Bispecific antibodies and uses thereof

Abstract

The invention belongs to the field of biological medicine, particularly relates to a recombinant antibody and application thereof, and provides a CD16-B7H7 bispecific antibody which at least comprises a first antigen binding unit for specifically recognizing an antibody or an antigen binding fragment of CD16 and a second antigen binding unit for specifically recognizing an antibody or an antigen binding fragment of B7H7. The bispecific antibody prepared by the invention can target CD16 and B7H7 at the same time so as to mediate killing of NK cells on tumor cells, and has relatively strong tumor inhibition capability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a CD16-B7H7 bispecific antibody and its application. Background Art

[0002] B7H7, also known as HHLA2, is a type I transmembrane protein, consisting of three extracellular Ig-like domains, a transmembrane region, and a cytoplasmic tail of 49 amino acids with no recognizable motif. B7H7 belongs to one of the members of the B7 family and has about 20%-30% similarity with other members of the B7 family. In normal tissues, B7-H7 is mainly expressed in tissues such as the gastrointestinal tract and bile duct, and is not expressed in other normal tissues. However, in many tumor types, such as breast cancer, lung cancer, colorectal cancer, pancreatic cancer, etc., B7H7 shows a high-expression state and is negatively correlated with the prognosis of patients. However, the currently publicly available data shows that only Hutchison MediPharma's HBM1020 monoclonal antibody drug is in the clinical phase I, and there is no bispecific or multispecific antibody targeting B7H7 entering clinical research.

[0003] Natural killer cells (NK cells) are important members of the innate immune system. Different from T cells, NK cells do not express antigen-specific receptors. NK cells themselves have broad-spectrum tumor-killing ability and also play an important role in enhancing antibody and T cell responses. Currently, there are various forms of tumor immunotherapy based on NK cells, and the means used are also different. The reduction in the number or impairment of the function of NK cells is related to the progression of various types of cancers. "Cold tumors" are immune-insensitive, with very few or no MHC class I molecules expressed on their surface, so they are hardly recognized by T cells, but can be recognized and killed by NK cells. The introduction of this concept has improved the status of NK cells in anti-tumor immunotherapy.

[0004] CD16 molecule is an important marker on the surface of NK cells, which can activate the immunoreceptor tyrosine activation motif (ITAM) of the IgE NK cell receptor (FcεRIγ) and CD3ζ to exert ADCC effect. Therefore, the CD16 target is preferentially selected in bispecific antibodies based on NK cell re-direction.

[0005] Bispecific antibodies are antibodies that can specifically bind to two antigen sites simultaneously. Bispecific antibodies used for tumor treatment can be classified into three categories according to their mechanism of action: re-directing effector cells; immune regulation; dual binding to target tumor cell receptors. Among them, antibodies used for the re-directing function account for the majority, and many currently marketed bispecific antibodies for tumor treatment are also based on cell re-direction.

[0006] Based on these understandings, the present invention designs a novel recombinant bispecific fusion protein, which can specifically bind to CD16 and specifically bind to B7H7. This fusion protein can targetedly bring B7H7 molecules near tumor cells, and by binding to B7H7 molecules on tumor cells, it promotes the formation of immune synapses between NK cells and tumor cells, thereby activating NK cells to kill B7H7+ tumor cells, providing new inspiration and ideas for antibody therapy of tumor immunity. Summary of the Invention

[0007] This application is made based on the inventor's discovery and understanding of the following facts and problems:

[0008] In the first aspect of the present invention, a recombinant antibody is provided, comprising:

[0009] The first binding region: The first binding region comprises an antibody or antigen-binding fragment that specifically recognizes CD16;

[0010] The second binding region: The second binding region comprises an antibody or antigen-binding fragment that specifically recognizes B7H7,

[0011] wherein,

[0012] The first binding region comprises the scFv region of a CD16 antibody and a first Fc region, and the second binding region comprises the Fab region of a B7H7 antibody and a second Fc region.

