CN115368464A

CN115368464A - Bispecific antibodies and uses thereof

Info

Publication number: CN115368464A
Application number: CN202210850693.8A
Authority: CN
Inventors: 曹国帅; 成赢; 李洋洋; 武玉伟
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2022-11-22

Abstract

The invention provides a bispecific antibody and application thereof, wherein the antibody comprises: the method comprises the following steps: a first antigen binding region having binding activity for a CD3 molecule; and a second antigen-binding region having binding activity for a CD33 molecule. The bispecific antibody prepared by the invention can simultaneously target CD3 and CD33, thereby mediating the killing of T cells on tumor cells and having stronger tumor inhibition capability.

Description

Bispecific antibodies and uses thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a bispecific antibody and application thereof, and more particularly relates to a recombinant antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a composition, application of the recombinant antibody or an antigen binding fragment or the nucleic acid molecule or the expression vector or the recombinant cell or the composition in preparation of a medicament, the medicament, application of the recombinant antibody in preparation of a kit and the kit.

Background

Cancer is a disease seriously threatening the life health of people, and the incidence rate and the death rate of cancer are increasing in the world in recent years. The existing cancer treatment methods comprise surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, macromolecule immunotherapy and other treatment methods, but the methods only play a limited role in part of cancer patients, and the cancer still has a problem of disturbing the life health of human beings.

Acute myeloid leukemia is a cancer caused by uncontrolled proliferation of myeloid precursor cells. The incidence of acute marrow is positively correlated with age, with the highest incidence among the 60-70 year old population. The standard treatment for acute marrow is bone marrow transplantation after chemotherapy, but the five-year productivity of patients over 60 years is only 10% due to the inability of elderly patients to tolerate the side effects of chemotherapy. Because gene mutation of tumor cells of acute myeloid patients has heterogeneity, the small molecule targeted drug can only be applied to a few patients with corresponding gene mutation, which severely limits the applicability of the drug.

CD33 is a type I transmembrane protein that is expressed on the surface of myeloid lineage cells, myeloid lineage precursor cells (tumor cells) in acute myeloid patients, but not on the surface of normal stem cells, and thus a therapy targeting CD33 is a potential universal therapy for acute myeloid patients. For example, mylotarg is an antibody-conjugated drug consisting of a CD 33-targeting monoclonal antibody linked to a cytotoxic calicheamicin, essentially using antibodies to target tumor cells for delivery of chemotherapeutic drugs. Clinically, the medicine is found to cause serious adverse reactions including hepatotoxicity (including venous occlusive diseases), infusion-related reactions (including anaphylactic reactions), bleeding and the like.

Thus, there is still a need to further develop bispecific antibodies with low immunogenicity, low toxicity and strong affinity.

Disclosure of Invention

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

CD33 is a type I transmembrane protein that is expressed on the surface of myeloid lineage cells, myeloid progenitor cells (tumor cells) in acute myeloid patients, but not on the surface of normal stem cells, and thus a therapy targeting CD33 is a potential therapy for myeloid leukemia.

The CD3 antibody single-chain variable fragment (ScFv) can be combined with T cells, the inventor designs a bispecific antibody targeting CD3 and CD33, and obtains the dominant bispecific antibody CD3 xCD 33 after a large amount of experimental screening, wherein the bispecific antibody can be simultaneously combined with CD33 positive tumor cells and T cells, effectively mediates the killing of the T cells on the tumor cells, and has stronger anti-cancer capability.

Accordingly, in a first aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the invention, comprising: a first antigen binding region having binding activity for a CD3 molecule; and a second antigen-binding region having binding activity for a CD33 molecule. The recombinant antibody provided by the embodiment of the invention can be combined with CD3 and CD33 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has strong tumor inhibition capability.

In a second aspect of the invention, a nucleic acid molecule is presented. According to an embodiment of the invention, the nucleic acid molecule encodes the recombinant antibody of the first aspect. The recombinant antibody coded by the nucleic acid according to the embodiment of the invention can be simultaneously combined with CD3 and CD33, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a third aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, the nucleic acid molecule of the second aspect is carried. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequence is one or more control sequences that direct the 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 suitable host cell, and the recombinant antibody can be simultaneously combined with CD3 and CD33, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a fourth aspect of the invention, a method of producing the recombinant antibody of the first aspect is provided. According to an embodiment of the invention, comprising: introducing the expression vector of the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion so as to obtain the recombinant antibody. The recombinant antibody can be effectively obtained according to the method provided by the embodiment of the invention, can be simultaneously combined with CD3 and CD33, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a fifth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell carries the nucleic acid of the second aspect, or the expression vector of the third aspect, or is capable of expressing the recombinant antibody of the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some embodiments of the present invention, the recombinant cell can efficiently express the recombinant antibody in a large amount under appropriate conditions, and the recombinant antibody can be simultaneously combined with CD3 and CD33, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor inhibition ability.

In a sixth aspect of the invention, a composition is provided. According to an embodiment of the invention, comprising: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and CD33 protein molecules, and effectively mediate killing effect of T cells on tumor cells, and a composition, such as a food composition, a pharmaceutical composition, and the like, comprising the recombinant antibody or a nucleic acid molecule capable of expressing the recombinant antibody, an expression vector, or a recombinant cell, also has a significant effect of treating or preventing tumors.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect, a nucleic acid molecule 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 for the manufacture of a medicament for the treatment or prevention of myeloid cell leukemia and CD33 positive cancer. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and CD33 protein molecules, effectively mediate the killing effect of T cells on tumor cells, and a series of drugs comprising the recombinant antibody also have significant effects on treating or preventing cancer.

In an eighth aspect of the invention, a medicament is presented. According to an embodiment of the invention, the medicament comprises: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 on the surface of immune cells and CD33 protein molecules on the surface of tumor cells, and effectively mediate the killing effect of T cells on tumor cells, so that a drug comprising the recombinant antibody or a series of substances capable of expressing the recombinant antibody also has a significant effect of treating or preventing cancer.

In a ninth aspect, the present invention proposes the use of a recombinant antibody, nucleic acid molecule, expression vector or recombinant cell as described above for the preparation of a kit for the detection of CD3 and/or CD33. The recombinant antibody can be combined with CD3 and/or CD33 protein, under appropriate conditions, the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody, and a kit can be prepared by the recombinant antibody, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting CD3 and/or CD33. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or CD33 protein in biological samples.

In a tenth aspect of the invention, a kit is presented. According to an embodiment of the invention, the kit comprises the recombinant antibody as described above. The recombinant antibody provided according to the embodiments of the present invention can bind to CD3 and/or CD33 protein, and the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody under suitable conditions, and a kit can be prepared therefrom, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting CD3 and/or CD33. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or CD33 protein in a biological sample, and can also be used for judging the state of an individual, such as judging whether the CD33 level of the individual is higher or lower than a normal level after obtaining the CD33 level of the individual.

