CN117510644A

CN117510644A - Recombinant antibodies and uses thereof

Info

Publication number: CN117510644A
Application number: CN202311357582.4A
Authority: CN
Inventors: 成赢; 曹国帅; 武玉伟; 李洋洋
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2023-10-18
Filing date: 2023-10-18
Publication date: 2024-02-06

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a recombinant antibody and application thereof, and the invention provides a CD3/CD155/CD16 trispecific antibody, which consists of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a variable region CDR sequence of the CD3 antibody and a variable region CDR sequence of the CD155 antibody, the second polypeptide chain comprises a variable region CDR sequence of the CD16 antibody and a variable region CDR sequence of the CD155 antibody, the variable region CDR sequence of the CD3 antibody is shown as SEQ ID NO. 1-6, the variable region CDR sequence of the CD155 antibody is shown as SEQ ID NO. 7-12, and the variable region CDR sequence of the CD16 antibody is shown as SEQ ID NO. 13-18. The trispecific antibody prepared by the invention can simultaneously target CD3, CD155 and CD16, can promote the killing of the antibody on tumor cells, and has stronger tumor inhibition capability.

Description

Recombinant antibodies and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a recombinant antibody and application thereof.

Background

CD155, also known as NECL-5 or PVR, is a type I transmembrane protein, the extracellular segment of which contains 1 IgV domain, 2 IgC domains and the intracellular segment of which contains the ITIM motif. CD155 expression is on the surface of tissue cells such as monocytes, dendritic cells, etc., and up-regulates expression in various tumor tissues. Studies have shown that CD155 promotes tumor invasion, metastasis, and that high expression of CD155 correlates with poor tumor prognosis. Until now, only one CD155 monoclonal antibody entered the clinical trial phase, i.e. the NTX-1088 antibody of the Nectin company, and no CD155 related bispecific or trispecific antibodies have entered the clinical trial.

Among bispecific antibodies based on T cell redirection, this target for CD3 is widely selected. Unlike immune checkpoint blocking antibodies, CD 3-related bispecific antibodies can mediate T cell activation across TCR and peptide-major histocompatibility complex (pMHC), but the molecules in the synapse function much like classical TCR-pMHC interactions. The activity of bispecific antibodies is affected by the affinity of CD3, and CD3 high affinity bispecific antibodies have better killing effects in vitro experiments, but are at higher risk of factor release syndrome in vivo. Also, very low affinity CD3 antibody sequences have been found to be effective in stimulating T cell activation after bispecific antibody construction. When the CD3 antibody affinity is in the appropriate range and the tumor target-associated antibody is of high affinity, the bispecific antibody will promote selective localization of T cells to tumor sites rather than to the peripheral circulation, avoiding systemic activation.

Natural killer cells (natural killer cells, NK cells) are important members constituting the innate immune system, unlike T cells, NK cells do not express antigen-specific receptors. NK cells themselves have broad-spectrum tumor killing capacity and play an important role in enhancing antibody and T cell responses. At present, tumor immunotherapy forms based on NK cells are various, and the adopted means are different. CD16 molecules are important markers on NK cell surfaces that activate the IgE NK cell receptor (FcεRIgamma) and the Immunoreceptor Tyrosine Activation Motif (ITAM) of CD3 zeta for ADCC. Thus, the CD16 target is preferentially selected among bispecific antibodies based on NK cell redirection.

Based on these findings, the present invention contemplates novel recombinant trispecific antibodies that specifically bind CD3, CD16 and CD155. The antibody can bring T cells and NK cells to the vicinity of tumor cells in a targeting way, and the T cells and the NK cells and the tumor cells are promoted to form immune synapses by combining with CD155 molecules on the tumor cells, so that the T cells and the NK cells activate and kill CD155+ tumor cells, and a new inspiring and thinking are provided for antibody therapy of tumor immunity.

Disclosure of Invention

The present application is made based on the discovery and recognition by the inventors of the following facts and problems:

the first aspect of the present invention provides a recombinant antibody comprising a first polypeptide chain and a second polypeptide chain:

the first polypeptide chain comprises a variable region CDR sequence of a CD3 antibody and a variable region CDR sequence of a CD155 antibody,

the second polypeptide chain comprises a variable region CDR sequence of a CD16 antibody and a variable region CDR sequence of a CD155 antibody,

the variable region CDR sequence of the CD3 antibody is shown as SEQ ID NO. 1-6 or has at least 85% of amino acid sequence with the amino acid shown as SEQ ID NO. 1-6, the variable region CDR sequence of the CD155 antibody is shown as SEQ ID NO. 7-12 or has at least 85% of amino acid sequence with the amino acid shown as SEQ ID NO. 7-12, and the variable region CDR sequence of the CD16 antibody is shown as SEQ ID NO. 13-18 or has at least 85% of amino acid sequence with the amino acid shown as SEQ ID NO. 13-18.

The present invention is based on a trispecific NK cell bridging method employing a trispecific binding protein with a binding arm for binding to CD3 on T cells, a binding arm for binding to CD16a on NK cells and a binding arm for binding to CD155 on the cell surface of tumor cells. By promoting simultaneous binding of T cells to tumor cells and NK cells to tumor cells, the trispecific proteins promote the formation of cellular synapses between the two immune cells and tumor cells and thus selectively redirect the activity of T cells and NK cells to target tumor cells, achieving the effect of mobilizing both immune cells simultaneously by one drug.

