CN114409797A

CN114409797A - Recombinant antibodies and uses thereof

Info

Publication number: CN114409797A
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: 2022-04-29
Anticipated expiration: 2041-12-24
Also published as: CN114409797B

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 and a first Fc region, said CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region; and a second peptidyl chain comprising a PD-1 extracellular region and a second Fc region; wherein the C end of the CD3 single-chain antibody is connected with the N end of the first Fc region, and the C end of the PD-1 extracellular region is connected with the N end of the second Fc region. The bispecific antibody prepared by the invention can simultaneously target CD3 and PD-L1, 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 biomedicine, and particularly relates 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 an antigen binding fragment or a nucleic acid molecule or an expression vector or a recombinant cell or a composition in preparation of a medicament, application of the recombinant antibody in preparation of a kit and a 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 means 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.

In recent years, bispecific antibodies have become a focus of research in immunotherapy. The bispecific antibody is an artificial antibody containing two specific antigen binding sites, and can bridge between a target cell (tumor cell) and an effector cell (immune cell) to generate an effect function of targeted killing of tumors. The immunosuppressive receptor PD-1 is expressed on the surfaces 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 anti-cancer function of immune cells through the interaction of PD-L1/PD-1. The relatively high expression of PD-L1 in tumor tissue makes it a potential tumor-associated antigen. However, because of (1) the risk of immunogenicity of bispecific antibody drugs: the antibody is not human self-owned, and is generated by the antigen immunization of animals such as mice and the like, the immunogenicity of the antibody can be only partially reduced through humanized modification, and the human immune system still generates the antibody aiming at the antibody, namely the anti-antibody, so that the effect reduction and even the failure of the medicine are caused. (2) If the antibody of the target tumor-associated antigen is in a light chain and heavy chain dimer structure, the production and purification difficulty exists; if the antibody targeting the tumor-associated antigen is an ScFv fragment, its affinity for the tumor-associated antigen is greatly reduced, affecting the anticancer effect.

Thus, there is still a need to further develop bispecific antibodies with low immunogenicity 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:

PD-L1 in tumor tissue binds with immune checkpoint PD-1, and then inhibit the anti-cancer function of the immune cell, CD3 antibody single-chain variable fragment (ScFv) can bind with T cell, the inventor has designed the bispecific antibody targeting CD3 and PD-L1, after a large amount of experimental screening, have obtained predominant bispecific antibody CD3 XPD-1 and CD3 XPD-1 mut, the bispecific antibody can bind with high-expression PD-L1 and T cell in tumor tissue at the same time, mediate the killing of T cell to tumor cell effectively, have stronger anticancer ability.

Accordingly, in a first aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the invention, comprising: a first peptide chain comprising a CD3 single chain antibody and a first Fc region, said CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region; and a second peptidyl chain comprising a PD-1 extracellular region and a second Fc region; wherein the C end of the CD3 single-chain antibody is connected with the N end of the first Fc region, and the C end of the PD-1 extracellular region is connected with the N end of the second Fc region. The recombinant antibody provided by the embodiment of the invention can be simultaneously combined with CD3 and PD-L1, effectively mediates the killing effect of T cells on tumor cells, and has strong tumor inhibition capacity.

In a second aspect of the invention, the invention features 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 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 third aspect of the invention, the invention features 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 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 PD-L1, 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 PD-L1, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capacity.

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. 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 under appropriate conditions, and the recombinant antibody can be simultaneously combined with CD3 and PD-L1, 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 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 invention can effectively combine with CD3 and PD-L1 protein molecules and promote the selective localization of T cells to the local part of a tumor rather than the peripheral circulation, so as to avoid the systemic activation.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody of the first aspect, a nucleic acid of the second aspect, an expression vector of the third aspect, a recombinant cell of the fifth aspect or a composition of the sixth aspect in the manufacture of a medicament for the treatment or prevention of cancer. As described above, the recombinant antibody of the present embodiment can effectively bind to CD3 and PD-L1 protein molecules and induce T cells to selectively localize to tumor rather than to circulate in the periphery, avoiding systemic activation, and the drug comprising a series of substances of the recombinant antibody also has 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 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 antibody of the present embodiment 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 rather than to the peripheral circulation, thereby avoiding systemic activation, and thus, the drug comprising a series of substances of the recombinant antibody also has a significant effect of treating or preventing cancer.