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 above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic structural view of a CD3 XCD 33 recombinant bispecific antibody according to an embodiment of the present invention, in which the linking site of a linking peptide is not shown, and a first heavy chain constant region (left side) and a second heavy chain constant region (right side) of the recombinant bispecific antibody are linked by a knob-into-hole structure;

FIG. 2 is a graph showing the results of the detection of the binding ability of the CD3 XCD 33 recombinant bispecific antibody to the CD3E & D protein according to the embodiment of the present invention;

FIG. 3 is a graph showing the results of measuring the binding ability of a CD3 XCD 33 recombinant bispecific antibody to Jurkat T cells according to an embodiment of the present invention;

FIG. 4 is a graph showing the results of measuring the binding ability of the CD3 XCD 33 recombinant bispecific antibody to human peripheral blood CD8+ T cells according to the example of the present invention;

FIG. 5 is a graph showing the results of measuring the binding ability of a CD3 XCD 33 recombinant bispecific antibody to CHO-K1-CD33 cells according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of examining the binding ability of a CD3 XCD 33 recombinant bispecific antibody to acute myeloid HL-60 tumor cells according to an embodiment of the present invention;

FIG. 7 is a graph showing the results of detection of activation of Jurkat-NFAT-lucia reporter cells by a CD3 XCD 33 recombinant bispecific antibody according to an embodiment of the present invention;

FIG. 8 is a graph of the results of a CD3 XCD 33 recombinant bispecific antibody that promotes PBMC killing of A375-CD33 tumor cells according to an embodiment of the 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 with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

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

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, 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.

Herein, "bispecific antibody" refers to a peptide obtained by linking a peptide chain specifically recognizing different protein molecules to a peptide chain of a heavy chain constant region and/or a light chain constant region, respectively, wherein both chains of the heavy chain constant region are linked by a knob-in-hole structure.

A "Knob-in-hole structure" herein is the formation of a Knob (Knob) knot (hole) mutation in the CH3 region of the antibody heavy chain constant region to facilitate heavy chain engagement and heterodimer formation, for example, as in the present application, by mutating the amino acid in the CH3 region of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y C mutation in one chain, i.e., "hole"; T366W, S C mutation in the other chain, i.e., "Knob").

As used herein, "operably linked" refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences, and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene.

In a first aspect of the invention, the invention provides a recombinant antibody comprising: a first antigen binding region having binding activity for a CD3 molecule; and a second antigen-binding region having binding activity for a CD33 molecule. The recombinant antibody provided by the embodiment of the invention can be combined with CD3 and CD33 simultaneously, effectively mediates the killing effect of T cells on tumor cells, and has strong tumor inhibition capability.

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 first antigen-binding region comprises a CD3 single-chain antibody, the CD3 single-chain antibody comprises a CD3 antibody heavy chain variable region and a CD3 antibody light chain variable region, the C-terminus of the CD3 antibody heavy chain variable region is linked to the N-terminus of the CD3 antibody light chain variable region; or the C end of the CD3 antibody light chain variable region is connected with the N end of the CD3 antibody heavy chain variable region.

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 1-3.

According to some embodiments of the invention, the CD3 antibody light chain variable region comprises: a light chain CDR set forth in any one of SEQ ID NOs 4-6.

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises a heavy chain variable region as set forth in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO:3, CDR1, CDR2, CDR3 sequences shown in.

GFTFNTYA(SEQ ID NO：1)。

IRSKYNNYAT(SEQ ID NO：2)。

VRHGNFGNSYVSWFAY(SEQ ID NO：3)。

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises a heavy chain variable region as set forth in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO:3, CDR1, CDR2, CDR3 sequences;

according to some embodiments of the invention, the CD3 antibody light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6, CDR1, CDR2, CDR3 sequences shown in;

TGAVTTSNY(SEQ ID NO：4)。

GTN(SEQ ID NO：5)。

ALWYSNLWV(SEQ ID NO：6)。

according to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises: amino acid sequence shown as SEQ ID NO. 28

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS(SEQ ID NO：28)。

According to some embodiments of the invention, the CD3 antibody light chain variable region comprises: 31, SEQ ID NO.

ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO：31)。

According to some embodiments of the invention, the CD3 single-chain antibody further comprises a linker peptide 1, wherein the N-terminus of the linker peptide 1 is linked to the C-terminus of the heavy chain variable region of the CD3 antibody, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the light chain variable region of the CD3 antibody; or the N end of the connecting peptide 1 is connected with the C end of the CD3 antibody light chain variable region, and the C end of the connecting peptide 1 is connected with the N end of the CD3 antibody heavy chain variable region.

According to some embodiments of the invention, the linker peptide 1 comprises SEQ ID NO: 16.

GGGGSGGGGSGGGGS(SEQ ID NO：16)。

According to some embodiments of the invention, the CD3 single chain antibody comprises the amino acid sequence shown in SEQ ID NO 13.

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO：13)。

According to some embodiments of the invention, the first antigen binding region further comprises a first heavy chain constant region, wherein the C-terminus of the CD3 single chain antibody is linked to the N-terminus of the first heavy chain constant region.

According to some embodiments of the invention, the first heavy chain constant region comprises a first hinge region and a first Fc peptide fragment.

According to some embodiments of the invention, the first Fc fragment comprises a first CH2 region and a first CH3 region, and the C-terminus of the first CH2 region is linked to the N-terminus of the first CH3 region.

According to some embodiments of the invention, the C-terminus of the first hinge region is linked to the N-terminus of the first Fc peptide fragment.

According to some embodiments of the invention, the first hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the first CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the first CH3 region has a T366W and/or S354C mutation compared to a CH3 region fragment of a human wild type IgG 1.

The amino acid sequence of the humanized wild-type IgG1 (L234A/L235A) antibody is as follows:

PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:30)。

according to some embodiments of the invention, the first antigen binding region further comprises a linker peptide 2, the N-terminus of the linker peptide 2 is linked to the C-terminus of the CD3 single chain antibody, and the C-terminus of the linker peptide 2 is linked to the N-terminus of the first heavy chain constant region.

According to some embodiments of the invention, the linker peptide 2 comprises SEQ ID NO:17, or a pharmaceutically acceptable salt thereof.

GGGGS(SEQ ID NO：17)。

According to some embodiments of the invention, the first heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 18.

PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：18)。

According to some embodiments of the invention, the second antigen binding region comprises a first peptide chain and a second peptide chain, the first peptide chain comprising: CD33 antibody heavy chain variable region.

According to some embodiments of the invention, the CD33 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 7-9.

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises a heavy chain variable region as set forth in SEQ ID NOs: 7. the amino acid sequence of SEQ ID NO: 8. SEQ ID NO:9, CDR1, CDR2, CDR3 sequences shown.

GYTITDSN(SEQ ID NO：7)。

IYPYNGGT(SEQ ID NO：8)。

VNGNPWLAY(SEQ ID NO：9)。

According to some embodiments of the invention, the variable region of the CD33 antibody heavy chain comprises the amino acid sequence set forth in SEQ ID NO 14.

EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS(SEQ ID NO：14)。

According to some embodiments of the invention, the first peptide chain further comprises a second heavy chain constant region, wherein the C-terminus of the CD33 antibody heavy chain variable region is linked to the N-terminus of the second heavy chain constant region.

According to some specific embodiments of the invention, the second heavy chain constant region comprises: a CH1 region, a second hinge region, and a second Fc peptide fragment.

