CN114409797B

CN114409797B - Recombinant antibodies and uses thereof

Info

Publication number: CN114409797B
Application number: CN202111599672.5A
Authority: CN
Inventors: 田志刚; 曹国帅; 肖卫华; 孙汭; 孙昊昱
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2024-02-20
Anticipated expiration: 2041-12-24
Also published as: CN114409797A

Abstract

The invention provides a bispecific antibody and application thereof, wherein the antibody is a double-chain antibody, and comprises the following components: a first peptide chain comprising a CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region, and a first Fc region; and a second peptide chain comprising a PD-1 extracellular region and a second Fc region; wherein the C-terminal of the CD3 single-chain antibody is connected with the N-terminal of the first Fc region, and the C-terminal of the PD-1 extracellular region is connected with the N-terminal of the second Fc region. The bispecific antibody prepared by the invention can target CD3 and PD-L1 at the same time, thereby mediating the killing of T cells on tumor cells and having stronger tumor inhibition capability.

Description

Recombinant antibodies and uses thereof

Technical Field

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

Background

Cancer is a disease that seriously threatens the life health of the masses, and the incidence rate and death rate of cancer have been increasing in recent years worldwide. Currently available cancer treatment methods, including surgical excision, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, macromolecular immunotherapy and the like, but the methods only play a limited role in part of cancer patients, and cancers still have the problem of puzzlement of human life and health.

In recent years, bispecific antibodies have become a focus of research in immunotherapy. Bispecific antibodies are artificial antibodies containing two specific antigen binding sites, which can bridge between target cells (tumor cells) and effector cells (immune cells) to produce effector functions that are directed to kill tumors. The immunosuppressive receptor PD-1 is expressed on the surface of T cells and NK cells; the main ligand PD-L1 of PD-1 is highly expressed in tumor tissues, such as lung cancer, and tumor cells promote immunosuppression and limit the anticancer function of immune cells through PD-L1/PD-1 interaction. The relatively high expression of PD-L1 in tumor tissue makes it a potential tumor-associated antigen. However, there is a risk of immunogenicity due to (1) bispecific antibody drugs: the antibody itself is not all of human body, and is generated by immunization of animals such as mice through antigen, the immunogenicity of the animal can only be partially reduced by humanization transformation, and the immune system of the human body still can generate antibodies against the antibody, namely anti-antibodies, so that the medicine is attenuated or even disabled. (2) If the antibody targeting the tumor-associated antigen is in a light chain dimer structure and a heavy chain dimer structure, the production and purification difficulties exist; if the antibody targeting the tumor-associated antigen is an ScFv fragment, its affinity for the tumor-associated antigen will be greatly reduced, affecting the anti-cancer effect.

Thus, there remains a need to further develop bispecific antibodies with low immunogenicity, strong affinity.

Disclosure of Invention

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

the PD-L1 in tumor tissue is combined with the immune checkpoint PD-1, so that the anticancer function of immune cells is inhibited, a CD3 antibody single-chain variable fragment (ScFv) can be combined with T cells, the inventor designs a bispecific antibody targeting CD3 and PD-L1, and after a large number of experimental screening, the dominant bispecific antibodies CD3 xPD-1 and CD3 xPD-1 mut are obtained, and the bispecific antibody can be combined with the high-expression PD-L1 and the T cells in the tumor tissue at the same time, so that the killing of the T cells on the tumor cells is effectively mediated, and the tumor cell has stronger anticancer capability.

Thus, in a first aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the invention, it comprises: a first peptide chain comprising a CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region, and a first Fc region; and a second peptide chain comprising a PD-1 extracellular region and a second Fc region; wherein the C-terminal of the CD3 single-chain antibody is connected with the N-terminal of the first Fc region, and the C-terminal of the PD-1 extracellular region is connected with the N-terminal of the second Fc region. The recombinant antibody according to the embodiment of the invention can be combined with CD3 and PD-L1 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a second 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 combined with CD3 and PD-L1 at the same time, 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 provides an expression vector. According to an embodiment of the invention, the nucleic acid according to the second aspect is carried. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The expression vector provided by the embodiment of the invention can efficiently express the recombinant antibody in a proper host cell, and the recombinant antibody can be combined with CD3 and PD-L1 at the same time, so that the killing effect of T cells on tumor cells is effectively mediated, and the recombinant antibody has stronger tumor inhibition capability.

In a fourth aspect of the invention, the invention provides a method of preparing a recombinant antibody according to the first aspect. According to an embodiment of the invention, it comprises: introducing the expression vector of the third aspect into a cell; the cells are cultured under conditions suitable for protein expression and secretion to obtain the recombinant antibodies. According to the method provided by the embodiment of the invention, the recombinant antibody can be effectively obtained, can be combined with CD3 and PD-L1 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a fifth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries a nucleic acid according to the second aspect, or an expression vector according to the third aspect. The recombinant cells are obtained by transfecting or transforming the expression vector. According to some specific embodiments of the invention, the recombinant cell can efficiently express the recombinant antibody under proper conditions, and the recombinant antibody can be combined with CD3 and PD-L1 at the same time, so that the T cell can effectively mediate the killing effect of the T cell on the tumor cell, and has stronger tumor inhibition capability.

In a sixth aspect of the invention, the invention provides a composition. According to an embodiment of the invention, it comprises: the recombinant antibody of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and PD-L1 protein molecules and promote T cells to selectively localize to tumor sites rather than circulating in the periphery, avoiding systemic activation, and compositions, such as food compositions, pharmaceutical compositions, etc., comprising the recombinant antibodies also have significant therapeutic or prophylactic effects.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect, a nucleic acid 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 cancer. As described above, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and PD-L1 protein molecules and promote T cells to selectively localize to tumor sites rather than to peripheral circulation, avoiding systemic activation, and drugs comprising a series of substances of the recombinant antibodies also have a remarkable therapeutic or prophylactic effect on cancer.

In an eighth aspect of the invention, the invention provides a medicament. According to an embodiment of the invention, the medicament comprises: the recombinant antibody of the first aspect, the nucleic acid 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. According to an embodiment of the invention, the medicament is for the treatment of cancer. As described above, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 on the surface of immune cells and PD-L1 protein molecules on the surface of tumor cells, and promote T cells to selectively localize to tumor sites rather than to the peripheral circulation, avoiding systemic activation, and thus, the drugs comprising a series of substances of the recombinant antibodies also have remarkable therapeutic or prophylactic effects on cancer.

In a ninth aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect for the preparation of a kit for the detection of CD3 and/or PD-L1. The recombinant antibodies can bind to CD3 and/or PD-L1 proteins, and therefore kits comprising the recombinant antibodies can be used to efficiently detect CD3 and/or PD-L1. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or PD-L1 proteins in biological samples.

In a tenth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises the recombinant antibody according to the first aspect. The recombinant antibody provided by the embodiment of the invention can be combined with CD3 and/or PD-L1 protein, so that the kit containing the recombinant antibody can be used for effectively detecting CD3 and/or PD-L1. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or PD-L1 protein in biological samples, and can also be used for judging the state of an individual, such as judging whether the PD-L1 level of the individual is higher than or lower than the normal level after the PD-L1 level of the individual is obtained.