In a ninth aspect, the invention provides the use of a recombinant antibody according to the first aspect in the preparation of a kit for the detection of CD3 and/or PD-L1. The recombinant antibody can bind to the CD3 and/or PD-L1 protein, and thus, a kit comprising the recombinant antibody can be used to effectively 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 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 of the first aspect. The recombinant antibody provided according to the embodiments of the present invention may bind to CD3 and/or PD-L1 protein, and thus, a kit comprising the recombinant antibody may be used to effectively 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 protein in a biological sample, and can also be used for judging the individual state, such as judging whether the PD-L1 level of the individual is higher or lower than the normal level after obtaining the PD-L1 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 recombinant bispecific antibody according to an embodiment of the present invention, in which the linking site of a linking peptide is not given, and a first Fc region (left side) and a second Fc 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 measurement of the binding ability of the recombinant bispecific antibody to Jurkat T cells according to the embodiment of the present invention, wherein isotype control represents isotype control IgG 1;

FIG. 3 is a graph showing the results of the detection of the binding ability of the recombinant bispecific antibody according to the embodiment of the present invention to CHO-K1-PD-L1;

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

FIG. 5 is a graph showing the result of measuring the expression level of PD-L1 on the surface of a tumor cell according to an embodiment of the present invention, wherein isotype control represents isotype control IgG 1;

FIG. 6 is a graph showing the results of the test of the ability of recombinant bispecific antibodies to promote the binding of Jurkat cells to tumor cells, wherein NCI-H358 indicates that the group of reaction systems comprises only NCI-H358 cells, NCI-H358 Jurkat (CFSE) indicates that the group of reaction systems comprises NCI-H358 cells and Jurkat cells, NCI-H358 Jurkat (CFSE) anti-CD3(OKT3) indicates that the group of reaction systems comprises NCI-H358 cells, Jurkat cells and CD3 antibodies, NCI-H358 Jurkat (CFSE) CD3 XPD 1mut indicates that the group of reaction systems comprises NCI-H358 cells, Jurkat cells and CD3 XPD 1mut bispecific antibodies according to the embodiment of the present invention;

FIG. 7 is a graph showing the results of a test in which a recombinant bispecific antibody promotes PBMC to kill lung cancer NCI-H358 cells according to an embodiment of the present invention;

FIG. 8 is a graph showing the results of detection of the recombinant bispecific antibody promoting 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 detection of the recombinant bispecific antibody promoting PBMC killing of ovarian cancer Ho8910 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 implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such 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, the "diabody" refers to a antibody obtained by linking peptide chains that can specifically recognize different protein molecules to two chains of an Fc region, respectively, wherein the two chains of the Fc region are linked by a knob-in-hole structure.

The "Knob-in-hole structure" herein is the formation of a Knob (Knob) mutation in the CH3 region of the antibody heavy chain Fc to facilitate heavy chain engagement and heterodimer formation, for example, by mutating amino acids in the human IgG1-Fc CH3 domain (T366S, L368A, Y407V mutations, i.e., "hole" in one chain; T366W mutations, i.e., "Knob" in the other chain) in the present application.

"operably linked" herein 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, which is a diabody, comprising: a first peptide chain comprising a CD3 single chain antibody and a first Fc region, said CD3 single chain antibody comprising a heavy chain variable region, a light chain variable region; and a second peptidyl chain comprising a PD-1 extracellular region and a second Fc region; wherein the C end of the CD3 single-chain antibody is connected with the N end of the first Fc region, and the C end of the PD-1 extracellular region is connected with the N end of the second Fc region. The recombinant antibody provided by the embodiment of the invention can be simultaneously combined with CD3 and PD-L1, effectively mediates the killing effect of T cells on tumor cells, and has strong tumor inhibition capacity.

According to some embodiments of the present 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 embodiments of the present invention, the recombinant antibody including the wild type PD-L1 extracellular domain or its mutant can effectively mediate the killing effect of T cells on tumors, and has strong tumor inhibition ability.

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

According to some embodiments of the invention, the wild-type PD-1 extracellular region comprises SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV(SEQ ID NO：18)。

According to some embodiments of the invention, the light chain variable region further comprises a linker peptide 1, wherein the N-terminal of the linker peptide 1 is connected to the C-terminal of the heavy chain variable region, and the C-terminal of the linker peptide 1 is connected to the N-terminal of the light chain variable region.

According to some embodiments of the invention, the linker peptide 1 comprises SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

GGGGSGGGGSGGGGS(SEQ ID NO：15)。

According to some embodiments of the invention, the Fc region further comprises a linker peptide 2, wherein 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 linker peptide 2 comprises SEQ ID NO: 16.

GGGGS(SEQ ID NO：16)。

According to some embodiments of the present invention, the Fc-Fc fragment further comprises a linker peptide 3, wherein the N-terminus of the linker peptide 3 is linked to the C-terminus of the extracellular domain of PD-1, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the second Fc domain;

according to some embodiments of the invention, the linker peptide 3 comprises SEQ ID NO: 19, or a pharmaceutically acceptable salt thereof.

GGGGS(SEQ ID NO：19)。

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

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

According to some specific embodiments of the invention, the first Fc region has at least one of the 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 the lack of a CH1 region, T366S, L368A, Y407V mutation compared to a wild-type IgG1 Fc region.

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

According to some embodiments of the invention, at least a portion of the first Fc region and the second Fc region is derived from a murine antibody, a human antibody, a primate IgG, or a mutant thereof.