According to an embodiment of the invention, the recombinant antibody consists of a first polypeptide chain and a second polypeptide chain:

the first polypeptide chain comprises a variable region CDR sequence of a CD3 antibody and a variable region CDR sequence of a CD155 antibody;

wherein the amino acid sequence of the light chain CDR1 of the CD3 antibody is shown as SEQ ID NO. 1, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 2, the light chain CDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 5, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 6,

The amino acid sequence of the light chain CDR1 of the CD155 antibody is shown as SEQ ID NO. 7, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 8, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 9, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 10, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 11, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 12,

the amino acid sequence of the light chain CDR1 of the CD16 antibody is shown as SEQ ID NO. 13, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 14, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 15, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 16, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 18.

According to an embodiment of the invention, the light chain variable region of the CD3 antibody has an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 19, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 20;

the light chain variable region of the CD155 antibody has an amino acid sequence shown as SEQ ID NO. 21 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 21, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 22 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 22;

The light chain variable region of the CD16 antibody has an amino acid sequence shown as SEQ ID NO. 23 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 23, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 24 or an amino acid sequence with at least 85% identity with the amino acid shown as SEQ ID NO. 24.

According to an embodiment of the invention, the first polypeptide chain comprises a scFv region of a CD3 antibody, a scFv region of a CD155 antibody and a first Fc region, the second polypeptide chain comprises a scFv region of a CD155 antibody, a scFv region of a CD16 antibody and a second Fc region, the first polypeptide chain and the second polypeptide chain further comprise a connecting peptide;

the C-terminal of the scFv region of the CD3 antibody in the first polypeptide chain is connected with the N-terminal of the CD155 antibody, the C-terminal of the scFv region of the CD155 antibody is connected with the N-terminal of the first Fc region, the C-terminal of the scFv region of the CD16 antibody in the second polypeptide chain is connected with the N-terminal of the CD155 antibody, and the C-terminal of the scFv region of the CD155 antibody is connected with the N-terminal of the second Fc region.

According to an embodiment of the invention, the C-terminus of the light chain variable region of the CD3 antibody in the first polypeptide chain is linked to the N-terminus of the heavy chain variable region of the CD3 antibody by a connecting peptide 1, the C-terminus of the heavy chain variable region of the CD3 antibody is linked to the N-terminus of the heavy chain variable region of the CD155 antibody by a connecting peptide 2, the C-terminus of the heavy chain variable region of the CD155 antibody is linked to the N-terminus of the light chain variable region of the CD155 antibody by a connecting peptide 3, the C-terminus of the light chain variable region of the CD155 antibody is linked to the N-terminus of the first Fc region,

The C-terminal of the light chain variable region of the CD16 antibody in the second polypeptide chain is connected with the N-terminal of the heavy chain variable region of the CD16 antibody through a connecting peptide 4, the C-terminal of the heavy chain variable region of the CD16 antibody is connected with the N-terminal of the heavy chain variable region of the CD155 antibody through a connecting peptide 5, the C-terminal of the heavy chain variable region of the CD155 antibody is connected with the N-terminal of the light chain variable region of the CD155 antibody through a connecting peptide 6, and the C-terminal of the light chain variable region of the CD155 antibody is connected with the N-terminal of the second Fc region.

According to an embodiment of the present invention, the amino acid sequences of the connecting peptides 1 to 6 may be the same or different, and the connecting peptide may be any connecting peptide known in the art for connecting amino acid peptide chains.

According to an embodiment of the present invention, the amino acid sequences of the connecting peptides 1 to 6 are identical, and the connecting peptide has the amino acid sequence shown as SEQ ID NO. 31.

According to an embodiment of the invention, the first and second Fc regions are linked by a knob-intoo-hole structure.

According to an embodiment of the invention, the first Fc region has at least one of the L234A, L235A mutations compared to the wild-type IgG1 Fc region and the second Fc region has at least one of the L234A, L235A mutations compared to the wild-type IgG1 Fc region.

According to an embodiment of the invention, at least a portion of the first and second Fc regions are derived from at least one of a murine antibody, a primates antibody or a mutant thereof.

According to an embodiment of the invention, at least a portion of the first and second Fc regions are derived from human IgG1 or a mutant thereof.

According to an embodiment of the invention, the first Fc region has an amino acid sequence as shown in SEQ ID NO. 25 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 25, and the second Fc region has an amino acid sequence as shown in SEQ ID NO. 26 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 26.

According to an embodiment of the invention, the first polypeptide chain has an amino acid sequence as shown in SEQ ID NO. 27 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 27, and the second polypeptide chain has an amino acid sequence as shown in SEQ ID NO. 28 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 28.

According to an embodiment of the invention, the recombinant antibody further comprises a signal peptide having an amino acid sequence as shown in SEQ ID NO. 32, the N-terminus of the first polypeptide chain and/or the second polypeptide chain.

In a second aspect the invention provides an isolated polynucleotide encoding the recombinant antibody of the first aspect.

In a third aspect the invention provides an expression vector carrying a polynucleotide according to the second aspect. According to an embodiment of the invention, the nucleic acid according to the second aspect is carried. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The expression vector provided by the embodiment of the invention can efficiently express the recombinant antibody in a proper host cell, and the recombinant antibody can be combined with CD3, CD155 and CD16 at the same time, so that the killing effect of T cells on tumor cells is effectively mediated, and the recombinant antibody has stronger tumor inhibition capability.

In a fourth aspect, the present invention provides a method for producing a recombinant antibody according to the first aspect, comprising:

introducing the expression vector of the third aspect into a cell;

culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibodies;

according to an embodiment of the invention, the cell is a eukaryotic cell.

In a fifth aspect, the invention provides a recombinant cell carrying a polynucleotide according to the second aspect or an expression vector according to the third aspect.

