CN114805592A - Design, preparation and application of trispecific antibody - Google Patents
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Abstract
The invention provides a design of a trispecific antibody, a preparation method and application thereof, the trispecific antibody comprises (a) anti-Her2 monoclonal antibody Herceptin, (b) anti-CD16a (FcgRIIa) single-chain antibody scFv, (c) SIRPa D1 protein, (D) flexible linker; the C end of an antibody Herceptin heavy chain is connected with an anti-CD16a single-chain antibody through a linker, and the C end of an antibody Herceptin light chain is connected with SIRPa D1 through a linker; the Fc segment of the antibody is modified to change the binding capacity of the Fc segment with a receptor and prolong the half-life period of the protein; tumor cells expressing CD47 can be targeted by SIRPa D1 and CD47 ligand binding; meanwhile, the signal of ' don't eat me ' can be blocked, so that macrophages are activated to phagocytose tumor cells; specifically bind to NK (Nature Kiling) cells by anti-CD16 a; macrophages and NK cells of the tumor microenvironment are recruited to kill tumor cells by SIRPa and anti-CD16 a; the present invention ensures the specificity of anti-CD16a, and does not cause side effects (Neutropenia) due to binding to CD16b on the surface of neutrophils.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to design, preparation and application of a trispecific antibody.
Background
With the continuous development and deepening of oncology and immunology, tumor immunotherapy has become the leading treatment means in the anti-tumor field. At the present stage, the main research direction for tumor immunotherapy at home and abroad is the immune checkpoint inhibitor. CD47 has been considered by the industry as the most important target in the field of tumor immunity after PD-1/PD-L1, and currently, countless candidate drugs targeting CD47 are in the preclinical and clinical development stage all over the world, but no approved anti-CD 47 therapy exists.
CD47 is widely expressed on the surface of many cancer cells, but is also widely expressed on the surface of erythrocytes to protect themselves from phagocytosis. This means that the target CD47 drug can kill tumor cells and simultaneously can not avoid injuring red blood cells by mistake, thereby causing the number of red blood cells and platelets to be reduced and causing serious anemia reaction.
The invention is a trispecific antibody directed against Her2, CD47 and CD16 a. Herceptin has become the standard therapy for Her2 positive tumors such as breast cancer; the CD47-SIRPa "Don't eat me" pathway has been well documented as a target for significant clinical efficacy; CD16a is expressed on the surface of NK cells. The mechanism of action of the present invention includes, but is not limited to, the following three: (1) the Her2 and CD47 double-positive tumor can be pulled to be together with the NK cells, and the channels are formed after the NK cells release the Perporin (perforin) to damage the tumor cell membranes, so that a large amount of Gramzyme B (granzyme B) of the NK cells enter the tumor cells to directly kill the tumor cells; (2) meanwhile, as the CD47 of the tumor cells is shielded by the combination of the three-antibody SIRPa D1, macrophages in the tumor microenvironment do not receive the inhibitory signals of 'eat me', thereby activating the phagocytosis of the macrophages and killing the tumor cells; (3) the anti-Her2 end can also directly inhibit the reproduction of Her2 positive tumor cells. FIG. 2 shows the mechanism of action of the drug of the present invention. Bispecific antibodies targeting Her2 and CD47 have been reported, but bispecific antibodies targeting Her2-CD47-CD16a have not been reported and belong to First-In-Class.
Since there are two subtypes of human CD16 protein, CD16a and CD16b, CD16a is mainly expressed in NK cells, and also expressed in monocytes and macrophages, and belongs to an activated receptor; CD16b is expressed on neutrophils and the two subtypes are highly homologous, therefore, antibodies against CD16a also typically bind to CD16b, often causing neutropenia. The anti-CD16a in this invention specifically binds to the CD16a receptor and not to CD16b, and thus does not cause neutropenia.
Disclosure of Invention
In order to solve the problems, the invention discloses the design, preparation and application of a trispecific antibody; the key technical problem to be solved by the invention is to ensure the specificity of anti-CD16a, not bind with CD16b on the surface of neutrophils, thereby not causing the side effect of neutropenia of some anti-CD16a antibodies, simultaneously maintaining the high specificity and high affinity of Herceptin antibodies and SIRPa D1 protein, and reducing the side effect caused by non-specific binding to the maximum extent.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an object of the present invention is to provide a trispecific antibody comprising (a) anti-Her2 monoclonal antibody Herceptin, (b) anti-CD16a single chain antibody scFv, (c) SIRPa D1 protein, (D) a flexible linker.
The trispecific antibody takes Herceptin as the basic structure of the trispecific antibody, and is connected with anti-CD16a single-chain antibody scFv or SIRPa D1 protein at the N-terminal or C-terminal of a Herceptin heavy chain through a flexible linker, and is connected with SIRPa D1 protein or anti-CD16a single-chain antibody scFv at the N-terminal or C-terminal of a Herceptin light chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin heavy chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin light chain via a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin heavy chain via a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin heavy chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin light chain via a flexible linker and the anti-CD16a scFv is linked to the N-terminus of the Herceptin heavy chain via a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin heavy chain via a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin light chain via a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin light chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin light chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin heavy chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin light chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin light chain through a flexible linker.
