CN114395044B

CN114395044B - Recombinant antibodies and their applications

Info

Publication number: CN114395044B
Application number: CN202111485379.6A
Authority: CN
Inventors: 田志刚; 成赢; 肖卫华; 曹国帅; 孙昊昱; 孙汭
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2023-12-29
Anticipated expiration: 2041-12-07
Also published as: CN114395044A

Abstract

本发明提出了一种重组抗体及其应用，该重组抗体含有选自下列至少之一的CDR序列或与其具有至少95％同一性的氨基酸序列：CD16抗体可变区CDR序列：SEQ ID NO:1～6；B7H6抗体可变区CDR序列：SEQ ID NO:7～12。本发明所制备的重组抗体能同时靶向CD16和B7H6，且半衰期得到显著延长，较单靶抗体表现出更强的肿瘤抑制能力。The present invention proposes a recombinant antibody and its application. The recombinant antibody contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity with it: CD16 antibody variable region CDR sequence: SEQ ID NO: 1 ~6; B7H6 antibody variable region CDR sequence: SEQ ID NO: 7 ~ 12. The recombinant antibody prepared by the present invention can target CD16 and B7H6 at the same time, has a significantly extended half-life, and exhibits stronger tumor inhibition ability than a single-target antibody.

Description

Recombinant antibodies and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a recombinant antibody and application thereof, and more particularly relates to a recombinant antibody, immune cells, nucleic acid, expression vectors, recombinant cells, a composition, application of the substances in preparation of medicines and a kit.

Background

B7H6 is a type I transmembrane protein belonging to the B7 family, which contains 2 immunoglobulin domains. B7H6 is also a ligand for NK cell activating receptor NKp 30. B7H6 is expressed on a variety of tumor cells, but mRNA of B7H6 is not detected in human normal tissue and healthy peripheral blood mononuclear cells. In the inflammatory environment, some pro-inflammatory cytokines such as IL-1β and TNF can stimulate cd14+ and cd16+ monocytes and neutrophils to up-regulate B7H6 expression. The tumor expression profile of B7H6 is very broad, including leukemia, lymphoma, colorectal cancer, non-small cell lung cancer, breast cancer, ovarian cancer, gastric cancer, liver cancer, and the like. The expression of B7H6 has also been linked to metastasis of some cancers, but the role of B7H6 in most tumors is currently unclear. Currently, although B7H6 is expressed in a variety of tumors, there are few tumor treatments for this molecule. Bispecific antibodies against B7H6 have been developed by researchers in the form of bites, which do not contain the constant regions of the native antibody, and thus have determined that the half-life of such molecules in vivo is very short, greatly limiting their use.

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.

Currently, both clinically and in the development stage, B7H6 bispecific antibodies were developed based on T cells, and no B7H6 bispecific antibodies have been developed based on NK cells. The research shows that the bispecific antibody of the T cell redirecting type can activate T cells to release Tumor Necrosis Factor (TNF), the TNF can activate monocytes to produce inflammatory cytokines such as IL-6, IL-1 beta and the like, and finally adverse reactions such as clinical hyperthermia and the like are caused, which is why a plurality of bispecific antibodies are clinically terminated due to side effects at present. NK cells have stronger killing ability than T cells, but lower levels of secreted cytokines, so that the frequency of occurrence of clinical adverse events is lower, and therefore, development of bispecific antibodies with strong targeting has great value for prevention and treatment of tumors.

Disclosure of Invention

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

in the invention, the inventor combines and screens CD16 and B7H6 antigen binding fragments, designs a recombinant bispecific antibody, the recombinant bispecific antibody can specifically bind CD16 and B7H6, further brings B7H6 molecules near tumor cells in a targeting way, promotes CD16+ lymphocytes and tumor cells to form immune synapses by combining with B7H6 molecules on the tumor cells, so that the lymphocytes activate and kill the B7H6+ tumor cells, and the recombinant bispecific antibody has higher CD16 and B7H6 binding activity, longer in vivo half-life and higher antitumor activity.