According to some embodiments of the invention, at least a portion of the first and second Fc regions are from human IgG1 or a mutant 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 an amino acid sequence as shown below:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：4)。

the second peptide chain (CD3 XPD-1) has the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：5)。

the second peptide chain (CD3 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 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 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 (CD3 XPD 1) has the nucleotide sequence shown below:

GATTCTCCTGATAGACCCTGGAATCCCCCTACTTTCAGCCCTGCTCTTCTGGTCGTCACCGAAGGTGATAATGCAACATTTACCTGTAGCTTCTCAAACACCAGCGAGAGCTTTGTTCTTAACTGGTACAGAATGAGCCCCTCCAACCAGACTGATAAGCTGGCCGCCTTTCCTGAGGACCGGTCTCAACCAGGCCAGGATTGTCGGTTTCGGGTTACCCAGCTCCCTAACGGCAGGGATTTTCACATGTCAGTCGTGAGGGCCAGAAGAAACGACAGCGGCACATACCTGTGCGGGGCAATTTCCCTTGCACCTAAGGCACAGATCAAGGAGAGTCTGAGAGCAGAACTGCGCGTTACAGAACGACGCGCCGAGGTGCCAACAGCACATCCTTCTCCCTCTCCTAGGCCCGCTGGTCAGTTCCAGACCTTGGTCGGAGGTGGGGGTAGCCCAAAGTCTTGTGACAAAACTCATACCTGCCCCCCTTGTCCCGCCCCAGAACTTCTTGGAGGGCCCTCAGTGTTCCTCTTCCCCCCAAAGCCAAAGGATACACTCATGATCAGTCGCACCCCAGAGGTGACATGCGTTGTAGTGGCTGTCTCCCACGAGGATCCCGAGGTGAAGTTTAATTGGTACGTCGACGGGGTTGAAGTGCACAATGCTAAGACCAAGCCCAGGGAGGAACAGTACGCCTCTACATACAGGGTGGTGTCCGTCTTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAATATAAGTGTAAGGTGTCCAATAAAGCACTCCCCGCCCCAATTGAGAAGACCATAAGCAAAGCAAAAGGCCAACCACGTGAACCACAGGTCTATACCCTTCCCCCATCTAGGGAGGAAATGACCAAGAATCAAGTCTCATTGTCCTGCGCCGTGAAGGGTTTCTACCCTAGCGACATCGCCGTGGAGTGGGAATCCAATGGGCAGCCCGAGAACAACTATAAAACCACCCCACCTGTCTTGGATTCCGACGGCTCATTCTTCCTCGTCAGCAAGCTGACCGTGGATAAGAGTCGGTGGCAGCAGGGTAACGTGTTCTCCTGCTCAGTGATGCATGAGGCACTGCATAACCACTACACACAGAAAAGTCTTAGTCTCTCCCCAGGCAAG(SEQ ID NO：2)。

the gene encoding the second peptide chain (CD3 XPD 1mut) has the nucleotide sequence shown below:

GATAGTCCTGATAGGCCTTGGAATCCTCCTACTTTCTCTCCCGCCTTGCTGGTCGTTACTGAGGGCGACAACGCAACCTTCACTTGCAGTTTTAGTAACACTAGTGAGTCCTTTCACGTGGTCTGGCACAGAGAGTCACCAAGTGGCCAGACTGATACTCTTGCAGCTTTTCCTGAAGATCGCTCCCAGCCAGGCCAGGATTGTCGGTTTAGGGTCACCCAGCTGCCCAATGGGAGAGACTTCCATATGTCTGTCGTGAGAGCACGCCGAAACGATAGTGGTACCTACGTATGCGGCGTCATCTCACTGGCACCCAAAATCCAGATTAAAGAGTCTCTTAGGGCTGAGTTGAGAGTTACCGAGAGAAGAGCAGAGGTACCCACAGCACATCCAAGTCCTAGTCCCAGGCCAGCCGGACAGTTCCAGACTTTGGTAGGAGGTGGCGGGTCACCTAAGTCATGCGACAAGACCCATACCTGTCCCCCATGTCCAGCTCCCGAATTGTTGGGTGGCCCTTCCGTATTTCTGTTCCCTCCCAAACCAAAAGACACACTGATGATAAGTAGAACTCCAGAGGTTACCTGCGTCGTCGTAGCAGTGTCACACGAAGACCCCGAGGTGAAGTTCAATTGGTATGTGGACGGCGTCGAAGTGCATAACGCTAAAACAAAACCAAGAGAGGAGCAGTATGCCAGCACATATCGCGTGGTGAGTGTGCTTACTGTGCTGCACCAGGACTGGCTGAACGGTAAAGAGTACAAGTGTAAAGTGAGTAACAAGGCCCTGCCTGCACCTATCGAAAAGACTATCAGCAAAGCTAAGGGCCAGCCACGTGAGCCACAGGTGTATACACTCCCCCCATCTAGAGAAGAGATGACAAAGAATCAGGTGTCTCTCAGCTGCGCAGTCAAGGGTTTCTACCCTAGCGACATTGCTGTGGAGTGGGAGTCCAATGGGCAGCCAGAAAACAATTACAAGACCACACCACCTGTCCTGGATAGTGACGGCAGCTTCTTCCTGGTGTCCAAACTCACCGTGGATAAGAGCCGGTGGCAGCAGGGTAACGTTTTTAGCTGCTCCGTCATGCATGAAGCTCTGCACAACCATTACACCCAGAAGAGTCTGTCACTGTCCCCAGGCAAA(SEQ ID NO:3)。