According to some embodiments of the invention, the C-terminus of the CH1 region is linked to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is linked to the N-terminus of the second Fc peptide fragment.

According to some specific embodiments of the invention, the second Fc peptide fragment comprises: a second CH2 and a second CH3 region, wherein the C-terminal of the second CH2 region is connected with the N-terminal of the second CH3 region.

According to some embodiments of the invention, the CH1 region is a CH1 region of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second CH3 region is a CH3 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some specific embodiments of the invention, the second CH3 region has at least one of the T366S, L368A, Y407V, Y C mutations compared to a CH3 region fragment of human wild type IgG 1.

According to some embodiments of the invention, the second heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 19.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：19)。

According to some embodiments of the invention, the second peptide chain comprises: CD33 antibody light chain variable region.

According to some embodiments of the invention, the CD33 antibody light chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 10-12.

According to some embodiments of the invention, the CD33 antibody light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO:12, CDR1, CDR2, CDR3 sequences.

ESLDNYGIR(SEQ ID NO：10)。

AAS(SEQ ID NO：11)。

QQTKEVPWS(SEQ ID NO：12)。

According to some embodiments of the invention, the CD33 antibody light chain variable region comprises: 15, or an amino acid sequence as shown in SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.

DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK(SEQ ID NO：15)。

According to some embodiments of the invention, the second peptide chain further comprises a light chain constant region, wherein the C-terminus of the CD33 antibody light chain variable region is linked to the N-terminus of the light chain constant region.

According to some embodiments of the invention, the light chain constant region is a wild-type light chain constant region of human, primate or murine origin.

According to some embodiments of the invention, the antibody light chain constant region is a human Kappa light chain constant region.

According to some embodiments of the invention, the light chain constant region is a light chain constant region of a human wild-type IgG 1.

According to some embodiments of the invention, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 29.

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：29)。

According to some embodiments of the invention the first antigen binding region and the second antigen binding region are linked by a knob-into-hole structure.

According to some embodiments of the invention the knob-into-hole structure is formed by a mutation of at least one of a T366W and/or S354C mutation of the first CH3 region and a T366S, L368A, Y407V, Y349 9C mutation of the second CH3 region.

According to some embodiments of the invention, the first peptide chain and the second peptide chain are linked by an inter-bond disulfide bond.

According to some embodiments of the invention, the recombinant antibody comprises the amino acid sequence shown in SEQ ID NO 20-22.

The first antigen binding region comprises the amino acid sequence:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：20)。

second antigen binding region-the first peptide chain has the amino acid sequence shown below:

EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：21)。

second antigen binding region-second peptide chain has the amino acid sequence shown below:

DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：22)。

in a second aspect of the invention, the invention provides a nucleic acid molecule encoding the recombinant antibody of the first aspect. The recombinant antibody coded by the nucleic acid molecule can be simultaneously combined with CD3 and CD33, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capacity.

According to some embodiments of the invention, the nucleic acid has the sequence of SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO: 25.

The nucleic acid molecule encoding the first antigen binding region comprises the nucleotide sequence:

gaagtgcaattgttggaaagtggaggtgggcttgtccaacctggcggttctctgaagttgagctgcgccgcatctggcttcaccttcaacacatatgccatgaactgggttcggcaggcaccaggaaaggggttggagtgggtcgcacggattaggtctaagtacaataattacgcaacttattacgccgactccgtgaaggacaggtttacaataagtcgtgacgattctaaaaatactgcctacttgcagatgaataatctcaaaacagaggacaccgcagtctactactgtgtgcgacacggaaactttggtaactcatacgtttcttggttcgcttattggggccaggggactttggtgaccgtttctagtggaggaggtggttctggaggtggagggtctggcggaggtggaagtgaattggtggtgactcaggaacctagcctcacagtgtctcctggcggaacagtcacattgacctgcagatcttccaccggcgccgtcaccacctccaattacgctaactgggtgcagcagaagcctggtcaggcacctaggggattgatcgggggcactaacaagagagctcctggaactccagccaggttcagtggttccttgctcggcgggaaggcagcacttactctgagcggagtccagcctgaggacgaggctgagtactactgtgccctgtggtacagcaatctgtgggtgtttggcgggggcacaaaactcaccgtgcttggaggtggcggatctccaaagtcttgcgacaagacacatacatgccctccatgtcctgcacctgaagctgccggtggcccaagtgttttcttgttcccccctaaacctaaagatacactgatgatttccaggacaccagaggtgacatgcgtagttgtggatgtatctcacgaggatccagaggtgaagttcaattggtacgtagacggcgtagaagtgcataacgccaagacaaagccacgggaggagcagtataattcaacctaccgcgtggtttctgtactgaccgtgctccatcaagattggctcaatggtaaggagtacaaatgcaaggtcagcaataaagcacttcccgcacccatcgagaaaacaatctccaaggcaaaaggacagccccgcgagcctcaggtttatactctccctccatgcagagaggagatgacaaagaaccaggtgtcactttggtgccttgtcaagggcttctatccatctgatattgccgttgagtgggagtccaacgggcagcctgagaataattataagactactccccccgttcttgattccgacggcagcttctttctctacagcaagcttaccgtcgataagtcaagatggcaacaggggaatgtgttctcttgctccgtgatgcacgaagctttgcataaccattatacccaaaagagtctgtctctctcccccggaaaa(SEQ ID NO：23)。

the nucleic acid molecule encoding the second antigen binding region-the first peptide chain comprises the following nucleotide sequence:

gaagtccaactggtacagagtggcgctgaagtgaagaaacccggatcttctgtcaaggtgtcctgcaaggcatccggatacaccatcactgactccaacattcattgggtgagacaagcaccaggccagtctctcgagtggatcggctacatatacccctacaacggaggtacagactacaaccagaagttcaagaatagggccacactgacagtggacaaccctactaacactgcttatatggagctgtccagtctgcgtagcgaggataccgccttttactattgtgtgaatggcaatccttggcttgcctactggggccagggaaccttggtaaccgtgagctccgcaagtaccaagggtccctccgtctttccattggccccatcttctaagtcaacatcaggcggtactgcagcactcgggtgtctggtgaaggactactttccagagcctgtcacagtcagctggaacagcggtgctctgacaagtggggtacacactttcccagctgtgcttcagagttccgggctctattcactgagcagtgtggtgactgtccccagcagtagcctgggtactcagacctacatctgtaacgtgaatcataaaccttcaaacacaaaggtcgataagaaggttgaaccaaagagctgcgacaaaactcacacttgtcctccatgtcccgcaccagaagctgctggtggaccctcagtctttctgtttccccccaaacccaaagataccctgatgatttccaggacccctgaggtaacctgcgtggtggtggacgtttctcacgaagaccccgaggtgaagttcaactggtacgtggacggcgtggaggtgcataacgccaagaccaagcctcgcgaagagcaatataactccacatacagagtggtgagcgttttgaccgtgttgcaccaggactggctcaacgggaaggagtacaaatgtaaagtttcaaataaagccttgcctgctccaatcgagaaaaccatctccaaggccaaaggccaaccccgagaaccacaggtgtgcaccttgcctcccagtcgagaggaaatgacaaagaatcaggtgtctttgagctgtgctgtgaaaggcttctatccatccgacatcgcagtggagtgggaaagtaacggtcagccagagaacaattataagaccaccccacccgtgttggattctgacggctcttttttcctggttagcaaattgactgtggacaagtcccgctggcagcaggggaacgtgttttcttgtagtgtcatgcatgaagctctgcataatcactatactcagaagagcctgagcctgtctccagggaag(SEQ ID NO：24)。