Thus, in a first aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the invention, the CDR sequence comprises or has at least 95% identity to an amino acid sequence selected from at least one of the following: CD16 antibody variable region CDR sequences: SEQ ID NO. 1-6; B7H6 antibody variable region CDR sequences: SEQ ID NO. 7-12. The recombinant antibody provided by the embodiment of the invention can be effectively combined with CD16 and B7H6, has stronger in-vivo and in-vitro combination activities, longer in-vivo half-life and remarkable anti-tumor effect.

GGHNIGSKNVH(SEQ ID NO:1)。

QDNKRPS(SEQ ID NO:2)。

QVWDNYSVL(SEQ ID NO:3)。

SYYMH(SEQ ID NO:4)。

IINPSGGSTSYAQKFQG(SEQ ID NO:5)。

GSAYYYDFADY(SEQ ID NO:6)。

KASQSVDYDGDSYMN(SEQ ID NO:7)。

AASTLHS(SEQ ID NO:8)。

QQSKEDPRT(SEQ ID NO:9)。

DYNMD(SEQ ID NO:10)。

DINPNNGGTLYNQKFRG(SEQ ID NO:11)。

SEVFYGNYADY(SEQ ID NO:12)。

In a second aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the invention, a polypeptide having the sequence of SEQ ID NO:20 and 21. The recombinant antibody provided by the embodiment of the invention can be effectively combined with CD16 and B7H6, has stronger in-vivo and in-vitro combination activities, longer in-vivo half-life and remarkable anti-tumor effect.

QPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：20)。

DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：21)。

In a third aspect of the invention, the invention provides a nucleic acid. According to an embodiment of the invention, the nucleic acid encodes the recombinant antibody of the first aspect. The recombinant antibody coded by the nucleic acid can be effectively combined with CD16 and B7H6, has stronger in-vivo and in-vitro combination activity, longer in-vivo half-life and remarkable anti-tumor effect.

In a fourth aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the third aspect. 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 can be further effectively used for specific treatment or prevention of tumors, in particular B7H 6-expressing tumors.

In a fifth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the nucleic acid molecule according to the third aspect, the expression vector according to the fourth aspect or the recombinant antibody according to the first or second aspect. The recombinant cells are obtained by transfecting or transforming the expression vector. According to embodiments of the present invention, the recombinant cells can efficiently express the recombinant antibodies described above under appropriate conditions, and the recombinant cells can be effectively used for the specific treatment or prevention of tumors, particularly, tumors expressing B7H6.

In a sixth aspect of the invention, the invention provides a composition. According to an embodiment of the invention, a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect or a recombinant cell according to the fifth aspect is included. As described above, the recombinant antibody of the embodiment of the invention can be effectively combined with CD16 or B7H6 protein molecules, so that tumor cells which express the B7H6 can be specifically identified, T cells are selectively positioned to the local part of the tumor rather than the peripheral circulation, the whole body activation is avoided, and the composition containing the recombinant antibody also has obvious effect of treating or preventing the tumor which expresses the B7H6, and has higher safety and less side effect.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of a tumor. As described above, the recombinant antibody of the embodiment of the invention can be effectively combined with CD16 and B7H6 proteins, and further can specifically identify tumor cells expressing B7H6 with high specificity, and the medicaments prepared by using the recombinant antibody and a series of corresponding substances have obvious effects of treating or preventing tumors expressing B7H6, and have higher safety and smaller side effects.

In an eighth aspect of the invention, the invention provides a medicament comprising: the recombinant antibody according to the first or second aspect, the nucleic acid molecule according to the third aspect, the expression vector according to the fourth aspect, the recombinant cell according to the fifth aspect or the composition according to the sixth aspect, for use in the treatment or prevention of cancer. As described above, the recombinant antibody of the embodiment of the invention can be effectively combined with CD16 and B7H6 protein molecules, so that tumor cells which express the B7H6 can be specifically identified, T cells are selectively positioned to the local part of the tumor instead of peripheral circulation, systemic activation is avoided, and medicines containing the recombinant antibody also have obvious effects of treating or preventing the tumor which expresses the B7H6, and the safety is higher and the side effect is smaller.

In a ninth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, it comprises: the recombinant antibody of the first or second aspect. The antibody according to the embodiment of the invention can be effectively combined with CD16 and B7H6 protein molecules, and can specifically identify tumor cells which specifically express the B7H6, so that the recombinant antibody can be used for preparing a kit for diagnosing or detecting at least one of rectal cancer, non-small cell lung cancer, breast cancer and liver cancer, and the kit can be used for scientific research, such as detecting whether a biological sample carries CD16 and/or B7H6 protein.