In certain embodiments, the Herceptin is the basic structure of a trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin heavy chain through a flexible linker.
In certain embodiments, the amino acid sequence of the flexible linker includes, but is not limited to GGGGSGGGGSGGGGS (SEQ ID NO:14) or GSASAPTLKLEEGEFSEARV (SEQ ID NO: 15).
In certain embodiments, the amino acid sequence of the heavy chain variable region of the Herceptin monoclonal antibody is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 1);
the amino acid sequence of the light chain variable region is:
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK(SEQ ID NO:6)。
in certain embodiments, the amino acid sequence of the heavy chain constant region of the Herceptin monoclonal antibody is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:2)
or the following steps:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:3)。
in certain embodiments, the amino acid sequence of the light chain constant region of the Herceptin monoclonal antibody is:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:7)。
in certain embodiments, the anti-CD16a single chain antibody scFv consists of a heavy chain variable region VH and a light chain variable region VL, which are linked together by a flexible peptide linker.
In certain embodiments, the amino acid sequence of said anti-CD16a single chain antibody scFv is:
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAHIWWDDDKRYSPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARINPAYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK(SEQ ID NO:11)
or the following steps:
EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGSASAPTLKLEEGEFSEARVQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:12)
or the following steps:
EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:13)
in certain embodiments, the amino acid sequence of the SIRPa D1 protein is:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK(SEQ ID NO:9)
or the following steps:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK(SEQ ID NO:10)。
in certain embodiments, the Fc region of the monoclonal antibody Herceptin introduces L234A or L235A mutations, while introducing M252Y, S254T or T256E mutations in the Fc region.
In certain embodiments, the Fc region of the monoclonal antibody Herceptin is wild-type, while introducing M252Y, S254T, or T256E mutations in the Fc region.
It is another object of the present invention to provide a method for preparing the above-mentioned trispecific antibody, comprising the steps of:
(1) the structure of the invention is a symmetrical structure as shown in figure 1. The anti-CD16a single-chain antibody scFv or SIRPa D1 protein is connected at the N-end or C-end of the Herceptin heavy chain through a flexible linker. The scFv consists of a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL are linked together by a flexible peptide linker. In scFv, the sequence of domains may be VH-linker-VL or VL-linker-VH. The Herceptin antibody light chain is connected with SIRPaD1 or anti-CD16a single-chain antibody scFv at the N end or C end through a flexible linker. Introducing L234A/L235A mutation into Fc segment of antibody Herceptin to reduce the binding capacity with receptors FcgRIII and FcgRIIa, and introducing M252Y/S254T/T256E mutation into Fc segment to prolong the half-life of protein;
(2) cloning the DNA fragment obtained in the step (1) to a pcDNA series vector or other vectors used in a mammalian cell expression system by conventional molecular biology means, and cloning the DNA fragment obtained in the step (1) to another pcDNA series vector or other vectors used in an expression system including but not limited to mammalian cells, wherein the vector of the expression system comprises a fusion DNA sequence connected with a suitable transcription and translation regulatory sequence;
(3) transfecting the recombinant vector obtained in the step (2) to a mammalian cell to express and purify the fusion protein, so as to obtain the tri-specific antibody. The transfection means may be chemical transfection or electroporation transfection, and the mammalian cells may be HEK293 cells or CHO (Chinese Hamster Ovary) cells or derived cells thereof or other expression systems (e.g., e. The resulting antibody was named TriAB01/TriAB02/TriAB 03/TriAB-C.
In certain embodiments, the step (1) specifically comprises the following steps:
(a) synthesizing DNA sequences of a heavy chain, a light chain and an anti-CD16a single-chain antibody scFv of a Herceptin antibody;
(b) designing primers, and respectively amplifying the heavy chain of the Herceptin antibody and the single-chain antibody scFv of anti-CD16a by using the plasmid in (a) as a template;
(c) taking the DNA fragment in the step (b) as a template, and carrying out Overlap PCR by using an upstream primer for amplifying a heavy chain DNA sequence of a Herceptin antibody and a downstream primer for amplifying an anti-CD16a single-chain antibody scFv DNA sequence to obtain a DNA sequence;
(d) designing a mutation primer by taking the mutation point as a boundary, amplifying a fragment before the mutation point by using an upstream primer and a downstream mutation primer for amplifying a heavy chain DNA sequence of the Herceptin antibody and amplifying a fragment after the mutation point by using an upstream mutation primer and a downstream primer for amplifying an anti-CD16a single-chain antibody scFv by using the fragment in (c) as a template;
(e) taking the two fragments obtained in the step (d) as templates, and amplifying by using an upstream primer for amplifying a heavy chain DNA sequence of a Herceptin antibody and a downstream primer for amplifying an anti-CD16a single-chain antibody scFv to obtain a mutated fragment;
(f) obtaining another DNA fragment in the same manner;
(g) connecting the fragments prepared in (e) and (f) to pcDNA series vectors or other vectors for expression systems including but not limited to mammalian cells, transforming into E.coli competent cells DH5a or other competent cells, preparing plasmids after picking single clones;
(h) the plasmid prepared in (g) was transfected into HEK293 cells at 37 ℃ with 8% CO 2 Culturing in a shaking table at 125rpm, performing protein A affinity chromatography on the supernatant after 7 days of transient expression, purifying to obtain a recombinant antibody, and determining the concentration of the antibody by UV280 combined with a theoretical extinction coefficient.