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

Drawings

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

FIG. 1 is a schematic diagram of the structure of a recombinant bispecific antibody according to an embodiment of the present invention, wherein the connection position of the connecting peptide is not shown, and the first Fc region (left side) and the second Fc region (right side) of the recombinant bispecific antibody are connected by a knob-into-hole structure;

FIG. 2 is a graph of the results of the detection of the binding capacity of recombinant bispecific antibodies to Jurkat T cells according to an embodiment of the present invention, wherein isotype control represents isotype control IgG1;

FIG. 3 is a graph of the results of detection of the binding capacity of recombinant bispecific antibodies to CHO-K1-PD-L1 according to an embodiment of the invention;

FIG. 4 is a graph showing the results of detection of the binding capacity of recombinant bispecific antibodies CD3 XPD 1mut, CD3 XPD 1 to CHO-K1-PDL1 cells according to the embodiments of the invention;

FIG. 5 is a graph showing the results of detection of the expression level of PD-L1 on the surface of tumor cells, wherein isotype control represents isotype control IgG1 according to the embodiment of the present invention;

FIG. 6 is a graph of the results of a test for the ability of recombinant bispecific antibodies to promote binding of Jurkat cells to tumor cells according to an embodiment of the present invention, wherein NCI-H358 indicates that the set of reaction systems comprises only NCI-H358 cells, NCI-H358 Jurkat (CFSE) indicates that the set of reaction systems comprises NCI-H358 cells and Jurkat cells, NCI-H358 Jurkat (CFSE) anti-CD3 (OKT 3) indicates that the set of reaction systems comprises NCI-H358 cells, jurkat cells and CD3 antibodies, NCI-H358 Jurkat (CFSE) CD 3X PD1mut indicates that the set of reaction systems comprises NCI-H358 cells, jurkat cells and CD 3X PD1mut bispecific antibodies;

FIG. 7 is a graph of the detection results of recombinant bispecific antibodies promoting PBMC killing of lung cancer NCI-H358 cells according to an embodiment of the present invention;

FIG. 8 is a graph of the detection results of recombinant bispecific antibodies promoting the killing of cervical cancer HeLa cells by PBMC according to an embodiment of the present invention; and

FIG. 9 is a graph of the results of a test of recombinant bispecific antibodies promoting PBMC killing of ovarian cancer Ho8910 cells 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.

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

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

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

The "Knob intohole structure" herein is a button (Knob) forming mutation in the CH3 region of the heavy chain Fc of an antibody, facilitating heavy chain occlusion, and formation of heterodimers, for example, by mutating amino acids in the human IgG1-Fc CH3 domain (in one chain, the T366S, L368A, Y V mutation, i.e. "hole", and in the other chain, the T366W mutation, i.e. "Knob") in this application.

"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene.

In a first aspect of the present invention, the present invention provides a recombinant antibody, which is a diabody comprising: a first peptide chain comprising a CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region, and a first Fc region; and a second peptide chain comprising a PD-1 extracellular region and a second Fc region; wherein the C-terminal of the CD3 single-chain antibody is connected with the N-terminal of the first Fc region, and the C-terminal of the PD-1 extracellular region is connected with the N-terminal of the second Fc region. The recombinant antibody according to the embodiment of the invention can be combined with CD3 and PD-L1 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger 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 PD-1 extracellular region comprises a wild-type PD-1 extracellular region or a mutant thereof. According to some specific embodiments of the invention, the recombinant antibodies comprising the wild type PD-L1 extracellular region or the mutant thereof can effectively mediate the killing effect of T cells on tumors, and have stronger tumor inhibition capability.

According to some embodiments of the invention, the mutant phase has a V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, a100V, a107I mutation compared to the wild type PD-1 extracellular domain.

According to some embodiments of the invention, the wild-type PD-1 extracellular region comprises SEQ ID NO:18, and a polypeptide having the amino acid sequence shown in seq id no.

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV(SEQ ID NO：18)。

According to some embodiments of the invention, further comprising a linker peptide 1, the N-terminus of the linker peptide 1 is linked to the C-terminus of the heavy chain variable region, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the light chain variable region.

According to some embodiments of the invention, the connecting peptide 1 comprises SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

GGGGSGGGGSGGGGS(SEQ ID NO：15)。

According to some embodiments of the invention, further comprising a linker peptide 2, the N-terminus of the linker peptide 2 is linked to the C-terminus of the light chain variable region, and the C-terminus of the linker peptide 2 is linked to the N-terminus of the first Fc region.

According to some embodiments of the invention, the connecting peptide 2 comprises SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

GGGGS(SEQ ID NO：16)。

According to some embodiments of the invention, further comprising a linker peptide 3, the N-terminus of the linker peptide 3 being linked to the C-terminus of the PD-1 extracellular region, the C-terminus of the linker peptide 3 being linked to the N-terminus of the second Fc region;

According to some embodiments of the invention, the connecting peptide 3 comprises SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no.

GGGGS(SEQ ID NO：19)。

According to some embodiments of the invention, the linker peptides 1, 2, 3 are not particularly limited, and conventional flexible or rigid linker peptides used in the art may be used.

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

According to some specific embodiments of the invention, the first Fc region has at least one of a lack of a CH1 region, T366W mutation compared to a wild-type IgG1 Fc region, and the second Fc region has at least one of a lack of a CH1 region, T366S, L368A, Y V mutation compared to a wild-type IgG1 Fc region.

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

According to some embodiments of the invention, at least a portion of the first and second Fc regions are derived from murine antibodies, human antibodies, primates 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 embodiments of the invention, the first antibody Fc region has an amino acid sequence as shown in SEQ ID NO. 17 and the second antibody Fc region has an amino acid sequence as shown in SEQ ID NO. 20.

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：17)。

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：20)。

According to some embodiments of the invention, the recombinant antibody has the amino acid sequence of SEQ ID NO:4, and SEQ ID NO:5 or 6.

The first peptide chain has the amino acid sequence shown below:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：4)。

the second peptide chain (CD 3 XPD-1) has the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：5)。

the second peptide chain (CD 3 XPD-1 mut) has the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：6)。

in a second aspect of the invention, the invention provides a nucleic acid encoding the recombinant antibody of the first aspect. The recombinant antibody coded by the nucleic acid can be combined with CD3 and PD-L1 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

According to some embodiments of the invention, the nucleic acid has the sequence of SEQ ID NO:1, and SEQ ID NO:2 or 3.