In a sixth aspect, the invention provides a composition comprising: at least one of the recombinant antibody of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. The composition of the invention comprises a food composition, a pharmaceutical composition and the like.

The seventh aspect of the present invention provides the 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 or the composition according to the sixth aspect in the manufacture of a medicament for the treatment or prevention of cancer, the cancer having positive CD155 on the surface of cancer cells.

According to an embodiment of the invention, the cancer comprises at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

In an eighth aspect the invention provides the use of a recombinant antibody according to the first aspect, a polynucleotide according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect in the preparation of a kit for detecting at least one of CD3, CD155, CD 16.

In a ninth aspect, the invention provides a kit comprising the recombinant antibody of the first aspect for detecting at least one of CD3, CD155, CD 16. The recombinant antibodies can bind to CD3, CD155, CD16 proteins, and therefore, kits comprising the recombinant antibodies can be used to effectively detect at least one of CD3, CD155, CD 16. The kit can be used in scientific research, such as qualitative or quantitative detection of CD3, CD155 and/or CD16 proteins in biological samples, and can also be used for judging the state of an individual, such as judging whether the level of CD155 of the individual is excessively higher or lower than the normal level after the level of CD155 of the individual is obtained.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a schematic structure of a trispecific binding protein (3-16-155) in example 1 of the invention;

FIG. 2A is a diagram showing the binding of the trispecific binding protein (3-16-155) to CD3 protein in example 3 of the present invention;

FIG. 2B is a diagram showing the binding of the trispecific binding protein (3-16-155) to CD16a protein in example 3 of the invention;

FIG. 2C is a diagram showing the binding of the trispecific binding protein (3-16-155) to CD155 protein in example 3 of the invention;

FIG. 3A shows the effect of trispecific binding protein (3-16-155) on the CD25 activation marker of CD4+ T cells in the absence of target cells in example 4 of the invention;

FIG. 3B shows the effect of trispecific binding protein (3-16-155) on the CD69 activation marker of CD4+ T cells in the absence of target cells in example 4 of the invention;

FIG. 3C shows the effect of trispecific binding protein (3-16-155) on CD4+ T cell survival in the absence of target cells in example 4 of the invention;

FIG. 3D shows the effect of trispecific binding protein (3-16-155) on the CD25 activation marker of CD8+ T cells in the absence of target cells in example 4 of the invention;

FIG. 3E shows the effect of trispecific binding protein (3-16-155) on the CD69 activation marker of CD8+ T cells in the absence of target cells in example 4 of the invention;

FIG. 3F shows the effect of trispecific binding protein (3-16-155) on CD8+ T cell survival in the absence of target cells in example 4 of the invention;

FIG. 3G shows the effect of trispecific binding protein (3-16-155) on the CD25 activation signature of CD56+ NK cells in the absence of target cells in example 4 of the invention;

FIG. 3H shows the effect of trispecific binding protein (3-16-155) on the CD69 activation marker of CD56+ NK cells in the absence of target cells in example 4 of the invention;

FIG. 3I shows the effect of trispecific binding protein (3-16-155) on CD56+ NK cell survival in the absence of target cells in example 4 of the invention;

FIG. 4A is a graph showing the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) with NK cells against colorectal cancer HCT-15 cells in example 5 of the present invention;

FIG. 4B is a graph showing the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) with T cells against colorectal cancer HCT-15 cells in example 5 of the present invention;

FIG. 5A shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 5 with PBMC against colorectal cancer HCT-15 cells (6 h assay);

FIG. 5B shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 5 with PBMC against colorectal cancer HCT-15 cells (24 h assay);

FIG. 6A shows the results of an in vitro cytotoxicity (6 h assay) of the trispecific binding protein (3-16-155) of example 5 of the invention against breast cancer MDA-MB-231 cells with PBMC;

FIG. 6B is a graph showing the results of an in vitro cytotoxicity (24 h assay) of the trispecific binding protein (3-16-155) of example 5 of the invention against breast cancer MDA-MB-231 cells with PBMC;

FIG. 7A shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 6 with PBMC against melanoma A375 cells, with efficacy targets vs. PBMC: a375 =10:1, 6h detection;

FIG. 7B shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 6 with PBMC against melanoma A375 cells, with efficacy targets vs. PBMC: a375 =5:1, 6h detection;

FIG. 7C shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 6 with PBMC against melanoma A375 cells, with efficacy targets versus PBMC: a375 =2.5:1, 6h detection;

FIG. 7D shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 6 with PBMC against melanoma A375 cells, with efficacy targets versus PBMC: a375 =10:1, 24h detection;

FIG. 7E shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 6 with PBMC against melanoma A375 cells, with efficacy targets vs. PBMC: a375 =5:1, 24h detection;

FIG. 7F shows the results of an in vitro cytotoxicity assay of the trispecific binding protein (3-16-155) of example 6 with PBMC against melanoma A375 cells, with efficacy targets vs. PBMC: a375 =2.5:1, 24h detection;

FIG. 8 shows the in vivo antitumor activity of the trispecific binding protein (3-16-155) of example 7 of the invention against CD155+ tumor (A375).

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

Antibodies or antigen binding fragments of the invention are typically prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment is numbered and defined using the Kabat numbering system. Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprising two light chains of relatively light molecular weight and two heavy chains of relatively heavy molecular weight, the heavy (H) and light (L) chains being linked by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V chains of the L chain and H chain are referred to as VL and VH, respectively. Certain regions of amino acid composition and arrangement in the variable region have a higher degree of variation, known as hypervariable regions (Hypervariable region, HVR), which are the sites of antigen and antibody binding and are therefore also known as determinant-complementary-determining region (CDR). The heavy chain variable region and the light chain variable region each have three CDR regions.