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 is a schematic structural diagram of a recombinant bispecific antibody according to an embodiment of the present invention;

FIG. 2 is a graph of the results of electrophoresis detection of purified CD16XB7H26 recombinant bispecific antibodies according to an embodiment of the present invention, wherein M represents a protein molecular weight marker; r represents a reducing SDS-PAGE buffer; NR represents a non-reducing SDS-PAGE buffer;

FIG. 3A is a graph of the results of an assay for binding of a CD16XB 7H6 recombinant bispecific antibody to human CD16a protein according to an embodiment of the present invention, wherein Nkmab represents a CD16XB 7H6 recombinant bispecific antibody;

FIG. 3B is a graph of the results of an assay for binding of a CD16XB7H2 recombinant bispecific antibody to human B7H6 protein according to an embodiment of the present invention, wherein Nkmab represents a CD16XB7H2 recombinant bispecific antibody; and

FIG. 4 is a graph showing the results of in vitro cytotoxicity test analysis of CD16 XB 7H6 recombinant bispecific antibody against NK cells against HCT-15 cells, wherein Nkmab represents CD16 XB 7H6 recombinant bispecific antibody according to an embodiment of the present invention.

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.

Recombinant antibodies

In a first aspect of the invention, the invention provides a recombinant antibody comprising a CDR sequence or an amino acid sequence having at least 95% identity thereto selected from at least one of the following: CD16 antibody variable region CDR sequences: SEQ ID NO. 1-6; B7H6 antibody variable region CDR sequences: SEQ ID NO. 7-12. The recombinant antibody provided by the embodiment of the invention can be effectively combined with CD16 and B7H6, has stronger in-vivo and in-vitro combination activities, longer in-vivo half-life and remarkable anti-tumor effect.

GGHNIGSKNVHXXX(SEQ ID NO:1)。

QDNKRPSXXX(SEQ ID NO:2)。

QVWDNYSVLXXX(SEQ ID NO:3)。

SYYMHXXX(SEQ ID NO:4)。

IINPSGGSTSYAQKFQGXXX(SEQ ID NO:5)。

GSAYYYDFADYXXX(SEQ ID NO:6)。

KASQSVDYDGDSYMN(SEQ ID NO:7)。

AASTLHS(SEQ ID NO:8)。

QQSKEDPRT(SEQ ID NO:9)。

DYNMD(SEQ ID NO:10)。

DINPNNGGTLYNQKFRG(SEQ ID NO:11)。

SEVFYGNYADY(SEQ ID NO:12)。

According to some embodiments of the invention, the recombinant antibody may further comprise at least one of the following additional technical features:

according to some embodiments of the invention, the variable region of a CD16 antibody is comprised of a light chain variable region as set forth in SEQ ID NO. 13 and a heavy chain variable region as set forth in SEQ ID NO. 14, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the amino acid sequences set forth in SEQ ID NO. 13 and SEQ ID NO. 14.

QPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:13)。

EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS(SEQ ID NO:14)。

According to some embodiments of the invention, the antibody comprises a variable region of a B7H6 antibody, the variable region of the B7H6 antibody having a light chain variable region as shown in SEQ ID NO. 15 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 16, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to the amino acid sequences shown in SEQ ID NO. 15 and SEQ ID NO. 16.

DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIK(SEQ ID NO:15)。

EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:16)。

According to some embodiments of the invention, the method further comprises a linking peptide.

According to some embodiments of the invention, the connecting peptide has the amino acid sequence shown in SEQ ID NO. 17.

GGGGSGGGGSGGGGS(SEQ ID NO:17)。

According to some embodiments of the invention, the N-terminus of the connecting peptide is linked to the C-terminus of the CD16 antibody light chain variable region and the C-terminus of the connecting peptide is linked to the N-terminus of the CD16 antibody heavy chain variable region.

According to some embodiments of the invention, the N-terminus of the connecting peptide is linked to the C-terminus of the B7H6 light chain variable region and the C-terminus of the connecting peptide is linked to the N-terminus of the B7H6 heavy chain variable region.