The gene encoding the first peptide chain has the nucleotide sequence shown below:

GAAGTACAACTGCTGGAATCAGGGGGAGGACTTGTTCAGCCAGGAGGGTCTCTCAAGCTGAGCTGCGCCGCTTCAGGGTTCACATTCAACACTTACGCAATGAATTGGGTGAGACAGGCCCCAGGTAAAGGGCTCGAATGGGTGGCTCGCATTCGCTCCAAATACAATAACTATGCCACCTACTACGCTGACTCTGTGAAAGATCGCTTCACAATCAGCAGGGACGACTCTAAAAATACCGCTTATCTGCAGATGAATAATCTGAAAACCGAAGACACCGCAGTCTATTACTGCGTCAGGCATGGCAACTTCGGAAACTCTTACGTAAGCTGGTTCGCTTACTGGGGCCAGGGAACCCTTGTGACCGTGTCTAGTGGTGGTGGTGGTTCTGGAGGTGGTGGAAGCGGAGGAGGTGGATCCGAACTGGTGGTGACACAGGAGCCTTCTCTCACAGTGAGTCCAGGAGGCACCGTGACCCTGACCTGTAGAAGTTCTACAGGAGCTGTCACCACATCCAATTACGCTAATTGGGTCCAGCAGAAGCCAGGCCAAGCACCTCGGGGTCTCATTGGGGGTACCAACAAGCGAGCTCCTGGGACTCCTGCCAGGTTTAGTGGGAGTCTGTTGGGCGGTAAGGCTGCTCTTACTCTTAGTGGAGTACAGCCCGAGGACGAGGCCGAATACTACTGTGCCCTGTGGTACTCAAACTTGTGGGTCTTCGGTGGTGGGACCAAACTTACTGTCCTGGGCGGAGGAGGATCTCCAAAGTCATGTGACAAGACTCACACATGTCCCCCATGTCCAGCCCCTGAGCTCCTGGGAGGACCATCTGTTTTCCTGTTTCCCCCTAAGCCCAAGGATACCCTGATGATCAGTAGAACCCCAGAGGTGACATGCGTTGTCGTGGCTGTTTCTCACGAAGACCCAGAGGTAAAGTTCAACTGGTACGTAGACGGCGTCGAAGTCCATAATGCCAAGACAAAGCCTCGTGAGGAGCAGTATGCCTCAACATATCGCGTCGTATCCGTGTTGACTGTCCTCCATCAGGACTGGCTGAACGGGAAAGAATACAAATGTAAGGTGAGCAATAAGGCTCTGCCCGCACCTATTGAGAAGACTATCTCTAAGGCTAAGGGCCAGCCAAGAGAACCTCAGGTTTATACCCTTCCTCCCTCTCGAGAGGAGATGACTAAGAACCAGGTGTCTCTTTGGTGCCTGGTCAAAGGTTTTTACCCAAGTGATATTGCCGTGGAGTGGGAAAGCAACGGTCAGCCCGAGAATAACTACAAAACAACCCCACCTGTGTTGGACTCTGACGGCTCCTTCTTTCTTTACTCCAAATTGACCGTGGACAAATCTCGCTGGCAGCAGGGGAATGTGTTCTCCTGTAGCGTCATGCACGAGGCCCTCCACAACCACTATACCCAGAAGAGCCTGTCTCTGAGTCCAGGAAAA(SEQ ID NO：1)。

the gene encoding the second peptide chain (CD 3 xpd 1) has the nucleotide sequence shown below:

GATTCTCCTGATAGACCCTGGAATCCCCCTACTTTCAGCCCTGCTCTTCTGGTCGTCACCGAAGGTGATAATGCAACATTTACCTGTAGCTTCTCAAACACCAGCGAGAGCTTTGTTCTTAACTGGTACAGAATGAGCCCCTCCAACCAGACTGATAAGCTGGCCGCCTTTCCTGAGGACCGGTCTCAACCAGGCCAGGATTGTCGGTTTCGGGTTACCCAGCTCCCTAACGGCAGGGATTTTCACATGTCAGTCGTGAGGGCCAGAAGAAACGACAGCGGCACATACCTGTGCGGGGCAATTTCCCTTGCACCTAAGGCACAGATCAAGGAGAGTCTGAGAGCAGAACTGCGCGTTACAGAACGACGCGCCGAGGTGCCAACAGCACATCCTTCTCCCTCTCCTAGGCCCGCTGGTCAGTTCCAGACCTTGGTCGGAGGTGGGGGTAGCCCAAAGTCTTGTGACAAAACTCATACCTGCCCCCCTTGTCCCGCCCCAGAACTTCTTGGAGGGCCCTCAGTGTTCCTCTTCCCCCCAAAGCCAAAGGATACACTCATGATCAGTCGCACCCCAGAGGTGACATGCGTTGTAGTGGCTGTCTCCCACGAGGATCCCGAGGTGAAGTTTAATTGGTACGTCGACGGGGTTGAAGTGCACAATGCTAAGACCAAGCCCAGGGAGGAACAGTACGCCTCTACATACAGGGTGGTGTCCGTCTTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAATATAAGTGTAAGGTGTCCAATAAAGCACTCCCCGCCCCAATTGAGAAGACCATAAGCAAAGCAAAAGGCCAACCACGTGAACCACAGGTCTATACCCTTCCCCCATCTAGGGAGGAAATGACCAAGAATCAAGTCTCATTGTCCTGCGCCGTGAAGGGTTTCTACCCTAGCGACATCGCCGTGGAGTGGGAATCCAATGGGCAGCCCGAGAACAACTATAAAACCACCCCACCTGTCTTGGATTCCGACGGCTCATTCTTCCTCGTCAGCAAGCTGACCGTGGATAAGAGTCGGTGGCAGCAGGGTAACGTGTTCTCCTGCTCAGTGATGCATGAGGCACTGCATAACCACTACACACAGAAAAGTCTTAGTCTCTCCCCAGGCAAG(SEQ ID NO：2)。

The gene encoding the second peptide chain (CD 3 XPD 1 mut) has the nucleotide sequence shown below:

GATAGTCCTGATAGGCCTTGGAATCCTCCTACTTTCTCTCCCGCCTTGCTGGTCGTTACTGAGGGCGACAACGCAACCTTCACTTGCAGTTTTAGTAACACTAGTGAGTCCTTTCACGTGGTCTGGCACAGAGAGTCACCAAGTGGCCAGACTGATACTCTTGCAGCTTTTCCTGAAGATCGCTCCCAGCCAGGCCAGGATTGTCGGTTTAGGGTCACCCAGCTGCCCAATGGGAGAGACTTCCATATGTCTGTCGTGAGAGCACGCCGAAACGATAGTGGTACCTACGTATGCGGCGTCATCTCACTGGCACCCAAAATCCAGATTAAAGAGTCTCTTAGGGCTGAGTTGAGAGTTACCGAGAGAAGAGCAGAGGTACCCACAGCACATCCAAGTCCTAGTCCCAGGCCAGCCGGACAGTTCCAGACTTTGGTAGGAGGTGGCGGGTCACCTAAGTCATGCGACAAGACCCATACCTGTCCCCCATGTCCAGCTCCCGAATTGTTGGGTGGCCCTTCCGTATTTCTGTTCCCTCCCAAACCAAAAGACACACTGATGATAAGTAGAACTCCAGAGGTTACCTGCGTCGTCGTAGCAGTGTCACACGAAGACCCCGAGGTGAAGTTCAATTGGTATGTGGACGGCGTCGAAGTGCATAACGCTAAAACAAAACCAAGAGAGGAGCAGTATGCCAGCACATATCGCGTGGTGAGTGTGCTTACTGTGCTGCACCAGGACTGGCTGAACGGTAAAGAGTACAAGTGTAAAGTGAGTAACAAGGCCCTGCCTGCACCTATCGAAAAGACTATCAGCAAAGCTAAGGGCCAGCCACGTGAGCCACAGGTGTATACACTCCCCCCATCTAGAGAAGAGATGACAAAGAATCAGGTGTCTCTCAGCTGCGCAGTCAAGGGTTTCTACCCTAGCGACATTGCTGTGGAGTGGGAGTCCAATGGGCAGCCAGAAAACAATTACAAGACCACACCACCTGTCCTGGATAGTGACGGCAGCTTCTTCCTGGTGTCCAAACTCACCGTGGATAAGAGCCGGTGGCAGCAGGGTAACGTTTTTAGCTGCTCCGTCATGCATGAAGCTCTGCACAACCATTACACCCAGAAGAGTCTGTCACTGTCCCCAGGCAAA(SEQ ID NO:3)。

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 DNA forms or RNA forms, one of which is disclosed, meaning that the other is also disclosed

In a third aspect of the invention, the invention provides an expression vector carrying a nucleic acid according to the second 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 the recombinant antibody can be combined with CD3 and PD-L1 at the same time, so that the killing effect of T cells on tumor cells is effectively mediated, and the recombinant antibody has stronger tumor inhibition capability.