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 invention provides an expression vector carrying the nucleic acid 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 PD-L1, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

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 invention can effectively obtain the recombinant antibody, and the recombinant antibody can be simultaneously combined with CD3 and PD-L1, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor inhibition capacity. 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 of the second aspect or the expression vector of the third 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 under appropriate conditions, and the recombinant antibody can be simultaneously combined with CD3 and PD-L1, 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, 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 antibody of the embodiment of the invention can effectively combine with CD3 and PD-L1 protein molecules and promote the selective localization of T cells to the local part of a tumor rather than the peripheral circulation, so as to avoid the systemic activation.

It is to be noted that the compositions include temporally and/or spatially separated combinations as long as they can work 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 of the first aspect, a nucleic acid of the second aspect, an expression vector of the third aspect, a recombinant cell of the fifth aspect or a composition of the sixth aspect in the manufacture of a medicament for the treatment or prevention of cancer. As described above, the recombinant antibody of the present embodiment can effectively bind to CD3 and PD-L1 protein molecules, and promote T cells to selectively localize to tumor rather than to circulate in the periphery, thereby avoiding systemic activation, and thus, the drug comprising a series of substances of the recombinant antibody also has significant tumor treatment or prevention effects.

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 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 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 antibody of the present embodiment can effectively bind to CD3 and PD-L1 protein molecules, and induce T cells to selectively localize to tumor rather than to circulate in the periphery, thereby preventing systemic activation, and thus, the drug comprising a series of substances of the recombinant antibody also has significant effects on 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 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 medicament preparation is matched with the administration mode generally, and the medicament of the invention is in the dosage forms of 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 the recombinant antibody of the first aspect. The recombinant antibody provided according to the embodiments of the present invention may bind to CD3 and/or PD-L1 protein, and thus, a kit comprising the recombinant antibody may be used to effectively 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 protein in a biological sample, and can also be used for judging the individual state, such as judging whether the PD-L1 level of the individual is higher or lower than the normal level after obtaining the PD-L1 level of the individual.

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

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 bispecific antibody molecules

The production of bispecific antibodies was carried out in this example, following the following experimental procedures: ExpicHO Medium (purchased from Thermo Fisher, A2910001) was used to culture ExpicHO cells (purchased from Thermo Fisher) at an adjusted cell concentration of 6X 10⁶Per mL, ExpicHO cell solution was obtained. pTT5 vector (synthesized by Kingonian, Suzhou Co., Ltd.) containing a-chain, b-chain (b1 and b 2) encoding genes (shown as SEQ ID NOS: 1, 2 and 3, respectively) including a nucleotide sequence encoding a CD3 single-chain antibody (SEQ ID NO: 7), a linker peptide 1(SEQ ID NO: 8), a linker peptide 2(SEQ ID NO: 9) and a first Fc region (SEQ ID NO: 10) was added to 2mL OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A, and b1 chain encoding geneIt comprises a nucleotide sequence encoding the extracellular domain of PD-1 (SEQ ID NO: 11), linker peptide 3(SEQ ID NO: 12) and a second Fc domain (SEQ ID NO: 13). 160 μ of LExpifactamineCHO transfection reagent (Thermofisiher, A29130) was added to 2mL of 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% CO₂After 1 day of incubation under conditions, 8mL of Feed, 300. mu. Lenhancer (Thermo Fisher, A29130) was added, and the mixture was transferred to a medium at 32 ℃ with 5% CO₂Culture 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), 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 the antibody CD3 XPD 1 has the amino acid sequence shown as SEQ ID NO 4 (fragment a, first peptide chain) and SEQ ID NO 5 (fragment b1, second peptide chain-wild type), and the antibody CD3 XPD 1mut has the amino acid sequence shown as SEQ ID NO 4 (fragment a, first peptide chain) and SEQ ID NO 6 (fragment b2, second peptide chain-mut), wherein said fragment a 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 a first Fc region (SEQ ID NO 17), and said fragment b1 comprises the amino acid sequence of the extracellular region of PD-1 (SEQ ID NO 18), linker peptide 3(SEQ ID NO 19) and a second Fc region (SEQ ID NO 20), the b2 fragment includes the amino acid sequence of PD-1 extracellular region mut (SEQ ID NO: 21), linker peptide 3(SEQ ID NO: 19) and second Fc region (SEQ ID NO: 20).