[0013] The present invention is based on a bispecific cell bridging method, which employs a bispecific binding protein having a binding arm that binds to CD16 on NK cells and a binding arm that binds to B7H7 on the cell surface of tumor cells. By promoting the simultaneous binding of NK cells and tumor cells, the bispecific protein promotes the formation of cell synapses between the two cells and thus selectively redirects NK cell activity towards targeted tumor cells.

[0014] According to a specific embodiment of the present invention, the first binding region comprises the CDR sequences of the variable regions of a CD16 antibody: the amino acid sequence of the light chain variable region CDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the light chain variable region CDR2 is as shown in SEQ ID NO:2, the amino acid sequence of the light chain variable region CDR3 is as shown in SEQ ID NO:3, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO:4, the amino acid sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO:6.

[0015] According to a specific embodiment of the present invention, the second binding region comprises the variable region CDR sequences of the B7H7 antibody: the amino acid sequence of the light chain variable region CDR1 is as shown in SEQ ID NO:7, the amino acid sequence of the light chain variable region CDR2 is as shown in SEQ ID NO:8, the amino acid sequence of the light chain variable region CDR3 is as shown in SEQ ID NO:9, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO:10, the amino acid sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO:12.

[0016] According to a specific embodiment of the present invention, the light chain variable region of the CD16 antibody has the amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:13, and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:14.

[0017] According to a specific embodiment of the present invention, the light chain variable region of the B7H7 antibody has the amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:15, and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:16.

[0018] According to a specific embodiment of the present invention, the C-terminus of the scFv region of the CD16 antibody in the first binding region is connected to the N-terminus of the first Fc region.

[0019] According to a specific embodiment of the present invention, the C-terminus of the light chain variable region in the scFv region of the CD16 antibody is connected to the N-terminus of the heavy chain variable region through a linker peptide 1, and the C-terminus of the heavy chain variable region is connected to the N-terminus of the first Fc region.

[0020] According to a specific embodiment of the present invention, the first Fc region and the second Fc region are connected through a knob-into-hole structure.

[0021] According to a specific embodiment of the present invention, the linker peptide 1 has the amino acid sequence as shown in SEQ ID NO:35.

[0022] According to a specific embodiment of the present invention, at least a part of at least one of the CH1 region of the Fab region of the B7H7 antibody, the first Fc region, and the second Fc region is derived from at least one of a murine antibody, a primate antibody, or a mutant thereof.

[0023] According to a specific embodiment of the present invention, at least a part of at least one of the CH1 region of the Fab region of the B7H7 antibody, the first Fc region, and the second Fc region is derived from human IgG1 or a mutant thereof.

[0024] According to a specific embodiment of the present invention, the first Fc region has the amino acid sequence shown in SEQ ID NO: 18, and the second Fc region has the amino acid sequence shown in SEQ ID NO: 19.

[0025] According to a specific embodiment of the present invention, the first binding region has the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO: 22, and the second binding region has the amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[0026] According to a specific embodiment of the present invention, the recombinant antibody further includes a signal peptide, which is connected to the N-terminus of the recombinant antibody, and the signal peptide has the amino acid sequence shown in SEQ ID NO: 34.

[0027] According to a specific embodiment of the present invention, the recombinant antibody further includes a third binding region, and the third binding region includes the Fab region of the B7H7 antibody.

[0028] According to a specific embodiment of the present invention, the C-terminus of the CH1 region of the Fab region of the B7H7 antibody in the third binding region is connected to the N-terminus of the scFv region of the CD16 antibody in the first binding region.

[0029] According to a specific embodiment of the present invention, the C-terminus of the CH1 region of the Fab region of the B7H7 antibody in the third binding region is connected to the N-terminus of the scFv region of the CD16 antibody through a linker peptide 2.

[0030] According to a specific embodiment of the present invention, the linker peptide 2 has the amino acid sequence shown in SEQ ID NO: 36.

[0031] According to a specific embodiment of the present invention, the amino acid sequence of the Fab region of the B7H7 antibody in the third binding region is the same as or different from the amino acid sequence of the Fab region of the B7H7 antibody in the second binding region.