In a fourth aspect of the invention, the invention provides a method for preparing a recombinant antibody according to the first aspect, comprising: introducing the expression vector of the third aspect into a cell; the cells are cultured under conditions suitable for protein expression and secretion to obtain the recombinant antibodies. According to the method provided by some specific embodiments of the invention, the recombinant antibody can be effectively obtained, can be combined with CD3 and PD-L1 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability. According to some embodiments of the present invention, the cell is not particularly limited, and either a prokaryotic cell or a eukaryotic cell may be used, and the expression efficiency of the recombinant antibody is high when the cell is a eukaryotic cell, such as a mammalian cell.

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 include an animal germ cell, fertilized egg or embryonic stem cell.

In a fifth aspect of the invention, the invention provides a recombinant cell carrying a nucleic acid according to the second aspect, or an expression vector according to the third aspect. The recombinant cells are obtained by transfecting or transforming the expression vector. According to some specific embodiments of the invention, the recombinant cell can efficiently express the recombinant antibody under proper conditions, and the recombinant antibody can be combined with CD3 and PD-L1 at the same time, so that the T cell can effectively mediate the killing effect of the T cell on the tumor cell, and has stronger tumor inhibition capability.

It should be noted that the recombinant cells of the present invention are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells can be fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the 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 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: the recombinant antibody of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and PD-L1 protein molecules and promote T cells to selectively localize to tumor sites rather than circulating in the periphery, avoiding systemic activation, and compositions, such as food compositions, pharmaceutical compositions, etc., comprising the recombinant antibodies also have significant therapeutic or prophylactic effects.

It is noted that the compositions include combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect, a nucleic acid 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 cancer. As described above, the recombinant antibodies of the embodiments of the present invention can be effectively combined with CD3 and PD-L1 protein molecules to promote T cells to selectively localize to tumor rather than circulate in the periphery, avoiding systemic activation, and thus, the drugs comprising a series of substances of the recombinant antibodies also have a remarkable therapeutic or prophylactic effect on tumors.

According to some embodiments of the invention, the above-mentioned use 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: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

In an eighth aspect of the invention, the invention provides a medicament comprising: the recombinant antibody of the first aspect, the nucleic acid 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. According to an embodiment of the invention, the medicament is for the treatment of cancer. As described above, the recombinant antibodies of the embodiments of the present invention can be effectively combined with CD3 and PD-L1 protein molecules to promote T cells to selectively localize to tumor rather than to circulate in the periphery, avoiding systemic activation, and thus, the medicaments comprising a series of substances of the recombinant antibodies also have a remarkable effect of treating or preventing cancer.

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, 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 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.

In a tenth aspect of the invention, the invention provides a kit comprising the recombinant antibody of the first aspect. The recombinant antibody provided by the embodiment of the invention can be combined with CD3 and/or PD-L1 protein, so that the kit containing the recombinant antibody can be used for effectively detecting CD3 and/or PD-L1. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or PD-L1 protein in biological samples, and can also be used for judging the state of an individual, such as judging whether the PD-L1 level of the individual is higher than or lower than the normal level after the PD-L1 level of the individual is obtained.

According to some embodiments of the invention, the kit is for detecting CD3 and/or PD-L1.

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

EXAMPLE 1 preparation of bispecific antibody molecules

The production of bispecific antibodies was performed in this example, and the specific experimental procedures were as follows: expiCHO cells (from Thermo Fisher) were cultured using ExpiCHO Expression Medium medium (from Thermo Fisher, A2910001) to adjust the cell concentration to 6X 10 ⁶ /mL, obtain an ExpiCHO cell solution. pTT5 vector (synthesized by Suzhou Jin Wei Intelligence Co.) containing the a-chain, b-chain (b 1 and b 2-chain) encoding genes (shown as SEQ ID NO:1, 2, 3, respectively) comprising the nucleotide sequences encoding the CD3 single chain antibody (SEQ ID NO: 7), the linker peptide 1 (SEQ ID NO: 8), the linker peptide 2 (SEQ ID NO: 9) and the first Fc region (SEQ ID NO: 10) was added to 2mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A, wherein the b 1-chain encoding gene comprises the nucleotide sequences encoding the PD-1 extracellular region (SEQ ID NO: 11), the linker peptide 3 (SEQ ID NO: 12) and the second Fc region (SEQ ID NO: 13). 160. Mu. LExPifectamine CHO transfection reagent (thermofiser, A29130) was added to 2mL of OptiSFM medium to obtain solution B. Solution a and solution B were then mixed to obtain a transfection mixture, and the transfection mixture was added to 50mL of an expiho cell solution throughout the course of 5 minutes. At 37℃with 5% CO ₂ After 1 day of incubation, 8mL Feed, 300. Mu. LEnhancer (Thermo Fisher, A29130) was added and transferred to 32℃and 5% CO ₂ Culture supernatants were harvested after 9 days of culture under conditions, with 8mL Feed added on day 5. Affinity from culture supernatants Using Protein A purification columns (GE) The bispecific antibody was purified, and the specific detection results are shown in fig. 1, and 2 antibodies were obtained in total: CD3 XPD 1 and CD3 XPD 1mut, wherein antibody CD3 XPD 1 has the amino acid sequence shown as SEQ ID NO:4 (a fragment, first peptide chain) and SEQ ID NO:5 (b 1 fragment, second peptide chain-wild type), antibody CD3 XPD 1mut has the amino acid sequence shown as SEQ ID NO:4 (a fragment, first peptide chain) and SEQ ID NO:6 (b 2 fragment, second peptide chain-mut), wherein the a fragment comprises the amino acid sequence of a CD3 single chain antibody (SEQ ID NO: 14), linker peptide 1 (SEQ ID NO: 15), linker peptide 2 (SEQ ID NO: 16) and first Fc region (SEQ ID NO: 17), the b1 fragment comprises the amino acid sequence of a PD-1 extracellular region (SEQ ID NO: 18), linker peptide 3 (SEQ ID NO: 19) and second Fc region (SEQ ID NO: 20), and the b2 fragment comprises the amino acid sequence of PD-1 extracellular region mut (SEQ ID NO: 21), linker peptide 1 (SEQ ID NO: 15) and first Fc region (SEQ ID NO: 17).