According to some embodiments of the invention, the antibody is further derived from at least one of a murine antibody, a human antibody, a primates antibody, or a mutant thereof.

According to some specific embodiments of the invention, at least a portion of the first and second Fc regions are derived from human IgG or mutants thereof.

According to some specific embodiments of the invention, at least a portion of the first and second Fc regions are derived from human IgG1 or mutants thereof.

According to some specific embodiments of the invention, the first Fc region has at least one of the S380C mutation and the T392W mutation compared to the wild-type IgG1 Fc region.

According to some specific embodiments of the invention, the second Fc region has at least one of a Y380C mutation, a T397S mutation, an L399A mutation, and a Y408V mutation compared to a wild type IgG1 Fc region.

According to some embodiments of the invention, the first antibody Fc region has an amino acid sequence as shown in SEQ ID NO. 18 and the second antibody Fc region has an amino acid sequence as shown in SEQ ID NO. 19.

EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:18)。

EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19)。

Pharmaceutical composition

In a third aspect of the invention, the invention provides a nucleic acid encoding a recombinant antibody as described above. The recombinant antibody coded by the nucleic acid according to some specific embodiments of the invention can be effectively combined with CD16 and B7H6, has stronger in-vivo and in-vitro combination activity, longer in-vivo half-life and remarkable anti-tumor effect.

According to some embodiments of the invention, the nucleic acid has the sequence of SEQ ID NO:26 and 27.

It should be noted that, for the nucleic acids mentioned in the present specification and claims, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences in the present application include either a DNA form or an RNA form, one of which is disclosed, meaning the other is also disclosed.

In a fourth aspect of the invention, the invention provides an expression vector carrying a nucleic acid molecule as hereinbefore described. 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 vectors according to some embodiments of the present invention can efficiently express the recombinant antibodies in a suitable host cell, and thus can be effectively used for specific treatment or prevention of tumors, particularly tumors expressing B7H6.

In a fifth aspect of the invention, the invention provides a recombinant cell carrying the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect or the recombinant antibody of the first or second aspect. The recombinant cells are obtained by transfecting or transforming the expression vector. According to some embodiments of the invention, the recombinant cells may be highly expressing the recombinant antibodies described above under suitable conditions, and the recombinant cells may be effective for the specific treatment or prevention of tumors, particularly B7H6 expressing tumors.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the recombinant antibodies described herein. Those skilled in the art will readily appreciate that conditions suitable for expression of recombinant antibodies include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal expression of the recombinant antibody according to the specific environment of the laboratory.

In a sixth aspect of the invention, the invention provides a composition comprising a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect or a recombinant cell according to the fifth aspect. As described above, the recombinant antibody of the embodiment of the invention can be effectively combined with CD16 and B7H6 protein molecules, so that tumor cells which express B7H6 can be specifically identified, T cells can be selectively positioned to tumor parts rather than peripheral circulation, systemic activation is avoided, and the composition containing the recombinant antibody, such as food composition, pharmaceutical composition and the like, has obvious effect of treating or preventing the tumor which expresses B7H6, and has higher safety and less side effect.

According to some embodiments of the present invention, the above-mentioned medicament may further comprise at least one of the following additional technical features:

according to some embodiments of the invention, the cancer comprises at least one of: rectal cancer, non-small cell lung cancer, breast cancer and liver cancer.

A medicament according to some embodiments of the present invention comprises a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient.

As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The medicament of the invention contains safe and effective amount of the active ingredients of the invention and pharmaceutically acceptable carriers. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation is matched with the administration mode, and the dosage forms of the medicine are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The medicament is preferably manufactured under aseptic conditions.

The effective amount of the active ingredient described herein may vary depending upon the mode of administration, the severity of the condition being treated, and the like. 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.

Pharmaceutically acceptable carriers described herein include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration and will be well known to those of ordinary skill in the art.

Use of the same

In a seventh aspect, the invention provides the use of a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect for the preparation of a medicament for the treatment or prevention of a tumor. As described above, the recombinant antibodies provided in some embodiments of the present invention can be effectively combined with CD16 protein and B7H6 protein, so that tumor cells expressing B7H6 with high specificity can be specifically identified, and the medicines prepared by using the recombinant antibodies and a series of corresponding substances have the obvious effect of treating or preventing tumors expressing B7H6, and the safety is higher and the side effects are smaller.