Antibodies of the invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

Herein, "diabody" means a peptide chain capable of specifically recognizing different protein molecules, which is obtained by linking two chains of an Fc region, respectively, wherein the two chains of the Fc region are linked by a knob intohole structure.

As used herein, the term "Knob intonation hole structure" refers to the formation of a Knob (Knob) button (hole) mutation in the CH3 region of an antibody heavy chain Fc, which facilitates heavy chain occlusion to form a heterodimer.

As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. It will be appreciated by those skilled in the art that the nucleic acid molecules encoding the antibodies or antigen binding fragments may be inserted separately into different vectors, typically into the same vector. The usual vectors may be, for example, plasmids, phages and the like. Such as Plasmid-X.

The term "identity" is used herein to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., 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: support.3 (National Biomedical Research Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs available in Peason et al (1988) Proc.Natl.Acad.Sci.85:2444, smith-Waterman algorithm (Meth.mol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.mol.biol.215:403-410), and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs available in BLAST-5:35, and the programs available in the company of Witsin the open area, inc. 35, and the methods of the company, inc. 35, and the methods provided by the methods of the company, inc. Proc.Natl.Acad.Sci.85:2444, and the methods of the invention.

anti-CD 3/CD155/CD16 antibodies or antigen binding fragments

The invention proposes a recombinant antibody consisting of a first polypeptide chain and a second polypeptide chain:

the first polypeptide chain comprises a variable region CDR sequence of a CD3 antibody and a variable region CDR sequence of a CD155 antibody, or an amino acid sequence having at least 85% identity to the above CDR sequences,

the second polypeptide chain comprises a variable region CDR sequence of a CD16 antibody and a variable region CDR sequence of a CD155 antibody, or an amino acid sequence having at least 85% identity to the above CDR sequences,

the variable region CDR sequence of the CD3 antibody is shown as SEQ ID NO. 1-6, the variable region CDR sequence of the CD155 antibody is shown as SEQ ID NO. 7-12, and the variable region CDR sequence of the CD16 antibody is shown as SEQ ID NO. 13-18.

According to one embodiment of the invention, the invention provides a binding protein comprising two polypeptide chains: the first polypeptide chain comprises, in order from the N-terminus to the C-terminus, a CD3 light chain variable region (SEQ ID NO: 19), a connecting peptide (SEQ ID NO: 31), a CD3 heavy chain variable region (SEQ ID NO: 20), a connecting peptide (SEQ ID NO: 31), a CD155 heavy chain variable region (SEQ ID NO: 22), a connecting peptide (SEQ ID NO: 31), a CD155 light chain variable region (SEQ ID NO: 21) and a first Fc region (SEQ ID NO: 25); the second polypeptide chain comprises, in order from the N-terminus to the C-terminus, a CD16 light chain variable region (SEQ ID NO: 23), a connecting peptide (SEQ ID NO: 31), a CD16 heavy chain variable region (SEQ ID NO: 24), a connecting peptide (SEQ ID NO: 31), a CD155 heavy chain variable region (SEQ ID NO: 22), a connecting peptide (SEQ ID NO: 31), a CD155 light chain variable region (SEQ ID NO: 21) and a second Fc region (SEQ ID NO: 26).

According to a specific embodiment of the present invention, there is provided a binding protein comprising two polypeptide chains, wherein a signal peptide is added to the N-terminus of the first peptide chain of the binding protein and/or the second peptide chain of the binding protein, so that an antibody can be efficiently expressed in a cell and secreted into a culture medium.

According to particular embodiments of the invention, one skilled in the art may replace, 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 invention to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). They are all considered to be included within the scope of the present invention. Such as substitution of amino acids with similar properties in the variable region. The sequences of the variants of the invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to a reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present invention are all shown in N-terminal to C-terminal fashion. It will be appreciated by those skilled in the art that the CDR sequences analyzed by different databases may vary, but that such variations are intended to be within the scope of the present invention.

Nucleic acid molecules, recombinant vectors, recombinant cells, immunoconjugates

In preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies may be used, linked to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

Thus, the present invention also provides an isolated nucleic acid encoding the above antibody or antigen-binding fragment thereof, and recombinant vectors and transformants containing the nucleic acid. The nucleic acid molecule encodes the antibody or antigen binding fragment thereof described above, preferably the nucleic acid is an expression cassette obtained by genetic engineering means.

The present invention provides isolated nucleic acid molecules encoding a first antigen binding unit and/or a second antigen binding unit of a protein of the invention. Other aspects provided herein are expression vectors comprising the nucleic acid molecules of the invention, host cells transfected with such expression vectors, and methods of making the proteins of the invention.

The present invention provides novel binding proteins that can more efficiently treat cancers that express CD155, such as colorectal cancer, melanoma, non-small cell lung cancer, pancreatic cancer, liver cancer, and the like that express CD 155.

Recombinant vectors may be referred to as cloning vectors, or as expression vectors, and may be obtained by operably linking the nucleic acids to commercially available vectors (e.g., plasmid or viral vectors), commonly used plasmids include pSeTag2, PEE14, pMH3, and the like.

In some preferred embodiments, the nucleic acid molecule is species optimized for expression in mammalian cells.

The invention also provides an expression vector comprising the isolated nucleic acid molecule described above. In the case of ligating the above isolated polynucleotide to a vector, the polynucleotide may be directly or indirectly ligated to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the polynucleotide. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the polynucleotide may be operably linked to a control element.