the gene encoding the second antigen-binding region-the first peptide chain has a nucleotide sequence shown below:

gacattcagctgactcagagcccttccacactgagtgcttctgttggcgacagggtgaccataacttgccgagcttccgagtccttggataactacggcattaggtttctcacttggtttcaacagaagccaggcaaggctccaaagctcctgatgtacgccgctagcaaccagggttctggtgtgccttctcggttctctggcagcgggagcgggacagaattcacactgacaatatcatccttgcagccagatgacttcgccacttattactgtcagcagaccaaggaggtgccctggagcttcggccaggggaccaaagtggaggtcaagcgcaccgtggctgctccaagcgttttcatttttccaccaagtgatgagcaactgaagtcaggaacagccagcgtggtgtgtctcttgaacaatttctatccaagagaggcaaaagtgcagtggaaggtggataatgctcttcagagcggtaactcacaggagtctgtaaccgaacaggactccaaagactccacttactccctctcctccaccctcactctgagtaaagccgactacgagaaacacaaagtttacgcctgcgaggtcacccatcagggcttgtccagccctgtgaccaagtcctttaacagaggcgagtgc(SEQ ID NO:25)。

it is to be noted that, with respect to the nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that any one or two of the complementary double strands are actually included. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences in the present application include DNA forms or RNA forms, one of which is disclosed, meaning that the other is also disclosed.

In a third aspect, the present invention provides an expression vector carrying the nucleic acid molecule of the second aspect. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequence is one or more control sequences that direct the 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 suitable host cell, and the recombinant antibody can be simultaneously combined with CD3 and CD33, effectively mediates the accurate and efficient killing of T cells on tumor cells, and has stronger tumor inhibition capacity.

In a fourth aspect of the invention, there is provided a method of producing the recombinant antibody of the first aspect, comprising: introducing the expression vector of the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion so as to obtain the recombinant antibody. The method provided by some specific embodiments of the present invention can effectively obtain the recombinant antibody, and the recombinant antibody can be simultaneously combined with CD3 and CD33, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor inhibition capability. According to some embodiments of the present invention, the cell is not particularly limited, and both prokaryotic cells and eukaryotic cells can be used, and the expression efficiency of the recombinant antibody is high when the cell is eukaryotic, such as mammalian cells.

According to some embodiments of the invention, the cell is a eukaryotic cell.

According to some embodiments of the invention, the eukaryotic cell is a mammalian cell. According to some embodiments of the invention, the recombinant antibody is expressed more efficiently when the cell is a eukaryotic cell, such as a mammalian cell.

According to some embodiments of the invention, the eukaryotic cell does not comprise an animal germ cell, a fertilized egg, or an embryonic stem cell.

In a fifth aspect, the present invention provides a recombinant cell carrying the nucleic acid molecule of the second aspect, or the expression vector of the third aspect, or expressing the recombinant antibody of the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some embodiments of the present invention, the recombinant cell can efficiently express a large amount of the recombinant antibody under appropriate conditions, and the recombinant antibody can be simultaneously combined with CD3 and CD33, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor inhibition ability.

It is to be noted that the recombinant cell of the present invention is not particularly limited, and may be a prokaryotic cell, a eukaryotic cell or a phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomyces or proteus mirabilis and the like. The eukaryotic cell can be fungi such as Pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces and trichoderma, insect cells such as meadow armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells and myeloma cells. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

The term "suitable conditions" as used herein means conditions suitable for the expression of the recombinant antibody as described herein. It will be readily understood by those skilled in the art that suitable conditions for recombinant antibody expression 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, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for the expression of the recombinant antibody optimally according to the specific circumstances in the laboratory.

In a sixth aspect of the invention, there is provided a composition comprising: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant antibody according to the embodiments of the present invention can effectively bind to CD3 and CD33 protein molecules and promote T cells to kill tumor cells accurately and efficiently, and compositions, such as food compositions, pharmaceutical compositions, and the like, comprising the recombinant antibody also have significant effects of treating or preventing tumors.

It is to be noted that the compositions include combinations that are separated in time and/or space, as long as they can act together to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in bulk, or separately. When the ingredients contained in the composition are administered separately to a subject, the individual ingredients may be administered to the subject simultaneously or sequentially.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect, a nucleic acid molecule 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 for the manufacture of a medicament for the treatment or prevention of myeloid cell leukemia and CD33 positive cancer. As described above, the recombinant antibody of the embodiment of the invention can effectively bind to CD3 and CD33 protein molecules, so as to promote T cells to kill tumor cells accurately and efficiently, and therefore, drugs containing a series of substances of the recombinant antibody also have significant effects of treating or preventing tumors.

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

according to some specific embodiments of the invention, the CD 33-positive cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

In an eighth aspect of the invention, there is provided a medicament comprising: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and CD33 protein molecules, and promote T cells to kill tumor cells accurately and efficiently, so that a series of drugs containing the recombinant antibody also have significant effects of treating or preventing myeloid leukemia and CD33 positive tumors.

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 medicament is for the treatment or prevention of myeloid leukemia and CD33 positive cancer.

According to some specific embodiments of the invention, the cancer comprises at least one of: the CD 33-positive cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

According to some embodiments of the invention, a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient are included.

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

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at 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 a safe and effective amount of the active ingredient of the invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The dosage form of the medicament of the invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent and sustained release agent. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The medicament is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a 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 by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

In a tenth aspect of the invention, the invention provides a kit comprising a recombinant antibody, nucleic acid molecule, expression vector or recombinant cell as described above. The recombinant antibody provided according to the embodiments of the present invention can bind to CD3 and/or CD33 protein, and the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody under suitable conditions, and a kit can be prepared therefrom, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting CD3 and/or CD33. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or CD33 protein in a biological sample, and can also be used for judging the individual state, such as judging whether the CD33 level of the individual is higher or lower than the normal level after obtaining the CD33 level of the individual.

According to some embodiments of the invention, the kit is for detecting CD3 and/or CD33.