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 as follows:

GATTCTCCTGATAGACCCTGGAATCCCCCTACTTTCAGCCCTGCTCTTCTGGTCGTCACCGAAGGTGATAATGCAACATTTACCTGTAGCTTCTCAAACACCAGCGAGAGCTTTGTTCTTAACTGGTACAGAATGAGCCCCTCCAACCAGACTGATAAGCTGGCCGCCTTTCCTGAGGACCGGTCTCAACCAGGCCAGGATTGTCGGTTTCGGGTTACCCAGCTCCCTAACGGCAGGGATTTTCACATGTCAGTCGTGAGGGCCAGAAGAAACGACAGCGGCACATACCTGTGCGGGGCAATTTCCCTTGCACCTAAGGCACAGATCAAGGAGAGTCTGAGAGCAGAACTGCGCGTTACAGAACGACGCGCCGAGGTGCCAACAGCACATCCTTCTCCCTCTCCTAGGCCCGCTGGTCAGTTCCAGACCTTGGTCGGAGGTGGGGGTAGCCCAAAGTCTTGTGACAAAACTCATACCTGCCCCCCTTGTCCCGCCCCAGAACTTCTTGGAGGGCCCTCAGTGTTCCTCTTCCCCCCAAAGCCAAAGGATACACTCATGATCAGTCGCACCCCAGAGGTGACATGCGTTGTAGTGGCTGTCTCCCACGAGGATCCCGAGGTGAAGTTTAATTGGTACGTCGACGGGGTTGAAGTGCACAATGCTAAGACCAAGCCCAGGGAGGAACAGTACGCCTCTACATACAGGGTGGTGTCCGTCTTGACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAATATAAGTGTAAGGTGTCCAATAAAGCACTCCCCGCCCCAATTGAGAAGACCATAAGCAAAGCAAAAGGCCAACCACGTGAACCACAGGTCTATACCCTTCCCCCATCTAGGGAGGAAATGACCAAGAATCAAGTCTCATTGTCCTGCGCCGTGAAGGGTTTCTACCCTAGCGACATCGCCGTGGAGTGGGAATCCAATGGGCAGCCCGAGAACAACTATAAAACCACCCCACCTGTCTTGGATTCCGACGGCTCATTCTTCCTCGTCAGCAAGCTGACCGTGGATAAGAGTCGGTGGCAGCAGGGTAACGTGTTCTCCTGCTCAGTGATGCATGAGGCACTGCATAACCACTACACACAGAAAAGTCTTAGTCTCTCCCCAGGCAAG(SEQ ID NO:2)。

the b2 chain coding gene comprises the nucleotide sequence shown as follows:

the a fragments of both CD3 × PD1 and CD3 × PD1mut antibodies include the amino acid sequences shown below:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。

the b1 fragment of CD3 × PD1 antibody includes the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:5)。

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

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:6)。

the gene encoding the CD3 single-chain antibody comprises the nucleotide sequence shown as follows:

GAAGTACAACTGCTGGAATCAGGGGGAGGACTTGTTCAGCCAGGAGGGTCTCTCAAGCTGAGCTGCGCCGCTTCAGGGTTCACATTCAACACTTACGCAATGAATTGGGTGAGACAGGCCCCAGGTAAAGGGCTCGAATGGGTGGCTCGCATTCGCTCCAAATACAATAACTATGCCACCTACTACGCTGACTCTGTGAAAGATCGCTTCACAATCAGCAGGGACGACTCTAAAAATACCGCTTATCTGCAGATGAATAATCTGAAAACCGAAGACACCGCAGTCTATTACTGCGTCAGGCATGGCAACTTCGGAAACTCTTACGTAAGCTGGTTCGCTTACTGGGGCCAGGGAACCCTTGTGACCGTGTCTAGTGGTGGTGGTGGTTCTGGAGGTGGTGGAAGCGGAGGAGGTGGATCCGAACTGGTGGTGACACAGGAGCCTTCTCTCACAGTGAGTCCAGGAGGCACCGTGACCCTGACCTGTAGAAGTTCTACAGGAGCTGTCACCACATCCAATTACGCTAATTGGGTCCAGCAGAAGCCAGGCCAAGCACCTCGGGGTCTCATTGGGGGTACCAACAAGCGAGCTCCTGGGACTCCTGCCAGGTTTAGTGGGAGTCTGTTGGGCGGTAAGGCTGCTCTTACTCTTAGTGGAGTACAGCCCGAGGACGAGGCCGAATACTACTGTGCCCTGTGGTACTCAAACTTGTGGGTCTTCGGTGGTGGGACCAAACTTACTGTCCTG(SEQ ID NO:7)。