[0032] According to a specific embodiment of the present invention, the third binding region has the amino acid sequences shown in SEQ ID NO: 30 and SEQ ID NO: 37 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO: 30 or SEQ ID NO: 37.

[0033] The second aspect of the present invention provides an isolated polynucleotide encoding the recombinant antibody described above.

[0034] The third aspect of the present invention provides an expression vector carrying the polynucleotide described in the second aspect.

[0035] The fourth aspect of the present invention provides a method for preparing the recombinant antibody described in the first aspect, comprising: introducing the expression vector described in the third aspect into a cell.

[0036] According to a specific embodiment of the present invention, the cell is cultured under conditions suitable for protein expression and secretion to obtain the recombinant antibody.

[0037] According to a specific embodiment of the present invention, the cell is a eukaryotic cell.

[0038] The fifth aspect of the present invention provides a recombinant cell carrying the polynucleotide described in the second aspect or the expression vector described in the third aspect.

[0039] The sixth aspect of the present invention provides a composition comprising:

[0040] at least one of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect.

[0041] Use of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for treating or preventing cancer, wherein the cancer cells have positive B7H7 on their surface.

[0042] According to a specific embodiment of the present invention, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, pancreatic cancer, oral squamous cell carcinoma, and head and neck cancer.

[0043] Use of the recombinant antibody according to the first aspect, the polynucleotide according to the second aspect, the expression vector according to the third aspect, the recombinant cell according to the fifth aspect, and the composition according to the sixth aspect in the preparation of a kit for detecting CD16 and / or B7H7.

[0044] The seventh aspect of the present invention provides a kit comprising the recombinant antibody according to the first aspect for detecting CD16 and / or B7H7. The recombinant antibody can bind to CD16 and / or B7H7 protein. Therefore, the kit containing the recombinant antibody can be used to effectively detect CD16 and / or B7H7. The kit can be used for scientific research, such as qualitatively or quantitatively detecting CD16 and / or B7H7 protein in biological samples, and can also be used to judge the individual status. For example, after obtaining the B7H7 level of the individual, it can be judged whether the level is too high or too low compared with the normal level.

[0045] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0047] Figure 1 Shows a schematic diagram of the bispecific antibody BB2-N16-V6 in Example 1 of the present invention;

[0048] Figure 2 Shows a schematic diagram of the bispecific antibody BB3-N16-V6 in Example 1 of the present invention;

[0049] Figure 3 Shows a diagram of the binding of the bispecific antibody to CD16 protein in Example 3 of the present invention;

[0050] Figure 4 Shows a diagram of the binding of the bispecific antibody to B7H7 protein in Example 3 of the present invention;

[0051] Figure 5 Shows a diagram of the simultaneous binding of the bispecific antibody to CD16 and B7H7 proteins in Example 3 of the present invention;

[0052] Figure 6 Shows a result diagram of the binding of the bispecific antibody to overexpressing non-small cell lung cancer HCC827 cells in Example 4 of the present invention;

[0053] Figure 7 Shows a result diagram of the binding of the bispecific antibody to human NK cells in Example 4 of the present invention;

[0054] Figure 8 Shows the in vitro cell killing experimental result graph of the bispecific binding protein and PBMC in Example 6 of the present invention against non-small cell lung cancer HCC827 cells;

[0055] Figure 9 Shows the in vitro cell killing experimental result graph of the bispecific binding protein and PBMC in Example 6 of the present invention against colorectal cancer SW620-B7H7 cells;

[0056] Figure 10 Shows the in vitro cell killing experimental result graph of the bispecific binding protein and PBMC in Example 6 of the present invention against CHO-K1 cells;

[0057] Figure 11 Shows the in vitro cytotoxic experimental result graph of the bispecific binding protein and NK cells against non-small cell lung cancer HCC827 cells in Example 6 of the present invention;

[0058] Figure 12 Shows the effect of the bispecific binding protein on the activation marker CD69 of NK cells in the absence of target cells in Example 7 of the present invention;

[0059] Figure 13 Shows the effect of the bispecific binding protein on the activation marker CD69 of NK cells in the presence of HCC827 target cells in Example 7 of the present invention. Detailed implementation manners

[0060] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0061] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0062] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0063] To facilitate the understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0064] As used herein, the term "comprising" or "including" is an open-ended expression, meaning including the content specified in the present invention, but not excluding other aspects.