The invention also provides a recombinant cell, which comprises the expression vector. The expression vector may be introduced into mammalian cells, constructed to obtain recombinant cells, and these recombinant cells may be used to express the antibodies or antigen-binding fragments provided by the present invention. The recombinant cells are cultured to obtain the corresponding antibodies. The host cell of the invention may be a prokaryotic host cell, a eukaryotic host cell or a phage. The prokaryotic host cell can be escherichia coli, bacillus subtilis, streptomycete, proteus mirabilis or the like. The eukaryotic host cell can be fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the host cell of the invention is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell or COS cell.

The immunoconjugates provided herein comprise a therapeutic agent coupled to an antibody or antigen-binding fragment thereof as described previously. The manner in which the antibody or antigen-binding fragment thereof is conjugated to the therapeutic agent may be in a conventional manner.

The present invention provides compositions comprising an antibody or antigen-binding fragment thereof as described above, and/or an immunoconjugate as described above, and a pharmaceutically acceptable carrier. In certain embodiments, the compositions comprise combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

Medicine, kit and pharmaceutical application and application in preparation of kit

The invention also provides a medicine which comprises the antibody or the antigen binding fragment thereof and a pharmaceutically acceptable carrier, and can also comprise the immunoconjugate, the nucleic acid molecule, the expression carrier and the recombinant cell.

In some embodiments, these pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, including any solvents, solid excipients, diluents, binders, disintegrants, or other liquid excipients, dispersing agents, flavoring or suspending agents, surfactants, isotonic agents, thickening agents, emulsifying agents, preservatives, solid binders, glidants or lubricants, and the like, suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

The compositions of the invention may also be administered in combination with each other, or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The antibodies, or antigen binding fragments thereof, or immunoconjugates of the invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit CD155 activity (including other antibodies or antigen binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or agents that interfere with CD155 upstream or downstream signaling.

Typically, 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, e.g., an amount that causes prevention or alleviation of symptoms associated with a disease being treated, such as a disease associated with abnormal CD155 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, age, sex, weight and tolerance to drugs; the skilled artisan will be able to determine the appropriate dosage based on these factors, etc., will also depend on the severity and type of disease.

The effective amount of the antibodies or antigen binding fragments of the 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 one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: 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 weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

In some embodiments of the invention, the kits provided herein for detecting CD3, CD155, CD16 in a sample comprise an antibody or antigen binding fragment thereof as described above, a pharmaceutically acceptable carrier, an immunoconjugate, a nucleic acid molecule, an expression vector, a recombinant cell. In some embodiments, the sample may be tissue of a patient suffering from a CD155 mediated disease. The kit may also include reagents conventionally used for detecting CD3, CD16, CD155, such as coating solutions and the like.

Nucleic acids encoding the heavy and/or light chains of the antibodies of the invention are within the scope of the invention, and corresponding nucleic acid sequences can be readily obtained by the skilled artisan based on the amino acid sequences of the heavy and/or light chains, as shown in table 1.

TABLE 1

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The embodiments will be described in detail below. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 preparation of antibody molecules

In order to effectively kill solid tumor cells, the inventor selects two targets of CD3 and CD16 to target T cells and NK cells respectively, so that the anti-tumor activity of the T cells and the NK cells can be fully mobilized, and meanwhile, in order to avoid 'self-phase killing' between immune cells caused by bridging of the T cells and the NK cells due to the CD3 and the CD16, the inventor fully prolongs the space distance between the two sections of CD3 and the CD16 when designing an antibody structure, so that cell synapses cannot be well formed, and on the contrary, the CD3, the CD155, the CD16 and the CD155 can reach ideal space distances to realize biological functions. The trispecific antibody in heterodimeric configuration was finally determined and this antibody was designated 3-16-155. The antibody contains three monovalent units, the first of which is in the form of an anti-CD 3 scFv, the second of which is in the form of an anti-CD 16 scFv, and the third of which is in the form of an anti-CD 155 scFv, wherein the third has two copies. Wherein the Fc constant regions of the first polypeptide chain and the second polypeptide chain are derived from human antibody IgG1, and because the molecule has a special asymmetric structure, different amino acid mutations are introduced into the constant regions of the first polypeptide chain and the second polypeptide chain to form a knob-in-hole structure in order to reduce the generation of homodimers. Also, in order to prevent activation of crosslinking by the Fcγ receptor, a (L234A/L235A) mutation was introduced into the first and second polypeptide chain constant regions. See fig. 1 for a specific design configuration.

The first polypeptide chain and the second polypeptide chain are cloned in an expression vector pcDNA3.4 by using common molecular biology technology, meanwhile, in order to enable the first polypeptide chain and the second polypeptide chain to be efficiently expressed in CHO cells and secreted into a culture medium, a leader peptide of a heavy chain of a murine antibody is selected to be inserted into the expression vector as a secretion signal peptide, the signal peptide is positioned at the N end of an antibody variable region, and the amino acid sequence is as follows: MGWSCIILFLVATATGVHS.