The a-chain coding gene comprises the nucleotide sequence shown as follows:

GAAGTACAACTGCTGGAATCAGGGGGAGGACTTGTTCAGCCAGGAGGGTCTCTCAAGCTGAGCTGCGCCGCTTCAGGGTTCACATTCAACACTTACGCAATGAATTGGGTGAGACAGGCCCCAGGTAAAGGGCTCGAATGGGTGGCTCGCATTCGCTCCAAATACAATAACTATGCCACCTACTACGCTGACTCTGTGAAAGATCGCTTCACAATCAGCAGGGACGACTCTAAAAATACCGCTTATCTGCAGATGAATAATCTGAAAACCGAAGACACCGCAGTCTATTACTGCGTCAGGCATGGCAACTTCGGAAACTCTTACGTAAGCTGGTTCGCTTACTGGGGCCAGGGAACCCTTGTGACCGTGTCTAGTGGTGGTGGTGGTTCTGGAGGTGGTGGAAGCGGAGGAGGTGGATCCGAACTGGTGGTGACACAGGAGCCTTCTCTCACAGTGAGTCCAGGAGGCACCGTGACCCTGACCTGTAGAAGTTCTACAGGAGCTGTCACCACATCCAATTACGCTAATTGGGTCCAGCAGAAGCCAGGCCAAGCACCTCGGGGTCTCATTGGGGGTACCAACAAGCGAGCTCCTGGGACTCCTGCCAGGTTTAGTGGGAGTCTGTTGGGCGGTAAGGCTGCTCTTACTCTTAGTGGAGTACAGCCCGAGGACGAGGCCGAATACTACTGTGCCCTGTGGTACTCAAACTTGTGGGTCTTCGGTGGTGGGACCAAACTTACTGTCCTGGGCGGAGGAGGATCTCCAAAGTCATGTGACAAGACTCACACATGTCCCCCATGTCCAGCCCCTGAGCTCCTGGGAGGACCATCTGTTTTCCTGTTTCCCCCTAAGCCCAAGGATACCCTGATGATCAGTAGAACCCCAGAGGTGACATGCGTTGTCGTGGCTGTTTCTCACGAAGACCCAGAGGTAAAGTTCAACTGGTACGTAGACGGCGTCGAAGTCCATAATGCCAAGACAAAGCCTCGTGAGGAGCAGTATGCCTCAACATATCGCGTCGTATCCGTGTTGACTGTCCTCCATCAGGACTGGCTGAACGGGAAAGAATACAAATGTAAGGTGAGCAATAAGGCTCTGCCCGCACCTATTGAGAAGACTATCTCTAAGGCTAAGGGCCAGCCAAGAGAACCTCAGGTTTATACCCTTCCTCCCTCTCGAGAGGAGATGACTAAGAACCAGGTGTCTCTTTGGTGCCTGGTCAAAGGTTTTTACCCAAGTGATATTGCCGTGGAGTGGGAAAGCAACGGTCAGCCCGAGAATAACTACAAAACAACCCCACCTGTGTTGGACTCTGACGGCTCCTTCTTTCTTTACTCCAAATTGACCGTGGACAAATCTCGCTGGCAGCAGGGGAATGTGTTCTCCTGTAGCGTCATGCACGAGGCCCTCCACAACCACTATACCCAGAAGAGCCTGTCTCTGAGTCCAGGAAAA(SEQ ID NO:1)。

the b1 chain coding gene comprises the nucleotide sequence shown below:

GATTCTCCTGATAGACCCTGGAATCCCCCTACTTTCAGCCCTGCTCTTCTGGTCGTCACCGAAGGTGATAATGCAACATTTACCTGTAGCTTCTCAAACACCAGCGAGAGCTTTGTTCTTAACTGGTACAGAATGAGCCCCTCCAACCAGACTGATAAGCTGGCCGCCTTTCCTGAGGACCGGTCTCAACCAGGCCAGGATTGTCGGTTTCGGGTTACCCAGCTCCCTAACGGCAGGGATTTTCACATGTCAGTCGTGAGGGCCAGAAGAAACGACAGCGGCACATACCTGTGCGGGGCAATTTCCCTTGCACCTAAGGCACAGATCAAGGAGAGTCTGAGAGCAGAACTGCGCGTTACAGAACGACGCGCCGAGGTGCCAACAGCACATCCTTCTCCCTCTCCTAGGCCCGCTGGTCAGTTCCAGACCTTGGTCGGAGGTGGGGGTAGCCCAAAGTCTTGTGACAAAACTCATACCTGCCCCCCTTGTCCCGCCCCAGAACTTCTTGGAGGGCCCTCAGTGTTCCTCTTCCCCCCAAAGCCAAAGGATACACTCATGATCAGTCGCACCCCAGAGGTGACATGCGTTGTAGTGGCTGTCTCCCACGAGGATCCCGAGGTGAAGTTTAATTGGTACGTCGACGGGGTTGAAGTGCACAATGCTAAGACCAAGCCCAGGGAGGAACAGTACGCCTCTACATACAGGGTGGTGTCCGTCTTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAATATAAGTGTAAGGTGTCCAATAAAGCACTCCCCGCCCCAATTGAGAAGACCATAAGCAAAGCAAAAGGCCAACCACGTGAACCACAGGTCTATACCCTTCCCCCATCTAGGGAGGAAATGACCAAGAATCAAGTCTCATTGTCCTGCGCCGTGAAGGGTTTCTACCCTAGCGACATCGCCGTGGAGTGGGAATCCAATGGGCAGCCCGAGAACAACTATAAAACCACCCCACCTGTCTTGGATTCCGACGGCTCATTCTTCCTCGTCAGCAAGCTGACCGTGGATAAGAGTCGGTGGCAGCAGGGTAACGTGTTCTCCTGCTCAGTGATGCATGAGGCACTGCATAACCACTACACACAGAAAAGTCTTAGTCTCTCCCCAGGCAAG(SEQ ID NO:2)。

the b2 chain coding gene comprises the nucleotide sequence shown below:

the a-fragments of the CD3 xpd 1 and CD3 xpd 1mut antibodies each comprise the amino acid sequence shown below:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。

The b1 fragment of the CD3 xpd 1 antibody comprises the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:5)。

the b2 fragment of the CD3 x PD1mut antibody comprises the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:6)。

the gene encoding the CD3 single chain antibody includes the nucleotide sequence shown below:

GAAGTACAACTGCTGGAATCAGGGGGAGGACTTGTTCAGCCAGGAGGGTCTCTCAAGCTGAGCTGCGCCGCTTCAGGGTTCACATTCAACACTTACGCAATGAATTGGGTGAGACAGGCCCCAGGTAAAGGGCTCGAATGGGTGGCTCGCATTCGCTCCAAATACAATAACTATGCCACCTACTACGCTGACTCTGTGAAAGATCGCTTCACAATCAGCAGGGACGACTCTAAAAATACCGCTTATCTGCAGATGAATAATCTGAAAACCGAAGACACCGCAGTCTATTACTGCGTCAGGCATGGCAACTTCGGAAACTCTTACGTAAGCTGGTTCGCTTACTGGGGCCAGGGAACCCTTGTGACCGTGTCTAGTGGTGGTGGTGGTTCTGGAGGTGGTGGAAGCGGAGGAGGTGGATCCGAACTGGTGGTGACACAGGAGCCTTCTCTCACAGTGAGTCCAGGAGGCACCGTGACCCTGACCTGTAGAAGTTCTACAGGAGCTGTCACCACATCCAATTACGCTAATTGGGTCCAGCAGAAGCCAGGCCAAGCACCTCGGGGTCTCATTGGGGGTACCAACAAGCGAGCTCCTGGGACTCCTGCCAGGTTTAGTGGGAGTCTGTTGGGCGGTAAGGCTGCTCTTACTCTTAGTGGAGTACAGCCCGAGGACGAGGCCGAATACTACTGTGCCCTGTGGTACTCAAACTTGTGGGTCTTCGGTGGTGGGACCAAACTTACTGTCCTG(SEQ ID NO:7)。

the gene encoding the connecting peptide 1 includes the nucleotide sequence shown below:

GGTGGTGGTGGTTCTGGAGGTGGTGGAAGCGGAGGAGGTGGATCC(SEQ ID NO:8)。

the gene encoding the connecting peptide 2 includes the nucleotide sequence shown below:

GGCGGAGGAGGATCT(SEQ ID NO:9)。

the gene encoding the first Fc region comprises the nucleotide sequence shown below:

CCAAAGTCATGTGACAAGACTCACACATGTCCCCCATGTCCAGCCCCTGAGCTCCTGGGAGGACCATCTGTTTTCCTGTTTCCCCCTAAGCCCAAGGATACCCTGATGATCAGTAGAACCCCAGAGGTGACATGCGTTGTCGTGGCTGTTTCTCACGAAGACCCAGAGGTAAAGTTCAACTGGTACGTAGACGGCGTCGAAGTCCATAATGCCAAGACAAAGCCTCGTGAGGAGCAGTATGCCTCAACATATCGCGTCGTATCCGTGTTGACTGTCCTCCATCAGGACTGGCTGAACGGGAAAGAATACAAATGTAAGGTGAGCAATAAGGCTCTGCCCGCACCTATTGAGAAGACTATCTCTAAGGCTAAGGGCCAGCCAAGAGAACCTCAGGTTTATACCCTTCCTCCCTCTCGAGAGGAGATGACTAAGAACCAGGTGTCTCTTTGGTGCCTGGTCAAAGGTTTTTACCCAAGTGATATTGCCGTGGAGTGGGAAAGCAACGGTCAGCCCGAGAATAACTACAAAACAACCCCACCTGTGTTGGACTCTGACGGCTCCTTCTTTCTTTACTCCAAATTGACCGTGGACAAATCTCGCTGGCAGCAGGGGAATGTGTTCTCCTGTAGCGTCATGCACGAGGCCCTCCACAACCACTATACCCAGAAGAGCCTGTCTCTGAGTCCAGGAAAA(SEQ ID NO:10)。

the gene encoding the extracellular domain of wild-type PD-1 comprises the nucleotide sequence as shown below:

GATTCTCCTGATAGACCCTGGAATCCCCCTACTTTCAGCCCTGCTCTTCTGGTCGTCACCGAAGGTGATAATGCAACATTTACCTGTAGCTTCTCAAACACCAGCGAGAGCTTTGTTCTTAACTGGTACAGAATGAGCCCCTCCAACCAGACTGATAAGCTGGCCGCCTTTCCTGAGGACCGGTCTCAACCAGGCCAGGATTGTCGGTTTCGGGTTACCCAGCTCCCTAACGGCAGGGATTTTCACATGTCAGTCGTGAGGGCCAGAAGAAACGACAGCGGCACATACCTGTGCGGGGCAATTTCCCTTGCACCTAAGGCACAGATCAAGGAGAGTCTGAGAGCAGAACTGCGCGTTACAGAACGACGCGCCGAGGTGCCAACAGCACATCCTTCTCCCTCTCCTAGGCCCGCTGGTCAGTTCCAGACCTTGGTC(SEQ ID NO:11)。

the gene encoding the connecting peptide 3 includes the nucleotide sequence shown below:

GGAGGTGGGGGTAGC(SEQ ID NO:12)。

the gene encoding the second Fc region has the nucleotide sequence shown below:

CCAAAGTCTTGTGACAAAACTCATACCTGCCCCCCTTGTCCCGCCCCAGAACTTCTTGGAGGGCCCTCAGTGTTCCTCTTCCCCCCAAAGCCAAAGGATACACTCATGATCAGTCGCACCCCAGAGGTGACATGCGTTGTAGTGGCTGTCTCCCACGAGGATCCCGAGGTGAAGTTTAATTGGTACGTCGACGGGGTTGAAGTGCACAATGCTAAGACCAAGCCCAGGGAGGAACAGTACGCCTCTACATACAGGGTGGTGTCCGTCTTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAATATAAGTGTAAGGTGTCCAATAAAGCACTCCCCGCCCCAATTGAGAAGACCATAAGCAAAGCAAAAGGCCAACCACGTGAACCACAGGTCTATACCCTTCCCCCATCTAGGGAGGAAATGACCAAGAATCAAGTCTCATTGTCCTGCGCCGTGAAGGGTTTCTACCCTAGCGACATCGCCGTGGAGTGGGAATCCAATGGGCAGCCCGAGAACAACTATAAAACCACCCCACCTGTCTTGGATTCCGACGGCTCATTCTTCCTCGTCAGCAAGCTGACCGTGGATAAGAGTCGGTGGCAGCAGGGTAACGTGTTCTCCTGCTCAGTGATGCATGAGGCACTGCATAACCACTACACACAGAAAAGTCTTAGTCTCTCCCCAGGCAAG(SEQ ID NO:13)。

the CD3 single chain antibody includes the amino acid sequence shown below:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO:14)。

the linker peptide 1 comprises the amino acid sequence shown below:

GGGGSGGGGSGGGGS(SEQ ID NO:15)。

the connecting peptide 2 comprises the amino acid sequence shown below:

GGGGS(SEQ ID NO:16)。

the first Fc region comprises the amino acid sequence shown below:

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:17)。

the wild type PD1 extracellular region comprises the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV(SEQ ID NO:18)。

the linker peptide 3 comprises the amino acid sequence shown below:

GGGGS(SEQ ID NO:19)。

the second Fc region comprises the amino acid sequence shown below:

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20)。

PD-1 extracellular domain mut comprises the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV(SEQ ID NO:21)。

Example 2 identification of the binding Capacity of bispecific antibodies to Jurkat T cells

The present example uses flow cytometry to detect the binding properties of bispecific antibodies and uses the intensity of the signal after bispecific antibody addition to determine the binding properties of bispecific antibodies and Jurkat T. The specific experimental procedure is as follows:

jurkat T cells were diluted 1X 10 with PBS ⁶ Per mL, in a volume of 90. Mu.L/tube in a 1.5mL EP tube, 10. Mu.L/tube mouse serum was added thereto, and the tube was blocked at 4℃for 30min; after closing, a series of concentration gradients (10) ^-3 、10 ^-2 、10 ^-1 、10 ⁰ 、10 ¹ 、10 ² μg/mL), 10 μl/tube of CD3×pd1mut bispecific antibody, igG (control IgG1, biolegend, QA16a 12), after incubation at 4 ℃ for 30min, 1mL PBS was added to the EP tube, centrifuged at 4 ℃ for 5min at 100g, the supernatant was discarded, washed once with PBS for precipitation, centrifuged to discard the supernatant, cells were resuspended in 100 μl/tube PBS, 1 μl/tube Alexa-647 labeled mouse anti-human Fc antibody secondary antibody (Biolegend, HP 6017) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended with 200 μl/tube PBS and examined using a flow cytometer, and specific experimental results are shown in fig. 2, demonstrating that the bispecific antibodies of the present invention are capable of binding Jurkat T cells.