[0065] As used herein, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the cases where the event or condition occurs, as well as the cases where the event or condition does not occur.

[0066] The antibodies or antigen-binding fragments described in the present invention are generally prepared by biosynthetic methods. According to the nucleotide sequences described in the present invention, those skilled in the art can conveniently obtain the encoding nucleic acids of the present invention by various known methods. These methods include, for example but not limited to: PCR, DNA artificial synthesis, etc. For specific methods, reference can be made to J. Sambrook, "Molecular Cloning: A Laboratory Manual". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by segmentally synthesizing the nucleotide sequence and then performing overlap extension PCR. Among them, the antibody or antigen fragment is numbered and defined using the Kabat numbering system. As used herein, the term "antibody" is an immunoglobulin molecule capable of binding specifically to an antigen. It includes two light chains with relatively low molecular weights and two heavy chains with relatively high molecular weights. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a four-peptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly and is called the variable region (V region), while the carboxyl-terminal (C-terminal) is relatively stable and varies little, and is called the constant region (C region). The V regions of the L chain and the H chain are respectively called VL and VH. In the variable region, certain regions have a higher degree of amino acid composition and arrangement variation, and are called hypervariable regions (HVR). The hypervariable region is the position where the antigen and the antibody bind, and is therefore also called the complementarity-determining region (CDR). There are three CDR regions on both the heavy chain variable region and the light chain variable region.

[0067] The antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

[0068] In this article, the "bispecific antibody" refers to the one obtained by connecting peptide chains that can specifically recognize different protein molecules to the two chains of the Fc region respectively, wherein the two chains of the Fc region are connected through the knob into hole structure.

[0069] In this article, the "knob into hole structure" refers to the formation of knob and hole mutations in the CH3 region of the heavy chain Fc of the antibody, which facilitates the heavy chain to bite and form a heterodimer.

[0070] In this article, the term "operably linked" refers to the connection of a foreign gene to a vector such that control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the foreign gene. When connecting the above nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself. Those skilled in the art can understand that nucleic acid molecules used to encode antibodies or antigen-binding fragments can be inserted into different vectors separately and independently, and commonly are inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc. For example, Plasmid-X plasmid.

[0071] As used herein, the term "identity" when describing an amino acid sequence or nucleic acid sequence relative to a reference sequence refers to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0072] Anti-CD16-B7H7 antibody or antigen-binding fragment

[0073] The present invention provides a recombinant antibody, comprising:

[0074] First binding region: The first binding region includes an antibody or antigen-binding fragment that specifically recognizes CD16;

[0075] Second binding region: The second binding region includes an antibody or antigen-binding fragment that specifically recognizes B7H7,

[0076] wherein,

[0077] the first binding region includes the scFv region of the CD16 antibody and the first Fc region, and the second binding region includes the Fab region of the B7H7 antibody and the second Fc region.

[0078] The present invention is based on a bispecific cell bridging method that employs a bispecific binding protein having a binding arm that binds to CD16 on NK cells and a binding arm that binds to B7H7 on the cell surface of tumor cells. By promoting the simultaneous binding of NK cells and tumor cells, the bispecific protein promotes the formation of a cell synapse between the two cells and thus selectively redirects NK cell activity against targeted tumor cells.

[0079] According to a specific embodiment of the present invention, the first binding region includes the variable region CDR sequences of the CD16 antibody: the amino acid sequence of the light chain variable region CDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the light chain variable region CDR2 is as shown in SEQ ID NO:2, the amino acid sequence of the light chain variable region CDR3 is as shown in SEQ ID NO:3, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO:4, the amino acid sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO:6.