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

GGTGGTGGTGGTTCTGGAGGTGGTGGAAGCGGAGGAGGTGGATCC(SEQ ID NO:8)。

the gene encoding linker 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 region of wild-type PD-1 comprises the nucleotide sequence shown as follows:

GATTCTCCTGATAGACCCTGGAATCCCCCTACTTTCAGCCCTGCTCTTCTGGTCGTCACCGAAGGTGATAATGCAACATTTACCTGTAGCTTCTCAAACACCAGCGAGAGCTTTGTTCTTAACTGGTACAGAATGAGCCCCTCCAACCAGACTGATAAGCTGGCCGCCTTTCCTGAGGACCGGTCTCAACCAGGCCAGGATTGTCGGTTTCGGGTTACCCAGCTCCCTAACGGCAGGGATTTTCACATGTCAGTCGTGAGGGCCAGAAGAAACGACAGCGGCACATACCTGTGCGGGGCAATTTCCCTTGCACCTAAGGCACAGATCAAGGAGAGTCTGAGAGCAGAACTGCGCGTTACAGAACGACGCGCCGAGGTGCCAACAGCACATCCTTCTCCCTCTCCTAGGCCCGCTGGTCAGTTCCAGACCTTGGTC(SEQ ID NO:11)。

the gene encoding linker 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 comprises the amino acid sequence shown as follows:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO:14)。

linker peptide 1 comprises the amino acid sequence shown below:

GGGGSGGGGSGGGGS(SEQ ID NO:15)。

linker 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 as follows:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV(SEQ ID NO:18)。

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 region mut comprises the amino acid sequence shown below:

DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV(SEQ ID NO:21)。

example 2 characterization of the binding Capacity of bispecific antibodies to Jurkat T cells

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

jurkat T cells were diluted 1X 10 with PBS⁶mL, added to a 1.5mL EP tube in a volume of 90 μ L/tube, to which 10 μ L/tube mouse serum was added, and blocked at 4 ℃ for 30 min; after the sealing is finished, a series of concentration gradients (10) are respectively added^-3、10^-2、10^-1、10⁰、10¹、10²μ g/mL) of CD3 XPD 1mut bispecific antibody, hIgG (control IgG1, Biolegend, QA16A12)10 μ L/tube, incubated at 4 ℃ for 30min, added 1mL PBS to the EP tube, centrifuged at 4 ℃ and 100g for 5min, discarded the supernatant, washed once with PBS, centrifuged, discarded the supernatant, resuspended cells with 100 μ L/tube PBS, added 1 μ L/tube Alexa-647 labeled secondary mouse anti-human Fc antibody (Biolegend, HP6017), and incubated at 4 ℃ for 30min in the absence of light. Washing twice with PBS, centrifuging, and discardingAnd (6) supernatant fluid. The cells were resuspended in 200. mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 2, which illustrates that bispecific antibodies of the invention are capable of binding to Jurkat T cells.

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

This example uses flow cytometry to detect the binding properties of bispecific antibodies and uses the intensity of signal after bispecific antibody addition to determine the binding properties of bispecific antibodies and CHO-K1-PD-L1 cells. The specific experimental procedures were as follows:

HEK293T cells at 5X 10⁵Cells/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. The pLVX-PD-L1-IRES-puro (pLVX-EF1a-IRES-puro vector was inserted with PD-L1 protein (SEQ ID NO:22) the coding sequence of (1), pMD2G and psPAX2 vector (total 3 mug) are added into 200 muL serum-free DMEM medium according to the ratio of 2:1:1, then 12. mu.g of polyetherimide (PEI, Polysciences Co., Ltd.) was added, and the obtained PD-L1 protein had the amino acid sequence shown in SEQ ID NO: 23; standing for 16min after uniformly mixing, then adding all the liquid into the six-hole plate paved with HEK293T cells, culturing for 6h, after 48 hours of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore), to obtain a virus supernatant, and the whole virus supernatant was added to a medium containing 1X 10.⁴To a 6-well plate 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 cell is CHO-K1-PD-L1 cell.