According to some embodiments of the invention, the above-mentioned use may further comprise at least one of the following additional technical features:

Kit for detecting a substance in a sample

In a ninth aspect of the invention, the invention provides a kit comprising: the recombinant antibody of the first or second aspect. Some embodiments of the present invention provide that the recombinant antibodies bind to B7H6 protein, and thus, kits comprising the recombinant antibodies can be used to effectively diagnose or detect tumors that highly express the B7H6 protein.

According to some embodiments of the invention, the kit is for diagnosing or detecting at least one of rectal cancer, non-small cell lung cancer, breast cancer and liver cancer. The recombinant antibody provided by some embodiments of the invention can be effectively combined with CD16 protein and B7H6 protein molecules, and further can specifically identify tumor cells which specifically express the B7H6, so that the recombinant antibody can be used for preparing a kit for diagnosing or detecting at least one of rectal cancer, non-small cell lung cancer, breast cancer and liver cancer, and the kit can be used for scientific research, such as qualitative or quantitative detection of CD16 and/or B7H6 protein in biological samples.

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 design and construction of CD16×B7H2 bispecific antibody molecules

This example, by screening for various configurations, the inventors determined a bispecific antibody in the scFv-Fc configuration, which was designated as cd16xb 7H6. The antibody comprises two monovalent units, wherein one monovalent unit is in the form of an anti-CD 16 ScFv-Fc and the other monovalent unit is in the form of an anti-B7H 6 ScFv-Fc, the light chain variable region and the heavy chain variable region of the CD16 antibody are connected by a connecting peptide, the light chain variable region and the heavy chain variable region of the B7H6 antibody are connected by 1 connecting peptide as described above, and the connecting peptide has the amino acid sequence shown in SEQ ID NO:17, said CD16 antibody has the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region as set forth in SEQ ID NO:14, and the B7H6 antibody has a heavy chain variable region as set forth in SEQ ID NO:15, and a light chain variable region as set forth in SEQ ID NO:16, and a heavy chain variable region shown in figure 16. Wherein, the inventor screens the combination of different B7H6 antibody variable regions and CD16 antibody variable regions to construct a CD16 XB 7H6 recombinant antibody molecule; and amino acid mutation modification is carried out on the Fc of a monovalent unit, the amino acid of the Fc region of the anti-CD 16 is changed into cysteine at a position 384 (S384C) and is changed into tryptophan at a position 396 (T396W), the amino acid of the Fc region of the anti-B7H 6 is changed into cysteine at a position 380 (Y380C), is changed into serine at a position 397 (T397S), is changed into alanine (L399A) and is changed into valine at a position 408 (Y408V), so that homodimers are respectively difficult to form, and heterodimers are easy to form, wherein the specific structure is shown in figure 1, and the CD16 XB 7H6 recombinant antibody molecule has a structure shown in SEQ ID NO:22, a CD16 single chain antibody as set forth in SEQ ID NO:23, a first antibody Fc region shown as SEQ ID NO. 18, and a second antibody Fc region shown as SEQ ID NO. 19.

The nucleic acid sequences encoding the monovalent unit polypeptide chains of CD16 and B7H6 are cloned into the expression vector pTT5 (eubao organism, cat No. VT 2202), respectively, using common molecular biology techniques, the specific nucleic acid sequences being as set forth in SEQ ID NO:24 and 25, and in order to allow efficient expression and secretion into the culture medium in CHO cells, a leader peptide of the kappa chain of the murine antibody was selected as a secretion signal peptide, and the nucleotide encoding the signal peptide was inserted into an expression vector, wherein the signal peptide is located at the N-terminus of the variable regions of both antibodies, and the amino acid sequence thereof is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 24), the nucleotide sequence encoding the signal peptide is shown as SEQ ID NO:28, at the 3' end of the nucleotide encoding the variable region of the two antibodies.