[0080] According to a specific embodiment of the present invention, the second binding region includes the variable region CDR sequences of the B7H7 antibody: the amino acid sequence of the light chain variable region CDR1 is as shown in SEQ ID NO:7, the amino acid sequence of the light chain variable region CDR2 is as shown in SEQ ID NO:8, the amino acid sequence of the light chain variable region CDR3 is as shown in SEQ ID NO:9, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO:10, the amino acid sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO:12.

[0081] According to a specific embodiment of the present invention, the C-terminus of the scFv region of the CD16 antibody in the first binding region is connected to the N-terminus of the first Fc region.

[0082] According to a specific embodiment of the present invention, the C-terminus of the light chain variable region in the scFv region of the CD16 antibody is connected to the N-terminus of the heavy chain variable region through Linker 1, and the C-terminus of the heavy chain variable region is connected to the N-terminus of the first Fc region.

[0083] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising three polypeptide chains: the first polypeptide chain sequentially comprises the light chain variable region of the CD16 antibody, Linker 1, the heavy chain variable region of the CD16 antibody, and the first Fc region from the N-terminus to the C-terminus; the second polypeptide chain comprises the heavy chain of the B7H7 antibody; the third polypeptide chain comprises the light chain of the B7H7 antibody.

[0084] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising three polypeptide chains: the first polypeptide chain sequentially comprises the light chain variable region of the CD16 antibody (SEQ ID NO: 13), Linker 1 (SEQ ID NO: 35), the heavy chain variable region of the CD16 antibody (SEQ ID NO: 14), and the first Fc region (SEQ ID NO: 18) from the N-terminus to the C-terminus; the second polypeptide chain comprises the heavy chain of the B7H7 antibody (SEQ ID NO: 23); the third polypeptide chain comprises the light chain of the B7H7 antibody (SEQ ID NO: 24).

[0085] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising three polypeptide chains, wherein the first polypeptide chain has the amino acid sequence shown in SEQ ID NO: 22, the second polypeptide chain has the amino acid sequence shown in SEQ ID NO: 23, and the third polypeptide chain has the amino acid sequence shown in SEQ ID NO: 24.

[0086] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising four polypeptide chains: the first polypeptide chain comprises the light chain of the B7H7 antibody; the second polypeptide chain sequentially comprises the heavy chain variable region of the B7H7 antibody, the CH1 region of the B7H7 antibody, the light chain variable region of the CD16 antibody, Linker 2, the scFv region of the CD16 antibody, Linker 1, and the first Fc region from the N-terminus to the C-terminus; the third polypeptide chain comprises the heavy chain of the B7H7 antibody; the fourth polypeptide chain comprises the light chain of the B7H7 antibody.

[0087] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising four polypeptide chains: the first polypeptide chain comprises the light chain of the B7H7 antibody (SEQ ID NO: 30); the second polypeptide chain sequentially comprises the heavy chain variable region of the B7H7 antibody (SEQ ID NO: 16), the CH1 region of the B7H7 antibody (SEQ ID NO: 21), linker peptide 2 (SEQ ID NO: 36), the light chain variable region of the CD16 antibody (SEQ ID NO: 13), linker peptide 1 (SEQ ID NO: 35), the heavy chain variable region of the CD16 antibody (SEQ ID NO: 14), and the first Fc region (SEQ ID NO: 18) from the N-terminus to the C-terminus; the third polypeptide chain comprises the heavy chain of the B7H7 antibody (SEQ ID NO: 23); and the fourth polypeptide chain comprises the light chain of the B7H7 antibody (SEQ ID NO: 24).

[0088] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising four polypeptide chains: the first polypeptide chain has the amino acid sequence shown in SEQ ID NO: 30, the second polypeptide chain has the amino acid sequence shown in SEQ ID NO: 28, the third polypeptide chain has the amino acid sequence shown in SEQ ID NO: 29, and the fourth polypeptide chain has the amino acid sequence shown in SEQ ID NO: 30.