The embodiments will be described in detail below. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 preparation of CD3 × CD33 bispecific antibody

The production of bispecific antibodies was carried out in this example, following the following experimental procedures: expicCHO cells (purchased from Thermo Fisher) were cultured in ExpicCHO Expression Medium Medium (purchased from Thermo Fisher, A2910001) at a cell concentration adjusted to 6X 10 ⁶ Per mL, expicHO cell solution was obtained. pTT5 vector (synthesized by Jin Wei, suzhou, inc.) containing three strands of encoding genes (shown in SEQ ID NOS: 23, 24, and 25, respectively) encoding the first antigen-binding region and the second antigen-binding region was added to 2mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. Wherein the first antigen binding region encoding gene comprises a nucleotide sequence encoding a CD3 single-chain antibody (SEQ ID NO: 13), a linker 1 (SEQ ID NO: 16), a linker 2 (SEQ ID NO: 17) and a first heavy chain constant region (SEQ ID NO: 18), the second antigen binding region-first peptide chain encoding gene comprises a nucleotide sequence encoding a CD33 antibody heavy chain variable region (SEQ ID NO: 14) and a second heavy chain constant region (SEQ ID NO: 19), and the second antigen binding region-second peptide chain encoding gene comprises a CD33 antibody light chain variable region and a light chain constant region. 160 μ L Expifeactinamine CHO transfection reagent (Thermofisiher, A29130) was added to 2mL OptiSFM medium to obtain solution B. Then, solution a and solution B were mixed to obtain a transfection mixture, and the transfection mixture was added to 50mL of expichho cell solution in its entirety within 5 minutes. At 37 deg.C,5％CO ₂ After 1 day of incubation under the conditions, 8mL of Feed (Thermo Fisher, A29130), 300. Mu.L of Enhancer (Thermo Fisher, A29130) were added, and the mixture was stirred at 32 ℃ and 5% CO ₂ Culture supernatants were harvested after 9 days of culture under conditions, with 8mL of Feed added on day 5. Bispecific antibody was affinity purified from the culture supernatant using Protein a purification column (GE) to obtain antibody CD3 × CD33. The antibody CD3 XCD 33 has an amino acid sequence as shown in SEQ ID NO:20 (first antigen binding region), SEQ ID NO:21 (second antigen binding region-first peptide chain) and SEQ ID NO:22 (second antigen binding region-second peptide chain).

The first antigen binding region encoding gene comprises a nucleotide sequence as shown below:

gaagtgcaattgttggaaagtggaggtgggcttgtccaacctggcggttctctgaagttgagctgcgccgcatctggcttcaccttcaacacatatgccatgaactgggttcggcaggcaccaggaaaggggttggagtgggtcgcacggattaggtctaagtacaataattacgcaacttattacgccgactccgtgaaggacaggtttacaataagtcgtgacgattctaaaaatactgcctacttgcagatgaataatctcaaaacagaggacaccgcagtctactactgtgtgcgacacggaaactttggtaactcatacgtttcttggttcgcttattggggccaggggactttggtgaccgtttctagtggaggaggtggttctggaggtggagggtctggcggaggtggaagtgaattggtggtgactcaggaacctagcctcacagtgtctcctggcggaacagtcacattgacctgcagatcttccaccggcgccgtcaccacctccaattacgctaactgggtgcagcagaagcctggtcaggcacctaggggattgatcgggggcactaacaagagagctcctggaactccagccaggttcagtggttccttgctcggcgggaaggcagcacttactctgagcggagtccagcctgaggacgaggctgagtactactgtgccctgtggtacagcaatctgtgggtgtttggcgggggcacaaaactcaccgtgcttggaggtggcggatctccaaagtcttgcgacaagacacatacatgccctccatgtcctgcacctgaagctgccggtggcccaagtgttttcttgttcccccctaaacctaaagatacactgatgatttccaggacaccagaggtgacatgcgtagttgtggatgtatctcacgaggatccagaggtgaagttcaattggtacgtagacggcgtagaagtgcataacgccaagacaaagccacgggaggagcagtataattcaacctaccgcgtggtttctgtactgaccgtgctccatcaagattggctcaatggtaaggagtacaaatgcaaggtcagcaataaagcacttcccgcacccatcgagaaaacaatctccaaggcaaaaggacagccccgcgagcctcaggtttatactctccctccatgcagagaggagatgacaaagaaccaggtgtcactttggtgccttgtcaagggcttctatccatctgatattgccgttgagtgggagtccaacgggcagcctgagaataattataagactactccccccgttcttgattccgacggcagcttctttctctacagcaagcttaccgtcgataagtcaagatggcaacaggggaatgtgttctcttgctccgtgatgcacgaagctttgcataaccattatacccaaaagagtctgtctctctcccccggaaaa(SEQ ID NO:23)。

the first antigen binding region-first peptide chain encoding gene comprises a nucleotide sequence as shown below:

gaagtccaactggtacagagtggcgctgaagtgaagaaacccggatcttctgtcaaggtgtcctgcaaggcatccggatacaccatcactgactccaacattcattgggtgagacaagcaccaggccagtctctcgagtggatcggctacatatacccctacaacggaggtacagactacaaccagaagttcaagaatagggccacactgacagtggacaaccctactaacactgcttatatggagctgtccagtctgcgtagcgaggataccgccttttactattgtgtgaatggcaatccttggcttgcctactggggccagggaaccttggtaaccgtgagctccgcaagtaccaagggtccctccgtctttccattggccccatcttctaagtcaacatcaggcggtactgcagcactcgggtgtctggtgaaggactactttccagagcctgtcacagtcagctggaacagcggtgctctgacaagtggggtacacactttcccagctgtgcttcagagttccgggctctattcactgagcagtgtggtgactgtccccagcagtagcctgggtactcagacctacatctgtaacgtgaatcataaaccttcaaacacaaaggtcgataagaaggttgaaccaaagagctgcgacaaaactcacacttgtcctccatgtcccgcaccagaagctgctggtggaccctcagtctttctgtttccccccaaacccaaagataccctgatgatttccaggacccctgaggtaacctgcgtggtggtggacgtttctcacgaagaccccgaggtgaagttcaactggtacgtggacggcgtggaggtgcataacgccaagaccaagcctcgcgaagagcaatataactccacatacagagtggtgagcgttttgaccgtgttgcaccaggactggctcaacgggaaggagtacaaatgtaaagtttcaaataaagccttgcctgctccaatcgagaaaaccatctccaaggccaaaggccaaccccgagaaccacaggtgtgcaccttgcctcccagtcgagaggaaatgacaaagaatcaggtgtctttgagctgtgctgtgaaaggcttctatccatccgacatcgcagtggagtgggaaagtaacggtcagccagagaacaattataagaccaccccacccgtgttggattctgacggctcttttttcctggttagcaaattgactgtggacaagtcccgctggcagcaggggaacgtgttttcttgtagtgtcatgcatgaagctctgcataatcactatactcagaagagcctgagcctgtctccagggaag(SEQ ID NO:24)。

the first antigen binding region-second peptide chain encoding gene comprises a nucleotide sequence shown as follows:

example 2 identification of the binding Capacity of CD3 × CD33 bispecific antibodies to CD3E & D

An ELISA assay was used to determine the binding properties of the CD3 × CD33 antibody obtained in example 1. CD3E & D protein (Acro, CDD-H52W 1) is coated in a 96-well plate, and the strength of signals after the antibody is added is used for judging the binding property of the antibody and CD3E & D.