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCTCCACCGGC(SEQ ID NO:28)。

CD16 single chain antibody

QPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS(SEQ ID NO:22)。

B7H6-1 single chain antibody

DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:23)。

CD16 antibody nucleotide sequence

CAGCCCGTGCTGACCCAGCCCTCCTCCGTGTCCGTGGCCCCCGGCCAGACCGCCACCATCTCCTGCGGCGGCCACAACATCGGCTCCAAGAACGTGCACTGGTACCAGCAGAGGCCCGGCCAGTCCCCCGTGCTGGTGATCTACCAGGACAACAAGAGGCCCTCCGGCATCCCCGAGAGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCATGGACGAGGCCGACTACTACTGCCAGGTGTGGGACAACTACTCCGTGCTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGAGTCCCTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCTCCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCATCATCAACCCCTCCGGCGGCTCCACCTCCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCTCCACCTCCACCGTGTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTCCGCCTACTACTACGACTTCGCCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGAGCCCAAGTCCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGCAAG(SEQ ID NO:26)。

B7H6 nucleotide sequence

GACATCGTGCTGACCCAGACCCCCCTGTCCCTGTCCGTGACCCCCGGCCAGCCCGCCTCCATCTCCTGCAAGGCCTCCCAGTCCGTGGACTACGACGGCGACTCCTACATGAACTGGTACCTGCAGAAGCCCGGCCAGCCCCCCCAGCTGCTGATCTACGCCGCCTCCACCCTGCACTCCGGCATCCCCGACAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCCAGCAGTCCAAGGAGGACCCCAGGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACCGACTACAACATGGACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGACATCAACCCCAACAACGGCGGCACCCTGTACAACCAGAAGTTCAGGGGCAGGGTGACCCTGACCGTGGACACCTCCATCTCCACCGCCTACATGGAGCTGTCCAGGCTGAGGTCCGACGACACCGCCGTGTACTACTGCGCCAGGTCCGAGGTGTTCTACGGCAACTACGCCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCCGAGCCCAAGTCCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTGCACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGTCCTGCGCCGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGGTGTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGCAAG(SEQ ID NO:27)。

EXAMPLE 2 expression and purification of CD16×B7H2 bispecific antibodies

2.1CD16 XB 7H6 bispecific binding protein expression

Bispecific molecules binding to CD16 and B7H6 were prepared by transient transfection of expcho-S cells (Gibco, cat No. a 29127) with a pTT5 vector carrying the gene encoding the CD16 x B7H6 chain. The day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4). Times.10 ⁶ /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 Per mL, when the viability was greater than 95% the transfection was prepared and the cells were diluted to 6X 10 using freshly pre-warmed ExpiCHO medium (Gibco, cat. A2910002) ⁶ /mL, a plasmid capable of expressing the CD16 polypeptide chain and the B7H6 polypeptide chain was taken again at 1:1, transfected into ExpiCHO-S cells with ExpiFectamine CHO transfection reagent (Gibco, cat. No. A29129), at 37℃and 8% CO ₂ Shaking culture at 120 rpm. Mixing ExpiFectamine CHO Enhancer and ExpiCHO Feed for 18-22 hr, immediately adding transfected cells, mixing, and mixing at 32deg.C with 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 the cell number and the cell activity rate is observed every day, and the cells are harvested after the cell activity rate is reduced to below 80 percent or after the cells are cultured for 10 to 14 days by centrifugation, and the supernatant is purified or frozen at the temperature of minus 80 ℃ for standby.

2.2CD16 XB 7H6 bispecific binding protein purification

The supernatant obtained in the 2.1 experiment was filtered with a 0.22 μm filter, an antibody having an Fc domain was captured from the expressed supernatant by using a Mabselect prism A affinity chromatography column (GE company, cat# 17549854), the column was equilibrated with phosphate buffer at 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 95% or higher by SDS-PAGE. The specific experimental results are shown in fig. 2.

Example 3 detection of binding Activity of CD16×B7H2 6 bispecific antibody to antigen (ELISA)

The binding activity of the CD16 x B7H6 bispecific antibody to the B7H6 antigen or CD16 antigen was detected separately in this example, and the specific experimental procedure was as follows:

human B7H6 (ACRO biosystems, cat. No. B76-H52H 8), human CD16a antigen (ACRO biosystems, cat. No. CDA-H5220) were each coated with a coating buffer (35 mM NaHCO) ₃ ，15mM Na ₂ CO ₃ pH 9.6) was diluted to 2. 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 was added 300. Mu.L of blocking buffer (1% BSA,0.05% Tween20-PBS, pH 7.2), at room temperatureStanding for 2h. The mixture was washed 3 times with PBST. The corresponding bispecific 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-rabbit anti-human IgG secondary antibody (boster, cat. BA 1070) 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.