Example 2 binding protein expression and purification

Trispecific molecules binding to CD3, CD16 and CD155 were prepared by transient transfection of ExpiCHO-S cells (Gibco, cat. No. A29127) with pcDNA3.4 vector carrying the 3-16-155 coding gene. The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4). Times.10 ⁶ Individual/ml, 37 ℃,8% CO ₂ The culture was continued overnight with shaking at 120 rpm. On the day of transfection, cells grew to 7X 10 ⁶ –1×10 ⁷ Each ml, when the viability was greater than 95%, transfection was prepared and the cells were diluted to 6X 10 using freshly pre-warmed ExpiCHO medium (Gibco, cat. A2910002) ⁶ Taking plasmids containing the first polypeptide chain and the second polypeptide chain according to the mass ratio of 1:1 transfection into ExpiCHO-S cells with ExpiFectamine CHO transfection reagent (Gibco, cat. No. A29129), 37℃and 8% CO ₂ Shaking culture at 120 rpm. 18-22h post transfection, expifectamine was isolatedImmediately after mixing ne CHO Enhancer and expi CHO Feed, the transfected cells were added, mixed well, 32℃and 5% CO ₂ Shaking culture at 120 rpm. On day 5 after transfection, 8ml of ExpiCHO Feed was added to the cells again, and culture was continued after mixing. The change of cell number and cell viability was observed daily, and cells were harvested by centrifugation after the cell viability was reduced to less than 80% or after 10-14 days of culture, and the supernatant was purified or frozen at-80 ℃.

The expressed supernatant was filtered with a 0.22 μm filter membrane, an antibody having an Fc domain was captured from the expressed supernatant by using a Mabselect prism A affinity column (cytova, cat. No. 17549854), the column was equilibrated with a phosphate buffer of pH7.2, the supernatant was eluted with an elution buffer (100 mM citric acid, pH 2.7), and finally the purified antibody was concentrated and displaced with PBS buffer, and the purity of the purified antibody was 90% or higher by SDS-PAGE, to thereby obtain a 3-16-155 trispecific binding protein.

Example 3 determination of binding Activity of trispecific antibodies to antigen (ELISA)

Human CD3 antigen (ACRO biosystems, cat. No. CDD-H52W 1), CD16a antigen (ACRO biosystems, cat. No. CDA-H5220) or CD155 antigen (ACRO biosystems, cat. No. CD 5-H5223) was coated with a coating buffer (35 mM NaHCO ₃ ，15mM Na ₂ CO ₃ pH 9.6) was diluted to 1. Mu.g/mL and 100. Mu.L per well was added to the ELISA plate overnight at 4 ℃. Followed by 3 washes with PBST (0.05% Tween20-PBS, pH 7.2). To the plate, 300. Mu.L of blocking buffer (1% BSA,0.05% Tween20-PBS, pH 7.2) was added, and the mixture was allowed to stand at room temperature for 2 hours. The mixture was washed 3 times with PBST. The corresponding trispecific antibody was added to each well and incubated for 1 hour at room temperature. The mixture was washed 3 times with PBST. Mu.l of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-097) diluted with blocking buffer was added to each well and incubated for 1 hour at room temperature. Washing with PBST for 3 times, adding TMB into each hole, reacting at room temperature in a dark place for 2-5 minutes, stopping the reaction with 2M sulfuric acid in each hole, and finally reading the OD450 value with an ELISA reader. FIG. 2A shows that the 3-16-155 trispecific antibody of the present invention can bind CD3, FIG. 2B shows that the 3-16-155 trispecific antibody of the present invention can bind human CD16a, and FIG. 2C shows that the 3-16-155 trispecific antibody of the present invention can bind human CD155.

EXAMPLE 4 cell activation and self-death detection of trispecific antibodies

PBMCs were diluted and counted and 2 x 10 wells per well were taken ⁵ The individual cells were added to 96-well plates, followed by addition of gradient concentrations of 3-16-155 trispecific antibody, and mixing. After 24 hours incubation at 37 ℃, cells were collected by centrifugation, resuspended in PBS, blocked for 15 minutes at room temperature with mouse serum, then added with flow antibody for detection of surface molecules, incubated at 4 ℃ for 30 minutes in the absence of light, washed with PBS and detected with a flow cytometer.

In the absence of target cells, the increasing proportion of CD25 molecules (FIG. 3A,3D, 3G) and CD69 (FIG. 3B,3E, 3H) expressed on the surface of CD4+ T, CD8+T cells and CD56+NK cells, as the concentration of the 3-16-155 trispecific antibody increases, suggests that the 3-16-155 trispecific antibody itself may stimulate T cell activation and up-regulate the expression of the corresponding activation markers CD25 and CD69. While the 7-AAD+ ratio of these three cells was almost unchanged (FIGS. 3C,3F, 3I), indicating that the 3-16-155 trispecific antibodies did not promote mutual killing of autologous T cells and NK cells.

Example 5 in vitro killing assay of trispecific binding proteins

T cell and NK cell killing

The HCT-15 cells in the adherence culture are digested and counted, and the cell density is adjusted to 2 multiplied by 10 ⁵ And each ml. The RTCA instrument (agilent) was turned on, the experimental mode was selected, and the cell information and drug information were filled in. Entering a schedule setting experiment step, adding 50 mu L of fresh culture medium (89% RPMI 1640 culture medium+10% fetal bovine serum+1% green streptomycin) into the plates, putting the plates into an instrument, closing the plates, and clicking the plates to start in the first step. After Done, the plate was removed, 100 μl of cell suspension was added, and left standing at room temperature for 15-30min to prevent edge effects, and placed into the instrument and clicked on. After growth to log phase, suspension was performed, 50. Mu. L T cells (4X 10) ⁵ And/ml) and add a gradient of trispecific antibody, click on start, analysis after a period of time.

As the concentration of the 3-16-155 trispecific antibody increases, the killing efficiency of NK cells and T cells against colorectal cancer HCT-15 cells gradually increases (FIGS. 4A, 4B), indicating that 3-16-155 can well promote NK cells and T cells to kill CD155+ tumor cells, respectively.