The CD3E & D protein was diluted to 2. Mu.g/mL with PBS buffer, added to a 96-well plate at a volume of 100. Mu.L/well, and left overnight at 4 ℃. The 96-well plate was aspirated off PBS buffer, the plate was washed 6 times with PBST (0.1% Tween 20 in PBS pH 7.2), then blocked by adding 200. Mu.L/well PBS/10% BSA and incubating at 37 ℃ for 2h. After removing the blocking solution and washing the plate 6 times with PBST, the test CD 3X CD33 antibody and the negative control human IgG antibody were diluted to appropriate concentrations using 100. Mu.L/well of PBST/0.05% BSA, and then incubated at 37 ℃ for 1h. The reaction system was removed and after washing the plate 6 times with PBST, HRP (horseradish peroxidase) -labeled rabbit anti-human IgG secondary antibody (Boshide, BA 1070) was diluted with 100. Mu.L/well of PBST/0.05% BSA and incubated at 37 ℃ for 1h. After the completion of incubation, the plate was washed 6 times with PBST, 80. Mu.L/well of TMB (tetramethylbenzidine) was added, the plate was incubated at room temperature for 3min, and the reaction was terminated by adding 80. Mu.L/well of 4M sulfuric acid. The absorbance was read at 450mm using a microplate reader. The results of the specific experiments are shown in FIG. 2, which indicates that the CD 3X CD33 antibody of the present invention is capable of binding to CD3E & D.

Example 3 identification of the binding Capacity of CD3 × CD33 bispecific antibodies to Jurkat T cells

This example uses flow cytometry to examine the binding properties of the CD3 × CD33 bispecific antibodies described in examples 1 and 2 and is used to determine the binding properties of the bispecific antibody and Jurkat T based on the intensity of the signal after the addition of the bispecific antibody. The specific experimental procedures were as follows:

jurkat T cells were diluted 1X 10 with PBS ⁶ (ii)/mL, added to a 1.5mL EP tube in a volume of 90. Mu.L/tube, to which 10. Mu.L/tube rat serum was added, and blocked at 4 ℃ for 30min; after blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XCD 33 bispecific antibody and human IgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added, incubated at 4 ℃ for 30min, 1mL of PBS was added to the EP tube, centrifuged at 4 ℃ and 100G for 5min, the supernatant was discarded, the pellet was washed once with PBS, the supernatant was discarded after centrifugation, the cells were resuspended in 100. Mu.L/tube of PBS, 1. Mu.L/tube of Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 3, which illustrates that the bispecific CD 3X CD33 antibody of the present invention is capable of binding to Jurkat T cells.

Example 4 identification of the binding Capacity of CD3 × CD33 bispecific antibodies to human peripheral blood T cells

The present example uses flow cytometry to detect the binding characteristics of the above-mentioned bispecific antibody CD3 × CD33, and the signal intensity after the addition of the bispecific antibody is used to determine the binding characteristics of the bispecific antibody and human peripheral blood T cells. The specific experimental procedures were as follows:

human peripheral blood mononuclear cells (Chimaphila sp.) were diluted 5X 10 with PBS ⁶ (ii)/mL, added to a 1.5mL EP tube in a volume of 90. Mu.L/tube, to which 10. Mu.L/tube rat serum was added, and blocked at 4 ℃ for 30min; after blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XCD 33 bispecific antibody and hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added, incubated at 4 ℃ for 30min, 1mL of PBS was added to the EP tube, centrifuged at 4 ℃ and 100G for 5min, the supernatant was discarded, the pellet was washed once with PBS, the supernatant was discarded after centrifugation, the cells were resuspended in 100. Mu.L/tube of PBS, 1. Mu.L/tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M G05) and 1. Mu.L/tube of FITC-labeled mouse anti-human CD8 antibody (Invitrogen, OKT 8) were added thereto, and incubated at 4 ℃ for 30min in the dark. Washing with PBSTwice, centrifuge and discard the supernatant. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results of the assay are shown in FIG. 4, which illustrates that the inventive bispecific antibody CD3 × CD33 is capable of binding to human peripheral blood T cells.

Example 5 identification of the binding Capacity of a CD3 × CD33 bispecific antibody to CHO-K1-CD33 cells

This example uses flow cytometry to examine the binding characteristics of the CD3 XCD 33 bispecific antibody used in example 1 above and uses the intensity of the signal after the addition of the bispecific antibody to determine the binding characteristics of the bispecific antibody and CHO-K1-CD33 cells. The specific experimental procedures were as follows:

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM without double antibody. Media was discarded before transfection and 1mL of fresh DMEM media without double antibody was added. pLVX-EF1a-CD33-IRES-puro (the coding sequence of CD33 protein (SEQ ID NO: 26) is inserted between EcoRI and BamHI sites of pLVX-EF1a-IRES-puro vector), pMD2G, psPAX2 vector (total 3. Mu.g) are added to 200. Mu.L of serum-free DMEM medium in the ratio of 2; after mixing, the mixture was allowed to stand for 16min, and then the whole liquid was added to the above six-well plate in which HEK293T cells were spread. After 6h of culture, the medium was discarded and fresh complete DMEM medium was added for culture. After 48h of transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), to obtain a virus supernatant. Adding all the virus supernatant to the solution containing 1 × 10 ⁴ To 6-well plates of CHO-K1 cells, polybrene (Sigma) was added at a final concentration of 4. Mu.g/mL, and the cells were cultured for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cells were CHO-K1-CD33 cells.

ATGCCTTTGCTTCTGCTTTTGCCTTTGCTTTGGGCAGGAGCCCTTGCCATGGACCCAAACTTTTGGCTCCAGGTGCAGGAATCAGTGACCGTCCAGGAAGGTCTGTGCGTGCTGGTGCCCTGCACCTTTTTCCATCCTATCCCATATTACGACAAAAATAGCCCAGTACACGGATATTGGTTTAGGGAGGGGGCTATTATCTCACGAGATAGCCCCGTGGCTACCAATAAGCTGGACCAGGAGGTACAAGAAGAGACCCAGGGCCGCTTTAGATTGTTGGGTGACCCTTCCAGGAACAACTGTTCTTTGAGTATTGTTGACGCCCGAAGGCGCGACAACGGTAGCTACTTCTTCCGCATGGAGCGCGGATCCACCAAATACTCCTATAAGAGCCCTCAATTGAGTGTCCATGTGACAGACCTGACCCACAGGCCAAAAATACTTATCCCAGGCACCCTGGAGCCAGGGCACTCAAAGAATCTGACTTGCTCTGTCTCTTGGGCCTGTGAACAGGGTACACCCCCTATCTTTAGCTGGTTGTCAGCTGCCCCTACCTCCCTGGGACCTAGAACCACACATTCCAGCGTACTTATTATTACTCCCAGGCCCCAGGATCATGGCACCAACCTGACTTGCCAGGTTAAATTCGCCGGGGCTGGGGTAACAACAGAGAGAACCATCCAGTTGAATGTGACTTATGTGCCACAGAATCCTACCACCGGAATCTTTCCTGGAGACGGATCTGGCAAGCAAGAGACCAGGGCAGGAGTAGTACATGGCGCTATTGGCGGTGCTGGAGTGACAGCTCTGTTGGCCCTGTGTTTGTGTCTGATTTTCTTCATCGTCAAAACTCACCGTAGGAAGGCTGCACGAACTGCCGTCGGCCGTAACGATACTCATCCAACTACCGGTAGCGCTAGTCCTAAGCATCAGAAGAAGAGCAAGTTGCATGGACCAACAGAGACTAGCAGTTGTAGCGGGGCTGCACCTACTGTCGAAATGGATGAGGAACTTCACTATGCCTCACTGAACTTCCACGGCATGAACCCCTCTAAGGACACCTCAACTGAATACTCAGAAGTCCGAACTCAG(SEQ ID NO:26)。

MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ(SEQ ID NO:27)。

CHO-K1-CD33 cells were diluted 1X 10 with PBS ⁶ To 1.5mL of EP tube was added 90. Mu.L/tube, 10. Mu.L/tube of rat serum was added, and the mixture was blocked at 4 ℃ for 30min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XCD 33 bispecific antibody and human IgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100g at 4 ℃ for 5min, the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube of PBS, and after resuspension, a 1. Mu.L/tube of Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. Resuspending the cells in 200. Mu.L/tube PBS, detecting with flow cytometer, and the specific experimental results are shown in FIG. 5, further showing the dual-purpose of the present inventionThe hetero-antibody CD3 × CD33 is capable of binding to CHO-K1-CD33 cells.

Example 6 identification of the binding Capacity of CD3 × CD33 bispecific antibodies to human acute myeloid leukemia HL-60 tumor cells

This example uses flow cytometry to examine the binding properties of the CD3 × CD33 bispecific antibody described in the above examples, and is used to determine the binding properties of the bispecific antibody and human acute myeloid leukemia HL-60 tumor cells based on the intensity of the signal after the addition of the bispecific antibody. The specific experimental procedures were as follows:

HL-60 cells were diluted to 1X 10 with PBS ⁶ To 1.5mL of EP tube was added 90. Mu.L/tube, 10. Mu.L/tube of rat serum was added, and the mixture was blocked at 4 ℃ for 30min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XCD 33 bispecific antibody and human IgG antibody (IgG 1, biolegend, QA16A 12) 10. Mu.L/tube were added and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100g at 4 ℃ for 5min, the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube of PBS, and after resuspension, a 1. Mu.L/tube of Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 6, which further shows that the bispecific antibody CD 3X CD33 of the present invention can bind to human acute myeloid HL-60 cells.

Example 7: identification of CD3 × CD33 bispecific antibodies to promote activation of Jurkat-NFAT-lucia reporter cells

This example identifies the ability of the CD3 XCD 33 bispecific antibody to cross-link CD33 on the surface of target cells and CD3 on the surface of effector cells, thereby activating T cells, using the Jurkat-NFAT-lucia reporter method. The relative chemiluminescence signal (RLU) intensity was used to determine the ability of the bi-specific antibody to activate T cells by bridging CD 33-positive target cells and T cells:

(1) CHO-K1-CD33 cells described in example 5 were diluted to 1X 10 with complete RPMI-1640 medium ⁵ a/mL, added to a 96-well plate,the volume of addition was 100. Mu.L/well.

(2) CD3 XCD 33 bispecific antibody and negative control human IgG antibody (IgG 1, biolegend, QA16A 12) were diluted to 500. Mu.g/mL, 100. Mu.g/mL, 20. Mu.g/mL, 4. Mu.g/mL, 0.8. Mu.g/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, respectively, in complete RPMI-1640 medium and added to a 96-well plate at a volume of 20. Mu.L/well.

(3) Jurkat-NFAT-lucia cells (purchased from Invivogen, jktl-NFAT) were diluted to 1.25X 10 with complete RPMI-1640 medium ⁵ PermL, added to a 96-well plate in a volume of 80. Mu.L/well.

(4) The reaction system obtained in step (3) was subjected to 5% CO at 37 ℃ C ₂ Culturing in an incubator for 24h.

(5) mu.L of culture supernatant was aspirated and added to a 96-well plate, and then luciferase substrate (Invivogen) was added to the plate in a volume of 50. Mu.L/well.

(6) Chemiluminescence was detected using a multifunctional microplate reader.

The specific experimental results are shown in fig. 7, and further show that the bispecific antibody CD3 × CD33 of the present invention can bridge CD33 positive target cells and T cells, promoting T cell activation.

Example 8 CD3 × CD33 bispecific antibodies promote PBMC killing of CD 33-positive tumor cells

This example examined the effect of the bispecific antibody CD3 × CD33 obtained in example 1 on killing A375-CD33 cells by PBMC, and examined by constructing a reaction system of the tumor cells + PBMC + bispecific antibody at different concentrations, as follows:

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. Media was discarded before transfection and 1mL of fresh DMEM media without double antibody was added. pLVX-EF1a-CD33-IRES-puro (the coding sequence of CD33 protein (SEQ ID NO: 26) is inserted between EcoRI and BamHI sites of pLVX-EF1a-IRES-puro vector), pMD2G, psPAX2 vector (total 3. Mu.g) are added to 200. Mu.L of serum-free DMEM medium in the ratio of 2An amino acid sequence; after mixing, the mixture was left to stand for 16min, and then the whole liquid was added to the above six-well plate plated with HEK293T cells. After 6h of culture, the medium was discarded and fresh complete DMEM medium was added for culture. After 48h of transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), to obtain a virus supernatant. Adding the whole virus supernatant to a medium containing 1X 10 ⁴ A375 cells (purchased from Pronosaxil, CL-0014) were plated in 6-well plates and incubated for 12h with polybrene (Sigma) at a final concentration of 4. Mu.g/mL. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cells are A375-CD33 cells.

(1) Adding complete RPMI-1640 culture medium into a 16-well RTCA plate according to the volume of 50 mu L/well, and performing on-machine calibration;

(2) A375-CD33 cells were diluted to 2X 10 with complete RPMI-1640 medium ⁵ mL, added to the RTCA plate obtained in step (1) in a volume of 50. Mu.L/well, respectively, and then subjected to 5% CO at 37 ℃ ₂ Detecting the cell coefficient for 24h by using an xCELLigence RTCA TP device under the condition;

(3) Diluting CD3 XCD 33 bispecific antibody and control 4-1BB XCD 33 bispecific antibody (produced in this laboratory) to a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng/mL) in complete RPMI-1640 medium, adding to the RTCA plate obtained in step (2), and adding in a volume of 20. Mu.L/well;

(4) PBMC (Cyanin Biopsis) were diluted to 1.25X 10 with complete RPMI-1640 medium ⁶ Adding the solution to the RTCA plate obtained in the step (3) in a volume of 80 mu L/hole;

(5) The reaction system obtained in step (4) was subjected to 5% CO at 37 ℃ C ₂ The cell coefficient was measured using an xcelgene RTCA TP device for 48h.

The specific experimental results are shown in fig. 8, and further show that the CD3 × CD33 bispecific antibody of the present invention can promote the killing of CD 33-expressing tumor cells by PBMCs.

The experimental results show that the bispecific antibody obtained by the invention can be combined with T cells and tumor cells, bridge the T cells and the tumor cells, promote the activation of the T cells and promote PBMC to kill and express CD33 positive tumor cells.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A recombinant antibody, comprising:

a first antigen binding region having binding activity for a CD3 molecule; and

a second antigen-binding region having binding activity for a CD33 molecule.