The specific experimental results are shown in fig. 3A, which shows that the recombinant bispecific antibody molecule can bind to CD16 antigen, and fig. 3B, which shows that the cd16xb7h6 bispecific antibody molecule can bind to B7H6 antigen.

Example 4 in vitro killing assay of recombinant bispecific binding proteins

4.1 isolation of human Peripheral Blood Mononuclear Cells (PBMC)

Adding anticoagulated human peripheral blood into an equal volume of sterile PBS, taking a 50mL sterile centrifuge tube, lightly adding 25mL diluted peripheral blood onto 15mL lymph separation liquid (GE healthcare, product number 17144003), keeping the interface clear and not swaying in the whole process, centrifuging at 21 ℃ for 30min at 400g, and increasing the speed of the centrifuge by 1 and reducing the speed by 0. The inside of the tube is divided into three layers after centrifugation, the middle layer is a lymphocyte layer, the middle layer is sucked into a new 50mL centrifuge tube, PBS is filled up to 50mL after the suction is finished, and the tube is centrifuged for 10min with 500 g. The supernatant was discarded, 1mL of PBS was added and gently blown up, 40mL of PBS was added, and 250g was centrifuged for 10min.

After centrifugation, the supernatant was discarded and the count was diluted after resuspension with 1mL MACS buffer (0.5%BSA,2.5mM EDTA-PBS).

4.2 purification NK cell isolation

Human T cells were isolated from peripheral blood using a magnetic bead sorting (Miltenyi company, cat. 130092657). The PBMC were collected and discarded to the end of the supernatant every 10 ⁷ After 40. Mu.L of MACS buffer was added to resuspend the cells, 10. Mu.L of NK Cell Biotin-Antibody Cocktail was added, mixed well and incubated at 4℃for 10min. Every 10 ⁷ Each cell was then added with 30. Mu.L of MACS buffer and 20. Mu. L NK Cell MicroBead Cocktail, mixed well and incubated at 4℃for 15min. LS separation column was placed in magnetic field, the separation column was wetted with 3mL MACS buffer, the cell suspension was added to the separation column, 1mL MAnd (3) after the ACS buffer washes the centrifuge tube, adding a separation column, and collecting effluent. The separation column was washed with 3mL MACS buffer and the effluent was collected. After mixing, cells were counted. The cell suspension was centrifuged at 300g at 4℃for 10min and resuspended in medium.

4.3 construction of B7H 6-deficient HCT-15 cells

The sg DNA targeting B7H6 was designed using the crispr cas9 technology database and has the sequence: TACCCATAGACGTGATGTTG (SEQ ID NO: 25), the sg DNA is inserted into the pX330 plasmid by the common molecular biology cloning technology, the constructed plasmid is transfected into HCT-15 cells by the liposome (Invitrogen, cat. L3000008) transfection technology, the B7H6 expression level of the HCT-15 surface is detected by a flow cytometry 48 hours after transfection, limiting dilution is carried out after negative group cells appear, namely, the diluted density is 4 cells per milliliter after cell digestion, the cells are inoculated into 96-well flat bottom plates, 200 mu L of the cells are added into each well, after the cells are obviously formed into single cell clusters after 2 weeks of culture, the expression level of the B7H6 on the surface of each cloned cell is detected by the flow cytometry, and the full negative cells are HCT-15 cells meeting the requirements.

4.4 in vitro killing detection of adherent tumor cells

Wild type HCT-15 and B7H6 deficient HCT-15 cells were digested and counted to adjust the cell density to 2X 10 ⁵ /mL. The RTCA instrument (agilent) was turned on, instrument type DP was selected, experimental mode was selected, and 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 carried out, 50. Mu.L of purified NK cells (4X 10) ⁵ /mL) and add a gradient of bispecific antibody, click on, analyze after a period of time.