PBMC killing

The tumor cells in the adherent culture are counted after digestion, and the cell density is adjusted to 4 multiplied by 10 ⁵ And each ml. The RTCA instrument (agilent) was turned on, the experimental mode was selected, and the cell information and drug information were filled in. Entering a schedule setting experiment step, adding 50 mu L of fresh culture medium (89% RPMI 1640 culture medium+10% fetal bovine serum+1% green streptomycin) into the plates, putting the plates into an instrument, closing the plates, and clicking the plates to start in the first step. After Done, the plate was removed, 50 μl of cell suspension was added, and left standing at room temperature for 15-30min to prevent edge effects, and placed into the instrument and clicked on. After growth to log phase, suspension was performed, and 80. Mu.LPBMC (2.5X10) ⁶ And/ml) and add a gradient of trispecific antibody, click on start, analysis after a period of time.

As the concentration of the 3-16-155 trispecific antibody increases, the killing efficiency of PBMC on colorectal cancer HCT-15 cells increases gradually no matter in 6h detection (FIG. 5A) or 24h detection (FIG. 5B), and the killing efficiency of PBMC on breast cancer MDA-MB-231 cells increases gradually no matter in 6h detection (FIG. 6A) or 24h detection (FIG. 6B), which shows that 3-16-155 can well promote the PBMC to kill CD155+ tumor cells.

Example 6 different effective target specific killing contrast

To better compare the difference between trispecific and bispecific antibodies, we constructed CD3-CD155 bispecific antibodies and CD16-CD155 bispecific antibodies as controls. Both bispecific antibodies adopt a heterodimeric configuration. Wherein the CD3-CD155 bispecific antibody comprises two monovalent units, wherein the first monovalent unit is in the form of an scFv against CD3 and the second monovalent unit is in the form of an scFv against CD155, the antibody being designated 3-155. The antibody comprises two polypeptide chains, wherein the amino acid sequence of the first polypeptide chain is SEQ ID NO. 14, and the amino acid sequence of the second polypeptide chain is SEQ ID NO. 15. The CD16-CD155 bispecific antibody contains two monovalent units, the first of which is in the form of an scFv against CD16 and the second of which is in the form of an scFv against CD155, designated 16-155. The antibody comprises two polypeptide chains, wherein the amino acid sequence of the first polypeptide chain is SEQ ID NO. 18, and the amino acid sequence of the second polypeptide chain is SEQ ID NO. 19. These two bispecific antibodies were subjected to protein expression and purification by the method of example 2, and bispecific antibodies with a purity of 90% or more were obtained for experiments.

The wall-attached cultured melanoma A375 cells were digested and counted to adjust the cell density to 2X 10 ⁵ /mL. The RTCA instrument (agilent) was turned on, the experimental mode was selected, and the cell information and drug information were filled in. Entering a schedule setting experiment step, adding 50 mu L of fresh culture medium (89% RPMI 1640 culture medium+10% fetal bovine serum+1% green streptomycin) into the plates, putting the plates into an instrument, closing the plates, and clicking the plates to start in the first step. After Done, the plate was removed, 50 μl of cell suspension was added, and left standing at room temperature for 15-30min to prevent edge effects, and placed into the instrument and clicked on. After growth to log phase, suspension was carried out, 80. Mu.L of PBMC of different concentrations (1.25X10 ⁶ /mL，6.25×10 ⁵ /mL，3.125×10 ⁵ /mL) and add a gradient of either a trispecific antibody or a bispecific antibody, click on, and analyze after a period of time.

As shown in the results of FIGS. 7A-7F, the 3-16-155 trispecific antibody has obviously higher killing power to PBMC than the 3-155 and 16-155 bispecific antibody in 6h detection, and is equivalent to the combination of the 3-155 and 16-155 antibodies. When the effective target is higher (10:1) in 24h detection, the 3-16-155 trispecific antibody has obviously stronger killing ability to PBMC than 3-155 and 16-155 bispecific antibody, and is equivalent to the combination of 3-155 and 16-155 antibodies; and when the effective target is relatively low (2.5:1), the killing promotion capability of the 3-16-155 trispecific antibody is obviously stronger than that of the two bispecific antibodies which are singly used and combined. These results also demonstrate that the 3-16-155 trispecific antibody has a biological function significantly better than that of the bispecific antibody.

EXAMPLE 7 in vivo xenograft efficacy Studies

To test the in vivo antitumor activity of the 3-16-155 trispecific binding protein against A375 tumor cells. Intravenous injection of PBMC into female NOD.Cg-Prkdc ^scid Il2rg ^em1Smoc Il15 ^{em1(hIL15)Smoc} Mice, after 4 days, a375 melanoma cells (3 x 10 ⁶ ) Subcutaneous injection (s.c.) in mice, until the tumor grows to the point of45-90mm ³ Post-grouping followed by intravenous injection of 3-16-155 different doses of trispecific antibody (0.03 mg/kg,0.1mg/kg,0.3 mg/kg) or control PBS solvent twice weekly into mice for a total of 6 injections. Tumor length and width were measured by external caliper measurements and tumor volume was calculated using standard formulas.