ATGAGGATCTTTGCTGTGTTCATATTTATGACCTACTGGCACCTGCTGAACGCTTTTACTGTGACCGTGCCAAAGGATCTGTACGTGGTGGAATATGGAAGCAACATGACCATCGAGTGTAAGTTTCCCGTCGAAAAACAGCTCGATCTGGCAGCCCTGATTGTCTACTGGGAGATGGAGGATAAGAATATCATACAATTCGTGCATGGAGAAGAGGATTTGAAGGTTCAGCACTCATCTTATCGCCAGAGGGCTCGGCTGCTGAAGGATCAGCTGTCTCTGGGCAATGCTGCTCTTCAAATAACAGATGTGAAGTTGCAGGACGCAGGTGTCTACCGGTGTATGATCAGTTACGGCGGGGCTGATTACAAGCGCATTACAGTGAAGGTTAATGCTCCATACAATAAGATCAACCAGCGGATCCTGGTGGTAGACCCCGTCACAAGTGAGCATGAGTTGACATGCCAGGCCGAGGGGTACCCAAAAGCCGAAGTGATTTGGACCTCTTCTGATCACCAGGTCCTCTCTGGGAAAACCACTACTACCAACTCTAAGCGCGAAGAGAAACTGTTCAATGTCACTTCCACACTCAGAATAAACACTACAACTAATGAGATTTTCTACTGTACTTTTCGTAGACTGGATCCAGAGGAGAACCATACTGCCGAGCTCGTGATCCCTGAGCTTCCCCTGGCTCACCCACCTAACGAGCGCACTCATCTTGTGATTCTGGGAGCAATTCTTTTGTGTCTCGGAGTGGCTCTGACCTTTATCTTTCGGCTCAGAAAGGGAAGGATGATGGATGTCAAAAAGTGTGGTATCCAGGACACCAACTCCAAGAAGCAATCTGACACACATTTGGAAGAGACC(SEQ ID NO:22)。

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ ID NO:23)。

CHO-K1-PD-L1 cells were diluted 1X 10 with PBS⁶To 1.5mL of EP tube was added 90. mu.L/tube, 10. mu.L/tube of mouse serum was added, and the mixture was blocked at 4 ℃ for 30 min. Adding a series of concentration gradients (10) separately^-4、10^-3、10^-2、10^-1、10⁰、10¹、10²μ g/mL) of CD3 XPD 1, CD3 XPD 1mut bispecific antibody, hIgG (control IgG1, Biolegend, QA16A12)10 μ L/tube, incubated at 4 ℃ for 30 min. After the incubation, 1mL of PBS was added to the EP tube, and the mixture was centrifuged at 100g at 4 ℃ for 5min, and the supernatant was discarded, followed by washing with PBS to precipitate. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. mu.L/tube of PBS, and after resuspension, 1. mu.L/tube of Alexa-647-labeled secondary mouse anti-human Fc antibody (Biolegend, HP6017) 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 further shows that the bispecific antibody CD3 XPD 1mut of the present invention can bind to CHO-K1-PD-L1 cells. The results in FIG. 4 show that CD3 XPD 1 binds to CHO-K1-PDL1 relatively less, but equally effectively, than CD3 XPD 1 mut.

Example 4: tumor cell surface PDL1 expression detection

In the present example, the expression of PDL1 on the surface of tumor cells (lung cancer NCI-H358, cervical cancer Hela, ovarian cancer Ho8910) was detected by flow cytometry, and the specific experimental procedures are as follows:

NCI-H358(CRL-5807), HeLa (ATCC accession number CRM-CCL-2), and Ho8910 cells were each diluted to 1X 10 with PBS⁶(iv)/mL, the diluted cells were added to a 1.5mL EP tube at a volume of 90. mu.L/tube, then mouse serum was added to the EP tube at a volume of 10. mu.L/tube, and the mixture was blocked at 4 ℃ for 30 min; after the blocking was completed, 1. mu.L/tube of APC-labeled mouse anti-human PDL1 antibody (Biolegend) and isotype control (BD, 555751) were added thereto, and the mixture was 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 examined by flow cytometry, and the results of the experiment are shown in FIG. 5, where the abscissa represents fluorescence intensity, the ordinate represents cell mass, NCI-H358 cells express PDL1, and HeLa, Ho8910 cells express PDL1 at a lower level.

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

In this example, a total of 4 groups were set up for carrying out experiments, namely only NCI-H358 group, NCI-H358 cell and Jurkat cell group, NCI-H358 cell, Jurkat cell and CD3 antibody group, NCI-H358 cell, Jurkat cell and CD3 XPD 1mut bispecific antibody group, and the specific experimental procedures were as follows:

(1) NCI-H358 cells were diluted to 1X 10 with complete DMEM medium⁶PermL, into 12-well plates, at a volume of 500. mu.L/well, to obtain NCI-H358 group.

(2) Jurkat T cell markers: diluted to 2X 10 with serum-free DMEM medium⁶Perml, add final concentration of 5. mu.M CFSE (Sigma), incubate at 37 ℃ for 30 min. Adding 1mL complete DMEM medium, centrifuging at 4 deg.C and 100g for 5min, discarding supernatant, washing and precipitating twice with complete DMEM medium, and diluting cells to 2 × 10 with complete DMEM medium⁶mL, 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 the NCI-H358 cells + Jurkat cells group.

(3) The bispecific antibodies CD3 XPD 1mut and CD3 antibody (OKT3) were diluted to 100. mu.g/mL, respectively, in complete DMEM medium, 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 the NCI-H358 cells + Jurkat cells + CD3 antibody group and the NCI-H358 cells + Jurkat cells + CD3 XPD 1mut bispecific antibody group.

(4) The 4 groups of reaction systems obtained above were reacted at 37 ℃ with 5% CO₂Culturing for 24h under the condition.