EXAMPLE 3 identification of the binding Capacity of bispecific antibodies to CHO-K1-PD-L1 cells

The present example uses flow cytometry to detect the binding properties of bispecific antibodies and uses the strength of the signal after bispecific antibody addition to determine the binding properties of bispecific antibodies and CHO-K1-PD-L1 cells. The specific experimental procedure is as follows:

HEK293T cells according to 5X 10 ⁵ Six-well plates were plated with D without diabodiesMEM medium was incubated overnight. The medium was discarded before transfection and 1mL of fresh DMEM medium without diabody was added. The coding sequence of the PD-L1 protein (SEQ ID NO: 22) was inserted between the cleavage sites EcoRI and BamHI of the pLVX-PD-L1-IRES-puro vector, the pMD2G, psPAX2 vector (total 3. Mu.g) was added to 200. Mu.L of serum-free DMEM medium in a ratio of 2:1:1, then 12. Mu.g of polyetherimide (PEI, polysciences Co., ltd.) was added, the obtained PD-L1 protein had the amino acid sequence shown as SEQ ID NO:23, and after mixing, it was allowed to stand for 16min, then the whole solution was added to the above six-well plate with HEK293T cells spread thereon, after 6h of culture, the medium was discarded, and after 48h of transfection, the cell culture supernatant was harvested, and after 0.45 μm filter (Millipore) was used, the virus supernatant was all added to 1X 10-containing virus supernatant ⁴ Polycoagulamine (Sigma) was added to a 6-well plate of CHO-K1 cells at a final concentration of 4. Mu.g/mL and incubated for 12h. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cells are CHO-K1-PD-L1 cells.

ATGAGGATCTTTGCTGTGTTCATATTTATGACCTACTGGCACCTGCTGAACGCTTTTACTGTGACCGTGCCAAAGGATCTGTACGTGGTGGAATATGGAAGCAACATGACCATCGAGTGTAAGTTTCCCGTCGAAAAACAGCTCGATCTGGCAGCCCTGATTGTCTACTGGGAGATGGAGGATAAGAATATCATACAATTCGTGCATGGAGAAGAGGATTTGAAGGTTCAGCACTCATCTTATCGCCAGAGGGCTCGGCTGCTGAAGGATCAGCTGTCTCTGGGCAATGCTGCTCTTCAAATAACAGATGTGAAGTTGCAGGACGCAGGTGTCTACCGGTGTATGATCAGTTACGGCGGGGCTGATTACAAGCGCATTACAGTGAAGGTTAATGCTCCATACAATAAGATCAACCAGCGGATCCTGGTGGTAGACCCCGTCACAAGTGAGCATGAGTTGACATGCCAGGCCGAGGGGTACCCAAAAGCCGAAGTGATTTGGACCTCTTCTGATCACCAGGTCCTCTCTGGGAAAACCACTACTACCAACTCTAAGCGCGAAGAGAAACTGTTCAATGTCACTTCCACACTCAGAATAAACACTACAACTAATGAGATTTTCTACTGTACTTTTCGTAGACTGGATCCAGAGGAGAACCATACTGCCGAGCTCGTGATCCCTGAGCTTCCCCTGGCTCACCCACCTAACGAGCGCACTCATCTTGTGATTCTGGGAGCAATTCTTTTGTGTCTCGGAGTGGCTCTGACCTTTATCTTTCGGCTCAGAAAGGGAAGGATGATGGATGTCAAAAAGTGTGGTATCCAGGACACCAACTCCAAGAAGCAATCTGACACACATTTGGAAGAGACC(SEQ ID NO:22)。

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ ID NO:23)。

CHO-K1-PD-L1 cells were diluted 1X 10 with PBS ⁶ Per mL, add 90. Mu.L/tube to a 1.5mL EP tube, add 10. Mu.L/tube mouse serum thereto, and block at 4℃for 30min. Respectively adding a series of concentration gradients (10 ^-4 、10 ^-3 、10 ^-2 、10 ^-1 、10 ⁰ 、10 ¹ 、10 ² μg/mL), CD3 xpd 1mut bispecific antibody, hIgG (control IgG1, biolegend, QA16a 12) 10 μl/tube, incubated for 30min at 4 ℃. After the incubation, 1mL of PBS was added to the EP tube, the mixture was centrifuged at 100g for 5min at 4℃and the supernatant was discarded and the pellet was washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube PBS, after which a volume of 1. Mu.L/tube Alexa-647-labeled mouse anti-human Fc antibody secondary antibody (Biolegend, HP 6017) was added thereto and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. The cells were resuspended with 200. Mu.L/tube PBS and examined by flow cytometry, and the specific experimental results are shown in FIG. 3, further showing that the bispecific antibody CD3 XPD 1mut of the invention was able to bind CHO-K1-PD-L1 cells. The results in FIG. 4 show that CD3 XPD 1 has a relatively low binding capacity for CHO-K1-PDL1, but is equally capable of binding efficiently, compared to CD3 XPD 1 mut.

Example 4: tumor cell surface PDL1 expression detection

In this example, flow cytometry was used to detect PDL1 expression on the surface of tumor cells (lung cancer NCI-H358, cervical cancer Hela, ovarian cancer Ho 8910), and specific experimental procedures were as follows:

NCI-H358 (CRL-5807), heLa (ATCC No. CRM-CCL-2), ho8910 cells were diluted 1X 10 with PBS, respectively ⁶ Each of the diluted cells was placed in a 1.5mL EP tube at a volume of 90. Mu.L/tube, and mouse serum was added to the EP tube at a volume of 10. Mu.L/tube, and the tube was blocked at 4℃for 30 minutes; after the end of blocking, 1. Mu.L/tube of APC-labeled mouse anti-human PDL1 antibody (Biolegend) and isotype control (BD, 555751) were added thereto, and incubation was performed at 4℃for 30min under light-shielding conditions. Washing with PBS twice, centrifugingThe supernatant was discarded. Cells were resuspended with 200. Mu.L/tube PBS and examined by flow cytometry, the specific experimental results are shown in FIG. 5, with the abscissa representing fluorescence intensity, the ordinate representing cell mass, NCI-H358 cells expressing PDL1, and HeLa, ho8910 cells low expressing PDL1.

Example 5: bispecific antibodies promote binding of Jurkat T cells to NCI-H358 tumor cells

In this example, a total of 4 experiments were performed, including NCI-H358, NCI-H358 and Jurkat cells, NCI-H358, jurkat and CD3 antibodies, NCI-H358, jurkat and CD3 XPD 1mut bispecific antibodies, as follows:

(1) NCI-H358 cells were diluted 1X 10 in complete DMEM medium ⁶ Per mL, was added to a 12-well plate at a volume of 500 μl/well to obtain NCI-H358 group.

(2) Jurkat T cell markers: diluted to 2X 10 with serum-free DMEM medium ⁶ Per mL, 5. Mu.M CFSE (Sigma) was added and incubated at 37℃for 30min. Adding 1mL of complete DMEM medium, centrifuging at 4deg.C for 5min at 100g, discarding supernatant, washing the precipitate twice with complete DMEM medium, and diluting the cells to 2×10 with complete DMEM medium ⁶ Per mL, added to a portion of the wells of the 12-well plate obtained in step (1) in a volume of 500. Mu.L/well to obtain NCI-H358 cells+Jurkat cell groups.