[0089] According to the embodiments of the present disclosure, two different configurations of CD16-B7H7 bispecific antibodies are screened out in the present invention. Even if the antigen-binding domains are the same, the antigen-binding abilities among antibodies with different configurations are not the same. An object of the present invention is to screen out a set of better CD16-B7H7 bispecific antibodies with a better effect of promoting immune cell killing of tumors, which comprises a recombinant protein of three polypeptide chains: the first polypeptide chain sequentially comprises the light chain variable region of the CD16 antibody, linker peptide 1, the heavy chain variable region of the CD16 antibody, and the first Fc region from the N-terminus to the C-terminus; the second polypeptide chain comprises the heavy chain of the B7H7 antibody; and the third polypeptide chain comprises the light chain of the B7H7 antibody. This antibody not only has a better ability to promote immune cell killing of non-small cell lung cancer HCC827 cells, but also has a better effect when killing other B7H7-positive cells.

[0090] According to a specific embodiment of the present invention, a signal peptide is added to the N-terminus of the first binding region and / or the second binding region of the recombinant protein, which can enable the antibody to be highly expressed in cells and secreted into the culture medium.

[0091] According to the specific embodiments of the present invention, without substantially affecting the antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to the sequences of the present invention to obtain variants of the sequences of the antibody or its functional fragments. They are all considered to be included within the scope of protection of the present invention. Amino acids with similar properties are substituted in the variable regions. The sequences of the variants of the present invention can have at least 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner. Those skilled in the art should know that the CDR sequences analyzed by different databases may be different, but these variations should all be included within the scope of protection of the present invention.

[0092] Nucleic acid molecules, recombinant vectors, recombinant cells, immunoconjugates

[0093] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be utilized, ligated with different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0094] Therefore, the present invention also provides isolated nucleic acids encoding the above-mentioned antibodies or their antigen-binding fragments, as well as recombinant vectors and transformants containing such nucleic acids. The nucleic acid molecule encodes the above-mentioned antibody or its antigen-binding fragment, and the nucleic acid is preferably an expression cassette obtained by genetic engineering means.

[0095] The present invention provides isolated nucleic acid molecules that encode the first antigen-binding unit and / or the second antigen-binding unit of the protein of the present invention. Other aspects provided herein are expression vectors containing the nucleic acid molecules of the present invention, host cells transfected with such expression vectors, and methods for manufacturing the proteins of the present invention.

[0096] The present invention provides innovative binding proteins that can more efficiently treat cancers expressing B7H7, such as colorectal cancer, melanoma, non-small cell lung cancer, pancreatic cancer, and liver cancer expressing B7H7, etc.

[0097] The recombinant vector can refer to a cloning vector or an expression vector, and can be obtained by operably linking the nucleic acid with a commercially available vector (such as a plasmid or a viral vector). Commonly used plasmids include pSeTag2, PEE14, pMH3, etc.

[0098] In some preferred embodiments, the nucleic acid molecule is optimized for the species and is more easily expressed in mammalian cells.

[0099] The present invention also provides an expression vector, which contains the above-mentioned isolated nucleic acid molecule. When the above-mentioned isolated polynucleotide is ligated to a vector, the polynucleotide can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide, etc. Of course, these control elements can be directly from the vector itself or exogenous, that is, not from the vector itself. Of course, the polynucleotide and the control elements are operably linked.

[0100] The present invention also provides a recombinant cell, which contains the above-mentioned expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. By culturing these recombinant cells, the corresponding antibody can be obtained. The host cells of the present invention can be prokaryotic host cells, eukaryotic host cells or phages. The prokaryotic host cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic host cells can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells. In some embodiments, the host cells of the present invention are preferably mammalian cells, more preferably BHK cells, CHO cells, NSO cells or COS cells.

[0101] The immunoconjugate provided by the present invention contains a therapeutic agent and the above-mentioned antibody or its antigen-binding fragment conjugated to the therapeutic agent. The antibody or its antigen-binding fragment can be conjugated to the therapeutic agent in a conventional manner.

[0102] The composition provided by the present invention contains the above-mentioned antibody or its antigen-binding fragment, and / or the above-mentioned immunoconjugate, and a pharmaceutically acceptable carrier. In certain embodiments, the composition includes a combination separated in time and / or space, as long as they can act together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to a subject as a whole, or separately. When the components contained in the composition are administered to a subject separately, each component can be administered to the subject simultaneously or sequentially.

