSE + 7AAD + ) Antibody concentration in cells. ND: Not determined.

[0248] Table 16: In the case of 1:1 ET ratio (stimulated CD8 + T cells: Kasumi-3 cells) were incubated for 14 hours. When treated with different concentrations of bispecific antibodies at a 1:1 ET ratio for 14 h, CFSE-positive cells The frequency of cells and 7-AAD positive cells .

[0249]

[0250]

[0251] The half-maximal effective concentration (EC 50 ) value was calculated as the value that produced 50% maximal cytotoxicity (CFSE + 7AAD + ) Antibody concentration in cells. ND: Not determined

[0252] Table 17: In the case of 5:1 E:T ratio (stimulated CD8 + T cells: Kasumi-3 cells) were incubated for 14 hours. When treated with different concentrations of bispecific antibodies at a 5:1 ET ratio for 14 h, CFSE-positive cells The frequency of cells and 7-AAD positive cells .

[0253]

[0254] The half-maximal effective concentration (EC 50 ) value was calculated as the value that produced 50% maximal cytotoxicity (CFSE + 7AAD + ) Antibody concentration in cells. UD: Undetectable.

[0255] It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the broad inventive concept of the present invention. It will therefore be appreciated that the present invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention, as defined in this specification.

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

Claims

1. An isolated anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, comprising: a. First heavy chain (HC1); b. Second heavy chain (HC2); c. the first light chain (LC1); and d. Second light chain (LC2), wherein HC1 associates with LC1 and HC2 associates with LC2, and wherein HC1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, the heavy chain complementary determining regions being represented by the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LC1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, the light chain complementary determining regions being represented by the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, to form a binding site that specifically binds to a first antigen of Vβ17, and wherein HC2 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, the heavy chain complementary determining regions being represented by the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively, and LC2 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, the light chain complementary determining regions being represented by the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, respectively. The amino acid sequence of NO:39 is represented to form a binding site for a second antigen that specifically binds to CD123.

2. The isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein HC1 comprises the amino acid sequence of SEQ ID NO: 13 and LC1 comprises the amino acid sequence of SEQ ID NO: 14, and wherein HC2 comprises the amino acid sequence of SEQ ID NO: 15 and LC2 comprises the amino acid sequence of SEQ ID NO:

16.

3. The isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the Vβ17 is located on the surface of CD8+ T cells or CD4+ T cells. 4 . The isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to claim 1 , wherein the CD123 is located on the surface of cancer cells or CD34+ stem cells.

5. The isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the bispecific antibody or antigen-binding fragment thereof has an EC of less than 0.2 pM in vitro. 50 Induces CD8+ or CD4+ T cell-dependent cytotoxicity of cancer cells.

6. An isolated nucleic acid encoding the HC1 and the LC1 of the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. An isolated nucleic acid encoding the HC2 and the LC2 of the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. A vector comprising the isolated nucleic acid according to claim 6 or 7.

9. A host cell comprising the vector according to claim 8. 10 . A buffer composition comprising the isolated anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 .

11. Use of the anti-Vβ17 / anti-CD123 bispecific antibody or its antigen-binding fragment according to claim 1 in the preparation of a medicament for guiding CD8+T cells or CD4+T cells expressing Vβ17 to cancer cells, wherein CD123 is expressed on the surface of the cancer cells. 12 . Use of the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to claim 1 in the preparation of a medicament for inhibiting the growth or proliferation of cancer cells, wherein CD123 is expressed on the surface of the cancer cells. 13 . A kit comprising the anti-Vβ17 / anti-CD123 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and packaging thereof.

14. A method for producing an anti-Vβ17 / anti-CD123 bispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 9 to produce the anti-Vβ17 / anti-CD123 bispecific antibody or the antigen-binding fragment thereof, and recovering the anti-Vβ17 / anti-CD123 bispecific antibody or the antigen-binding fragment thereof from the cell or culture.

15. An isolated humanized Vβ17 monoclonal antibody or an antigen-binding fragment thereof, wherein the humanized Vβ17 monoclonal antibody or the antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:

28. 16 . An isolated nucleic acid encoding the isolated humanized Vβ17 monoclonal antibody or antigen-binding fragment thereof according to claim 15 .

17. A vector comprising the isolated nucleic acid according to claim 16.

18. A host cell comprising the vector according to claim 17.

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