(5) And (4) removing the culture supernatant of the cultured system obtained in the step (4), adding 1mL of PBS, washing for 3min at 100rpm/min of a decoloring shaker, then removing the supernatant, and washing and precipitating once with PBS.

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

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

Example 6 bispecific antibodies promote PBMC killing of tumor cells

This example examined the effect of bispecific antibody obtained in example 1 on tumor cell killing by PBMC (lung cancer NCI-H358 cells, cervical cancer HeLa cells, ovarian cancer Ho8910 cells), and examined by constructing a reaction system of the bispecific antibody of tumor cell + PBMC + CD3 XPD 1mut at different concentrations, the specific experimental procedures are as follows:

(1) adding a 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 medium⁵mL, 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 ℃₂Detecting the cell coefficient for 24h by using an xCELLigence RTCA TP device under the condition;

(3) diluting CD3 XPD 1mut bispecific antibody into a series of concentration gradients (0.1, 1, 10, 100 mu g/mL) by using complete DMEM medium, adding the concentration gradients into the RTCA plate obtained in the step (2), and adding the concentration gradients into the RTCA plate with the volume of 20 mu L/hole;

(4) PBMC (Cya organisms) were diluted to 1.25X 10 with complete DMEM medium⁶Adding the solution/mL into the RTCA plate obtained in the step (3), and addingAdding 80 mu L/hole;

(5) the reaction system obtained in the step (4) is treated at 37 ℃ and 5% CO₂The cell coefficient was measured for 72h using an xCelLigence RTCA TP instrument.

The 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, cervical cancer HeLa cells and ovarian cancer Ho8910 cells expressing PDL1 at a low level.

The experimental results show that 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 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. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

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, wherein the recombinant antibody is a diabody, comprising:

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

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

wherein the C end of the CD3 single-chain antibody is connected with the N end of the first Fc region, and the C end of the PD-1 extracellular region is connected with the N end of the second Fc region.

2. The recombinant antibody according to claim 1, wherein the PD-1 extracellular region comprises a wild-type PD-1 extracellular region or a mutant thereof;

optionally, the mutant has at least one of the V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, a100V, and a107I mutations compared to the wild-type PD-1 extracellular region;

optionally, the wild-type PD-1 extracellular region comprises SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

3. The recombinant antibody according to claim 1 or 2, further comprising 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, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the light chain variable region;

optionally, the linker peptide 1 comprises SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

4. The recombinant antibody according to any one of claims 1-3, further comprising a linker peptide 2, wherein 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;

optionally, the linker peptide 2 comprises SEQ ID NO: 16.

5. The recombinant antibody according to any one of claims 1-4, further comprising a linker peptide 3, wherein the N-terminus of the linker peptide 3 is linked to the C-terminus of the extracellular region of PD-1, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the second Fc region;

optionally, the linker peptide 3 comprises SEQ ID NO: 19, or a pharmaceutically acceptable salt thereof.

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

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

8. The recombinant antibody according to claim 6 or 7, wherein at least a portion of the first and second Fc regions is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof,

optionally, at least a portion of the first and second Fc regions is from a murine antibody, a human antibody, a primate IgG, or a mutant thereof;

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

9. The recombinant antibody according to any one of claims 6-8, wherein said first Fc region has the amino acid sequence shown in SEQ ID NO 17 and said second Fc region has the amino acid sequence shown in SEQ ID NO 20.

10. The recombinant antibody of any one of claims 1-9, wherein the recombinant antibody has the amino acid sequence of SEQ ID NO:4, and SEQ ID NO:5 or 6.

11. A nucleic acid encoding the recombinant antibody of any one of claims 1 to 10.

12. The nucleic acid of claim 11, wherein the nucleic acid has the sequence of SEQ ID NO:1, and SEQ ID NO:2 or 3.

13. An expression vector carrying the nucleic acid of claim 11 or 12.

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

introducing the expression vector of claim 13 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.

15. A recombinant cell carrying the nucleic acid of claim 11 or 12, or the expression vector of claim 13.

16. A composition, comprising:

the recombinant antibody of any one of claims 1 to 10, the nucleic acid of claim 11 or 12, the expression vector of claim 13 or the recombinant cell of claim 15.

17. Use of the recombinant antibody of any one of claims 1-10, the nucleic acid of claim 11 or 12, the expression vector of claim 13, the recombinant cell of claim 15, or the composition of claim 16 in the manufacture of a medicament for treating or preventing cancer.

18. The use according to claim 17, wherein the cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

19. A medicament, comprising: the recombinant antibody of any one of claims 1-10, the nucleic acid of claim 11 or 12, the expression vector of claim 13, the recombinant cell of claim 15, or the composition of claim 16 for use in the treatment of cancer.

20. The medicament of claim 19, wherein the cancer comprises at least one of: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

21. Use of the recombinant antibody of any one of claims 1-10 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 to 10.

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