2. The recombinant antibody according to claim 1, wherein the first antigen-binding region comprises a CD3 single-chain antibody, wherein the CD3 single-chain antibody comprises a CD3 antibody heavy chain variable region and a CD3 antibody light chain variable region, wherein the C-terminus of the CD3 antibody heavy chain variable region is linked to the N-terminus of the CD3 antibody light chain variable region; or the C end of the CD3 antibody light chain variable region is connected with the N end of the CD3 antibody heavy chain variable region;

optionally, the CD3 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 1-3;

optionally, the CD3 antibody light chain variable region comprises: a light chain CDR set forth in any one of SEQ ID NOs 4-6;

optionally, the CD3 antibody heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 1. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO:3, CDR1, CDR2, CDR3 sequences;

optionally, the CD3 antibody light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 4. the amino acid sequence of SEQ ID NO: 5. SEQ ID NO:6, CDR1, CDR2, CDR3 sequences shown in;

optionally, the CD3 antibody heavy chain variable region comprises: 28 in SEQ ID NO;

optionally, the CD3 antibody light chain variable region comprises: 31, SEQ ID NO.

3. The recombinant antibody according to claim 2, wherein the CD3 single-chain antibody further comprises a linker peptide 1, wherein the N-terminus of the linker peptide 1 is linked to the C-terminus of the variable region of the heavy chain of the CD3 antibody, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the variable region of the light chain of the CD3 antibody; or the N end of the connecting peptide 1 is connected with the C end of the CD3 antibody light chain variable region, and the C end of the connecting peptide 1 is connected with the N end of the CD3 antibody heavy chain variable region;

optionally, the linker peptide 1 comprises SEQ ID NO: 16;

optionally, the CD3 single-chain antibody comprises an amino acid sequence shown in SEQ ID NO. 13.

4. The recombinant antibody according to claim 2, wherein the first antigen binding region further comprises a first heavy chain constant region, wherein the C-terminus of the CD3 single chain antibody is linked to the N-terminus of the first heavy chain constant region;

optionally, the first heavy chain constant region comprises a first hinge region and a first Fc peptide segment;

optionally, the first Fc peptide fragment comprises a first CH2 region and a first CH3 region, the C-terminus of the first CH2 region is linked to the N-terminus of the first CH3 region;

optionally, the C-terminus of the first hinge region is linked to the N-terminus of the first Fc peptide fragment;

optionally, the first hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the first CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the first CH3 region has a T366W and/or S354C mutation compared to a CH3 region fragment of a human wild type IgG 1;

optionally, the first antigen binding region further comprises a linker peptide 2, the N-terminus of the linker peptide 2 is linked to the C-terminus of the CD3 single chain antibody, and the C-terminus of the linker peptide 2 is linked to the N-terminus of the first heavy chain constant region;

optionally, the linker peptide 2 comprises SEQ ID NO: 17;

optionally, the first heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 18.

5. The recombinant antibody according to claim 1, wherein the second antigen binding region comprises a first peptide chain and a second peptide chain, the first peptide chain comprising: a CD33 antibody heavy chain variable region;

optionally, the CD33 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 7-9;

optionally, the CD3 antibody heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 7. SEQ ID NO: 8. SEQ ID NO:9, CDR1, CDR2, CDR3 sequences;

optionally, the CD33 antibody heavy chain variable region comprises: 14, SEQ ID NO;

optionally, the first peptide chain further comprises a second heavy chain constant region, wherein the C-terminus of the CD33 antibody heavy chain variable region is linked to the N-terminus of the second heavy chain constant region;

optionally, the second heavy chain constant region comprises: a CH1 region, a second hinge region, and a second Fc peptide fragment;

optionally, the C-terminus of the CH1 region is linked to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is linked to the N-terminus of the second Fc peptide fragment;

optionally, the second Fc peptide fragment comprises: a second CH2 region and a second CH3 region, wherein the C end of the second CH2 region is connected with the N end of the second CH3 region;

optionally, the CH1 region is a CH1 region of a wild-type IgG1 of human, primate or murine origin;

optionally, the second hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH3 region is a CH3 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH3 region has at least one of T366S, L368A, Y407V, Y C mutations compared to a CH3 region fragment of a human wild type IgG 1;

optionally, the second heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 19.

6. The recombinant antibody according to claim 1, wherein said second peptide chain comprises: a CD33 antibody light chain variable region;

optionally, the CD33 antibody light chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 10-12;

optionally, the CD33 antibody light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO:12, CDR1, CDR2, CDR3 sequences;

optionally, the CD33 antibody light chain variable region comprises: 15, SEQ ID NO;

optionally, the second peptide chain further comprises a light chain constant region, wherein the C-terminus of the CD33 antibody light chain variable region is linked to the N-terminus of the light chain constant region;

optionally, the light chain constant region is a wild-type light chain constant region of human, primate, or murine origin;

optionally, the antibody light chain constant region is a human Kappa light chain constant region;

optionally, the light chain constant region is a light chain constant region of a human wild-type IgG 1;

optionally, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 29.

7. The recombinant antibody according to any one of claims 1 to 6, wherein the first antigen-binding region and the second antigen-binding region are linked by a knob-into-hole structure;

optionally, the knob-into-hole structure is formed by a mutation of at least one of the T366W and/or S354C mutations of the first CH3 region and T366S, L368A, Y V, Y C mutations of the second CH3 region;

optionally, the first peptide chain and the second peptide chain are linked by an inter-bond disulfide bond;

optionally, the recombinant antibody comprises an amino acid sequence shown in SEQ ID NO 20-22.

8. A nucleic acid molecule encoding the recombinant antibody of any one of claims 1 to 7.

9. The nucleic acid of claim 8, wherein the nucleic acid molecule has the sequence of SEQ ID NO: 23-25.

10. An expression vector carrying the nucleic acid molecule of claim 8 or 9.

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

introducing the expression vector of claim 10 into a cell;

culturing said cells under conditions suitable for protein expression and secretion so as to obtain said recombinant antibody;

optionally, the cell is a eukaryotic cell.

12. A recombinant cell carrying the nucleic acid molecule of claim 8 or 9, or the expression vector of claim 10, or capable of expressing the recombinant antibody of any one of claims 1-7.

13. A composition, comprising:

the recombinant antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8 or 9, the expression vector according to claim 10 or the recombinant cell according to claim 12.

14. Use of the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, the recombinant cell of claim 12, or the composition of claim 13 in the manufacture of a medicament for treating or preventing myeloid cell leukemia and CD33 positive cancer.

15. The use according to claim 14, wherein the CD 33-positive cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

16. A medicament, comprising: the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, the recombinant cell of claim 12, or the composition of claim 13.

17. The medicament of claim 16, further comprising a pharmaceutically acceptable excipient;

optionally, the medicament is for the treatment or prevention of myeloid leukemia and CD33 positive cancer;

optionally, the CD 33-positive cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

18. Use of the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10 or the recombinant cell of claim 12 for the preparation of a kit for the detection of CD3 and/or CD33.

19. A kit comprising the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, or the recombinant cell of claim 12.

20. The kit of claim 19, wherein the kit is for detecting CD3 and/or CD33.