FIG. 8 shows the in vivo antitumor activity of the 3-16-155 trispecific binding protein against A375 cells. The tumor growth can be obviously inhibited by the treatment of the 3-16-155 trispecific antibody, even the dosage of 0.03mg/kg has good tumor inhibiting effect, which proves that the 3-16-155 trispecific antibody has obvious in vivo anti-tumor activity.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A recombinant antibody comprising a first polypeptide chain and a second polypeptide chain:

2. The recombinant antibody according to claim 1, wherein the recombinant antibody consists of a first polypeptide chain and a second polypeptide chain:

the amino acid sequence of the light chain CDR1 of the CD16 antibody is shown as SEQ ID NO. 13, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO. 14, the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 15, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 16, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 18;

Optionally, the light chain variable region of the CD3 antibody has an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 19, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 20 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID NO. 20;

3. The recombinant antibody according to claim 1, wherein said first polypeptide chain comprises a scFv region of a CD3 antibody, a scFv region of a CD155 antibody and a first Fc region, said second polypeptide chain comprises a scFv region of a CD155 antibody, a scFv region of a CD16 antibody and a second Fc region, said first polypeptide chain and said second polypeptide chain further comprise a connecting peptide;

The C-terminal of the scFv region of the CD3 antibody in the first polypeptide chain is connected with the N-terminal of the CD155 antibody, the C-terminal of the scFv region of the CD155 antibody is connected with the N-terminal of the first Fc region, the C-terminal of the scFv region of the CD16 antibody in the second polypeptide chain is connected with the N-terminal of the CD155 antibody, and the C-terminal of the scFv region of the CD155 antibody is connected with the N-terminal of the second Fc region;

optionally, the C-terminus of the light chain variable region of the CD3 antibody in the first polypeptide chain is linked to the N-terminus of the heavy chain variable region of the CD3 antibody by a linker peptide 1, the C-terminus of the heavy chain variable region of the CD3 antibody is linked to the N-terminus of the heavy chain variable region of the CD155 antibody by a linker peptide 2, the C-terminus of the heavy chain variable region of the CD155 antibody is linked to the N-terminus of the light chain variable region of the CD155 antibody by a linker peptide 3, the C-terminus of the light chain variable region of the CD155 antibody is linked to the N-terminus of the first Fc region,

the C-terminal of the light chain variable region of the CD16 antibody in the second polypeptide chain is connected with the N-terminal of the heavy chain variable region of the CD16 antibody through a connecting peptide 4, the C-terminal of the heavy chain variable region of the CD16 antibody is connected with the N-terminal of the heavy chain variable region of the CD155 antibody through a connecting peptide 5, the C-terminal of the heavy chain variable region of the CD155 antibody is connected with the N-terminal of the light chain variable region of the CD155 antibody through a connecting peptide 6, and the C-terminal of the light chain variable region of the CD155 antibody is connected with the N-terminal of the second Fc region;

Optionally, the connecting peptide 1, the connecting peptide 2, the connecting peptide 3, the connecting peptide 4, the connecting peptide 5 and the connecting peptide 6 have the amino acid sequence shown in SEQ ID NO. 31.

4. The recombinant antibody according to claim 3, wherein the first Fc region and the second Fc region are linked by a knob-into-hole structure;

optionally, the first Fc region has at least one of the L234A, L235A mutations compared to the wild-type IgG1 Fc region and the second Fc region has at least one of the L234A, L a mutations compared to the wild-type IgG1 Fc region;

optionally, at least a portion of the first and second Fc regions are derived from at least one of a murine antibody, a primates antibody, or a mutant thereof;

optionally, at least a portion of the first and second Fc regions are from human IgG1 or mutants thereof.

5. The recombinant antibody according to claim 3, wherein said first Fc region has an amino acid sequence as set forth in SEQ ID No. 25 or an amino acid sequence having at least 85% identity to the amino acid set forth in SEQ ID No. 25 and said second Fc region has an amino acid sequence as set forth in SEQ ID No. 26 or an amino acid sequence having at least 85% identity to the amino acid set forth in SEQ ID No. 26.

6. The recombinant antibody according to claim 1, wherein said first polypeptide chain has an amino acid sequence as shown in SEQ ID No. 27 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID No. 27 and said second polypeptide chain has an amino acid sequence as shown in SEQ ID No. 28 or an amino acid sequence having at least 85% identity to the amino acid shown in SEQ ID No. 28.

7. The recombinant antibody according to claim 1, further comprising a signal peptide added to the N-terminus of the first polypeptide chain and/or the second polypeptide chain, the signal peptide having an amino acid sequence as shown in SEQ ID No. 32.

8. An isolated polynucleotide encoding the recombinant antibody of any one of claims 1-7.

9. An expression vector carrying the polynucleotide of claim 8.

10. A method of producing the recombinant antibody of any one of claims 1-7, comprising:

introducing the expression vector of claim 9 into a cell;

Optionally, the cell is a eukaryotic cell.

11. A recombinant cell carrying the polynucleotide of claim 8 or the expression vector of claim 9.

12. A composition, comprising:

at least one of the recombinant antibodies of any one of claims 1-7, the polynucleotide of claim 8, the expression vector of claim 9, or the recombinant cell of claim 11.

13. Use of the recombinant antibody of any one of claims 1-7, the polynucleotide of claim 8, the expression vector of claim 9, the recombinant cell of claim 11, or the composition of claim 12 in the manufacture of a medicament for treating or preventing cancer whose cancer cell surface is positive for CD 155.

14. The use according to claim 13, wherein the cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

15. Use of the recombinant antibody of any one of claims 1-7, the polynucleotide of claim 8, the expression vector of claim 9, the recombinant cell of claim 11, the composition of claim 12 in the preparation of a kit for detecting at least one of CD3, CD16, CD 155.

16. A kit comprising the recombinant antibody of any one of claims 1-7 for detecting at least one of CD3, CD16, CD 155.