[0103] Drugs, kits, pharmaceutical uses and uses in the preparation of kits

[0104] The present invention also provides a drug, which includes the above-mentioned antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, and may also include the above-mentioned immunoconjugate, nucleic acid molecule, expression vector, recombinant cell.

[0105] In some embodiments, these pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickening agent, emulsifying agent, preservative, solid binder, glidant or lubricant, etc., suitable for the particular target dosage form. Their use is also contemplated within the scope of the present invention, except to the extent that any conventional excipients are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a detrimental manner.

[0106] The compositions of the present invention may also be administered in combination with each other, or in combination with one or more other therapeutic compounds, for example, in combination with chemotherapeutic agents. Accordingly, the compositions may also contain chemotherapeutic agents. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit B7H7 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with B7H7 upstream or downstream signal transduction.

[0107] Generally, the antibody or antigen-binding fragment thereof is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, for example, an amount that causes prevention or alleviation of symptoms associated with the disease being treated, such as a disease associated with abnormal B7H7 expression. The effective amount of the composition administered to a subject will depend on the type and severity of the disease, as well as on the characteristics of the individual, such as general health status, age, sex, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, and those skilled in the art will be able to determine an appropriate dose based on these factors and the like.

[0108] The effective amount of the antibody or antigen-binding fragment described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment situation, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0109] In some embodiments of the present invention, the kit for detecting B7H7 and CD16 in a sample provided by the present invention contains the antibodies or their antigen-binding fragments as described above, a pharmaceutically acceptable carrier, an immunoconjugate, a nucleic acid molecule, an expression vector, and a recombinant cell. In some embodiments, the sample may be a tissue of a patient suffering from a B7H7-mediated disease. The kit may further include reagents routinely used for detecting B7H7 and CD16, such as coating solutions and the like.

[0110] The nucleic acids encoding the heavy chain and / or light chain of the antibody of the present invention are within the scope of the present invention. Based on the amino acid sequences of the heavy chain and / or light chain, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1.

[0111] Table 1

[0112]

[0113]

[0114]

[0115]

Claims

1. A recombinant antibody, characterized in that, Comprising: A first binding region: The first binding region comprises an antibody or antigen-binding fragment that specifically recognizes CD16; A second binding region: The second binding region comprises an antibody or antigen-binding fragment that specifically recognizes B7H7, wherein, the first binding region comprises the scFv region of a CD16 antibody and a first Fc region, and the second binding region comprises the Fab region of a B7H7 antibody and a second Fc region.

2. The recombinant antibody according to claim 1, wherein The first binding region comprises the CDR sequences of the variable regions of a CD16 antibody: the amino acid sequence of the light chain variable region CDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the light chain variable region CDR2 is as shown in SEQ ID NO:2, the amino acid sequence of the light chain variable region CDR3 is as shown in SEQ ID NO:3, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO:4, the amino acid sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO:6; Optionally, the second binding region comprises the CDR sequences of the variable regions of a B7H7 antibody: the amino acid sequence of the light chain variable region CDR1 is as shown in SEQ ID NO:7, the amino acid sequence of the light chain variable region CDR2 is as shown in SEQ ID NO:8, the amino acid sequence of the light chain variable region CDR3 is as shown in SEQ ID NO:9, the amino acid sequence of the heavy chain variable region CDR1 is as shown in SEQ ID NO:10, the amino acid sequence of the heavy chain variable region CDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain variable region CDR3 is as shown in SEQ ID NO:

12.

3. The recombinant antibody according to claim 1, characterized in that, The light chain variable region of the CD16 antibody has the amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:13, and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:14; Optionally, the light chain variable region of the B7H7 antibody has the amino acid sequence as shown in SEQ ID NO:15 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:15, and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 95% identity with the amino acids shown in SEQ ID NO:

16.