Specific experimental results are shown in fig. 4, and as the concentration of the CD16 xb 7H6 bispecific antibody increases, the killing efficiency of NK cells against wild type HCT-15 cells gradually increases, but the killing efficiency against B7H6 deficient HCT-15 cells is not significantly affected, which indicates that the recombinant CD16 xb 7H6 bispecific antibody can well promote NK cells to specifically kill b7h6+ tumor cells.

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 the amino acid sequence of the CDR sequences:

CD16 antibody variable region CDR sequences: a light chain CDR sequence shown in SEQ ID NO 1-3, and a heavy chain CDR sequence shown in SEQ ID NO 4-6; and

B7H6 antibody variable region CDR sequences: and the light chain CDR sequence shown in SEQ ID NO 7-9 and the heavy chain CDR sequence shown in SEQ ID NO 10-12.

2. The recombinant antibody according to claim 1, characterized in that it comprises a variable region of a CD16 antibody, the amino acid sequence of the light chain variable region of said CD16 antibody is shown in SEQ ID No. 13 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 14.

3. The recombinant antibody according to claim 1, wherein the recombinant antibody comprises a variable region of a B7H6 antibody, the amino acid sequence of the light chain variable region of the B7H6 antibody is shown in SEQ ID No. 15 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 16.

4. The recombinant antibody according to claim 1, further comprising a linking peptide.

5. The recombinant antibody according to claim 4, wherein the amino acid sequence of the connecting peptide is shown in SEQ ID NO. 17.

6. The recombinant antibody according to claim 4, wherein the N-terminus of the connecting peptide is linked to the C-terminus of the CD16 antibody light chain variable region and the C-terminus of the connecting peptide is linked to the N-terminus of the CD16 antibody heavy chain variable region.

7. The recombinant antibody according to claim 4, wherein the N-terminus of the linker peptide is linked to the C-terminus of the B7H6 antibody light chain variable region and the C-terminus of the linker peptide is linked to the N-terminus of the B7H6 antibody heavy chain variable region.

8. The recombinant antibody according to claim 1, further comprising a first Fc region and a second Fc region, at least a portion of said first Fc region and said second Fc region being derived from at least one of a murine antibody, a primate-source antibody, or a mutant thereof.

9. The recombinant antibody according to claim 8, wherein at least a portion of the first and second Fc regions are derived from human IgG or a mutant thereof.

10. The recombinant antibody according to claim 9, wherein at least a portion of the first and second Fc regions are derived from human IgG1 or a mutant thereof.

11. The recombinant antibody according to claim 8, wherein said first Fc region has at least one of a S384C mutation and a T396W mutation as compared to a wild-type IgG1 Fc region.

12. The recombinant antibody according to claim 8, wherein said second Fc region has at least one of a Y380C mutation, a T397S mutation, an L399A mutation, and a Y408V mutation compared to a wild type IgG1 Fc region.

13. The recombinant antibody according to any one of claims 8-12, wherein the amino acid sequence of said first Fc region is shown in SEQ ID No. 18 and the amino acid sequence of said second Fc region is shown in SEQ ID No. 19.

14. A recombinant antibody having the sequence of SEQ ID NO:20 and 21.

15. A nucleic acid molecule encoding the recombinant antibody of any one of claims 1-14.

16. The nucleic acid molecule of claim 15, having the sequence of SEQ ID NO:26 and 27.

17. An expression vector carrying the nucleic acid molecule of claim 15 or 16.

18. A recombinant cell carrying the nucleic acid molecule of claim 15 or 16, the expression vector of claim 17 or the recombinant antibody of any one of claims 1-14.

19. A composition comprising the recombinant antibody of any one of claims 1-14, the nucleic acid molecule of claim 15 or 16, the expression vector of claim 17, or the recombinant cell of claim 18.

20. Use of the recombinant antibody of any one of claims 1-14, the nucleic acid molecule of claim 15 or 16, the expression vector of claim 17, the recombinant cell of claim 18, or the composition of claim 19 in the manufacture of a medicament for treating or preventing cancer, which is colorectal cancer.

21. A medicament, comprising: the recombinant antibody of any one of claims 1-14, the nucleic acid molecule of claim 15 or 16, the expression vector of claim 17, the recombinant cell of claim 18, or the composition of claim 19, for use in the treatment or prevention of cancer, which is colorectal cancer.