(3) The bispecific antibodies CD3 XPD 1mut and CD3 antibody (OKT 3) were diluted to 100. Mu.g/mL with complete DMEM medium, respectively, and added to a portion of the wells of the 12-well plate obtained in step (2) in a volume of 10. Mu.L/well to obtain NCI-H358 cell +Jurkat cell +CD3 antibody group, NCI-H358 cell +Jurkat cell +CD3 XPD 1mut bispecific antibody group.

(4) The 4 groups of reaction systems obtained above were subjected to a reaction at 37℃with 5% CO ₂ Culturing for 24 hours under the condition.

(5) Discarding the culture supernatant of the cultured system obtained in the step (4), adding 1mL of PBS, washing for 3min at 100rpm/min by using a decolorizing shaker, discarding the supernatant, and washing the precipitate once again by using the PBS.

(6) PBS (500. Mu.L/well) was added to the product obtained in the step (5), and the resultant was photographed by using a fluorescence microscope.

The specific experimental results are shown in FIG. 6, further showing that the bispecific antibodies of the present invention are capable of promoting binding of Jurkat T cells to NCI-H358 tumor cells.

Example 6 bispecific antibody promotes PBMC killing tumor cells

The influence of the bispecific antibody obtained in the example 1 on the killing of tumor cells (lung cancer NCI-H358 cells, cervical cancer HeLa cells and ovarian cancer Ho8910 cells) by PBMC is detected by constructing a reaction system of the tumor cells+PBMC+different concentrations of CD3 XPD 1mut bispecific antibody, and the specific experimental operation is as follows:

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

(2) The tumor cells were diluted to 2X 10 in complete DMEM medium ⁵ Per mL, 50. Mu.L/well of each volume was added to the RTCA plate obtained in step (1) and then at 37℃with 5% CO ₂ Detecting cell coefficients for 24h using xCELLigence RTCA TP equipment;

(3) Diluting the CD3 XPD 1mut bispecific antibody to a series of concentration gradients (0.1, 1, 10, 100. Mu.g/mL) with complete DMEM medium, adding to the RTCA plate obtained in step (2) in a volume of 20. Mu.L/well;

(4) PBMC (Chimaphila) were diluted 1.25X10 with complete DMEM medium ⁶ Adding 80 mu L/hole of the RTCA plate obtained in the step (3) into each mL;

(5) The reaction system obtained in the step (4) is treated with 5 percent CO at 37 DEG C ₂ Cell factor 72h was measured using xCELLigence RTCA TP apparatus.

Specific experimental results are shown in fig. 7, 8 and 9, and further show that the bispecific antibody of the present invention can promote PBMC to kill lung cancer NCI-H358 cells expressing PD-L1 and cervical cancer HeLa cells and ovarian cancer Ho8910 cells with low expression of PDL 1.

From the experimental results, the bispecific antibody obtained by the invention can be combined with T cells and tumor cells, and promote the T cells to kill the 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, wherein the recombinant antibody is a diabody comprising:

a first peptide chain comprising a CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region, and a first Fc region; and

a second peptide chain comprising a PD-1 extracellular region and a second Fc region; and

a connecting peptide 1, wherein the N end of the connecting peptide 1 is connected with the C end of the heavy chain variable region, and the C end of the connecting peptide 1 is connected with the N end of the light chain variable region; and

a linker peptide 2, the N-terminus of the linker peptide 2 being linked to the C-terminus of the light chain variable region, the C-terminus of the linker peptide 2 being linked to the N-terminus of the first Fc region; and

a connecting peptide 3, wherein the N-terminal of the connecting peptide 3 is connected with the C-terminal of the PD-1 extracellular region, and the C-terminal of the connecting peptide 3 is connected with the N-terminal of the second Fc region;

wherein the C-terminus of the CD3 single chain antibody is linked to the N-terminus of the first Fc region and the C-terminus of the PD-1 extracellular region is linked to the N-terminus of the second Fc region, the PD-1 extracellular region having V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A V and a107I mutations compared to the wild type PD-1 extracellular region, which is SEQ ID NO:18, an amino acid sequence shown in seq id no;

The CD3 single chain antibody has the sequence as set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

2. The recombinant antibody according to claim 1, wherein said connecting peptide 1 comprises the amino acid sequence of SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

3. The recombinant antibody according to claim 1 or 2, wherein said connecting peptide 2 comprises the amino acid sequence of SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.

4. The recombinant antibody according to any one of claims 1-3, wherein said connecting peptide 3 comprises the amino acid sequence of SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no.

5. The recombinant antibody according to claim 1, wherein the first Fc region and the second Fc region are linked by a knob-into-hole structure.

6. The recombinant antibody according to claim 5, wherein said first Fc region has at least one of a lack of a CH1 region, a T366W mutation compared to a wild-type IgG1 Fc region, and said second Fc region has at least one of a lack of a CH1 region, a T366S, L368A, Y407V mutation compared to a wild-type IgG1 Fc region.

7. The recombinant antibody according to claim 5 or 6, wherein at least a portion of said first and second Fc regions are derived from at least one of a murine antibody, a primate-derived antibody, or a mutant thereof.

8. The recombinant antibody according to claim 7, wherein at least a portion of said first and second Fc regions are derived from a murine antibody, a primate-origin IgG, 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 IgG1 or a mutant thereof.

10. The recombinant antibody according to any one of claims 5-7, wherein said first Fc region has an amino acid sequence as set forth in SEQ ID No. 17 and said second Fc region has an amino acid sequence as set forth in SEQ ID No. 20.

11. The recombinant antibody according to any one of claims 1-10, wherein said recombinant antibody has the amino acid sequence of SEQ ID NO:4 and 6.

12. A nucleic acid encoding the recombinant antibody of any one of claims 1-11.

13. The nucleic acid of claim 12, wherein the nucleic acid has the sequence of SEQ ID NO:1 and 3.

14. An expression vector carrying the nucleic acid of claim 12 or 13.

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

Introducing the expression vector of claim 14 into a cell;

the cells are cultured under conditions suitable for protein expression and secretion to obtain the recombinant antibodies.

16. The method of claim 15, wherein the cell is a eukaryotic cell.

17. A recombinant cell carrying the nucleic acid of claim 12 or 13, or the expression vector of claim 14.

18. A composition, comprising:

the recombinant antibody of any one of claims 1-11, the nucleic acid of claim 12 or 13, the expression vector of claim 14, or the recombinant cell of claim 17.

19. Use of the recombinant antibody of any one of claims 1-11, the nucleic acid of claim 12 or 13, the expression vector of claim 14, the recombinant cell of claim 17, or the composition of claim 18 in the manufacture of a medicament for treating a cancer comprising at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

20. A medicament, comprising: the recombinant antibody of any one of claims 1-11, the nucleic acid of claim 12 or 13, the expression vector of claim 14, the recombinant cell of claim 17, or the composition of claim 18, the medicament for treating a cancer comprising at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

21. Use of the recombinant antibody of any one of claims 1-11 in the preparation of a kit for detecting CD3 and/or PD-L1.

22. A kit comprising the recombinant antibody of any one of claims 1-11.

23. The kit of claim 22, wherein the kit is for detecting CD3 and/or PD-L1.