4. The recombinant antibody according to claim 1, characterized in that, The C-terminus of the scFv region of the CD16 antibody in the first binding region is connected to the N-terminus of the first Fc region; Optionally, the C-terminus of the light chain variable region in the scFv region of the CD16 antibody is connected to the N-terminus of the heavy chain variable region through a linker peptide 1, and the C-terminus of the heavy chain variable region is connected to the N-terminus of the first Fc region.

5. The recombinant antibody according to claim 1, characterized in that, The first Fc region and the second Fc region are connected by a knob-into-hole structure; Optionally, the linker peptide 1 has the amino acid sequence shown in SEQ ID NO:35; Optionally, at least a part of at least one of the CH1 region of the Fab region of the B7H7 antibody, the first Fc region, and the second Fc region is derived from at least one of a murine antibody, a primate antibody, or a mutant thereof; Optionally, at least a part of at least one of the CH1 region of the Fab region of the B7H7 antibody, the first Fc region, and the second Fc region is derived from human IgG1 or a mutant thereof; Optionally, the first Fc region has the amino acid sequence shown in SEQ ID NO:18, and the second Fc region has the amino acid sequence shown in SEQ ID NO:19; Optionally, the first binding region has the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 95% identity with the amino acid shown in SEQ ID NO:22, and the second binding region has the amino acid sequences shown in SEQ ID NO:23 and SEQ ID NO:24 or an amino acid sequence having at least 95% identity with the amino acid shown in SEQ ID NO:23 or SEQ ID NO:24; Optionally, the recombinant antibody further comprises a signal peptide, which is linked to the N-terminus of the recombinant antibody, and the signal peptide has the amino acid sequence shown in SEQ ID NO:

34.

6. The recombinant antibody according to any one of claims 1-5, characterized in that, The recombinant antibody further comprises a third binding region, and the third binding region comprises the Fab region of the B7H7 antibody.

7. The recombinant antibody according to claim 6, wherein, The C-terminus of the CH1 region of the Fab region of the B7H7 antibody in the third binding region is linked to the N-terminus of the scFv region of the CD16 antibody in the first binding region; Optionally, the C-terminus of the CH1 region of the Fab region of the B7H7 antibody in the third binding region is linked to the N-terminus of the scFv region of the CD16 antibody through linker peptide 2; Optionally, the linker peptide 2 has the amino acid sequence shown in SEQ ID NO:36; Optionally, the amino acid sequence of the Fab region of the B7H7 antibody in the third binding region is the same as or different from that of the Fab region of the B7H7 antibody in the second binding region; Optionally, the third binding region has the amino acid sequences shown in SEQ ID NO:30 and SEQ ID NO:37 or an amino acid sequence having at least 95% identity with the amino acid shown in SEQ ID NO:30 or SEQ ID NO:

37.

8. An isolated polynucleotide, characterized in that, The polynucleotide encodes the recombinant antibody according to any one of claims 1-7.

9. An expression vector, characterized in that, Carrying the polynucleotide according to claim 8.

10. A method for preparing the recombinant antibody according to any one of claims 1-7, characterized in that, Comprising: introducing the expression vector according to claim 9 into a cell, or culturing the cell under conditions suitable for protein expression and secretion to obtain the recombinant antibody; Optionally, the cell is a eukaryotic cell.

11. A recombinant cell, characterized in that, The recombinant cell carries the polynucleotide according to claim 8 or the expression vector according to claim 9.

12. A composition, characterized in that, Comprising: At least one of the recombinant antibody according to any one of claims 1-7, the polynucleotide according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 11.

13. Use of the recombinant antibody according to any one of claims 1-7, the polynucleotide according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 11, or the composition according to claim 12 in the preparation of a medicament for treating or preventing cancer, wherein the cancer cells have positive B7H7 on the surface.

14. The use according to claim 13, characterized in that, The cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, pancreatic cancer, oral squamous cell carcinoma, and head and neck cancer.

15. Use of the recombinant antibody according to any one of claims 1-7, the polynucleotide according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 11, or the composition according to claim 12 in the preparation of a kit for detecting CD16 and / or B7H7.

16. A kit, characterized in that, The kit contains the recombinant antibody according to any one of claims 1-7 and is used for detecting CD16 and / or B7H7.

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