In certain embodiments, antibody Herceptin sequences include, but are not limited to:
heavy chain sequence 1:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:4)
heavy chain sequence 2:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPK(SEQ ID NO:5)
the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:8)
in certain embodiments, SIRPa D1 protein sequences include, but are not limited to:
sequence 1:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK(SEQ ID NO:9)
sequence 2:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK(SEQ ID NO:10)
in certain embodiments, Anti-CD16a scFv sequences include, but are not limited to
Sequence 1:
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAHIWWDDDKRYSPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARINPAYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK(SEQ ID NO:11)
sequence 2:
EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGSASAPTLKLEEGEFSEARVQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:12)
and (3) sequence:
EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL(SEQ ID NO:13)
the invention also aims to provide the application of the trispecific antibody in preparing antitumor drugs. The trispecific antibody can be used for treating any tumor which expresses Her2 and CD47 simultaneously, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, biliary tract cancer, ovarian cancer and the like. Uses also include monotherapy for the treatment of tumors or in combination with other methods of tumor treatment.
It is still another object of the present invention to provide the use of the trispecific antibody prepared by the above method for the preparation of a bispecific, trispecific or multispecific antibody medicament. Bispecific, trispecific, or multispecific antibodies may be used for the treatment of any tumor that simultaneously expresses Her2 and CD47, including but not limited to lung, breast, gastroesophageal, biliary, and ovarian cancer, among others. Uses also include monotherapy for the treatment of tumors or in combination with other methods of tumor treatment.
The invention has the characteristics that:
1. to overcome the defect of poor specificity of the targeted CD16a, the anti-CD16a scFv specifically binds to CD16a but not to CD16b, so that neutropenia caused by the combination of neutrophils and antibodies is avoided.
2. Tumor cells that express CD47 can be linked by SIRPa D1 and CD47 ligand binding; meanwhile, the signal of ' Don't eat me ' can be blocked, so that macrophages are activated to phagocytose tumor cells;
3. the binding capacity of the Fc segment of the antibody and the receptor FcgRIIa is reduced by mutation means, so that neutropenia caused by the binding of neutrophils and the antibody is avoided.
4. And simultaneously introducing M252Y/S254T/T256E mutation into the Fc segment to prolong the half life of the protein.
5. The antibody structure is similar to that of the traditional IgG, and the symmetrical structure design is adopted, so that the antibody has no knob-into-hole mutation, and homologous isomers are avoided. Has obvious advantages in the aspects of purification process development and production cost.
Herceptin itself has a function of directly inhibiting tumor growth.
Drawings
FIG. 1 is a schematic diagram of the structure of a trispecific antibody of the present invention;
FIG. 2 is a schematic diagram of the mechanism of action of a trispecific antibody of the present invention;
FIG. 3 is a graph depicting the binding capacity of a trispecific antibody of the present invention to Her2 protein;
FIG. 4 is a graph that shows the binding ability of a trispecific antibody of the present invention to CD47 protein;
FIG. 5 is a graph that shows the binding ability of a trispecific antibody of the present invention to CD16a protein;
FIG. 6 is a graph that shows the binding ability of a trispecific antibody of the present invention to CD16b protein;
FIG. 7 is a graph showing the binding ability of the trispecific antibody of the present invention to SK-BR-3 cells;
FIG. 8 is a graph showing the binding ability of the trispecific antibodies of the present invention to SK-OV-3 cells.
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
Example 1
ELISA detection of binding of trispecific antibody to Her2 protein
Coating Her2 protein (10004-H08H1, Chinese-wangshen) with different concentrations (10000ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.0625ng/ml, 9.765625ng/ml, 2.44140625ng/ml, 0) at 100 ul/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 1ug/ml trispecific antibody and other control samples into each well, 100ul each, and incubating at 37 deg.C for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 3.
Example 2
ELISA for detecting the binding of a trispecific antibody to CD47 protein
Coating CD47 protein (12283-H08H, Chinese, Yinqian) with different concentrations (10000ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.0625ng/ml, 9.765625ng/ml, 2.44140625ng/ml, 0) at 100 ul/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100ul of each trispecific antibody with the concentration of 1ug/ml into each well, and incubating at 37 ℃ for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 4.
Example 3
ELISA for detecting the binding of a trispecific antibody to CD16a protein
Coating CD16a protein (10389-H08C, Chinese Hooker) with different concentrations (10000ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.0625ng/ml, 9.765625ng/ml, 2.44140625ng/ml, 0) at 100 ul/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100ul of each trispecific antibody with the concentration of 1ug/ml into each well, and incubating at 37 ℃ for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 5.
Example 4
ELISA for detecting the binding of a trispecific antibody to CD16b protein
Coating CD16a protein (11046-H08H, Chinese warrior) with different concentrations (10000ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.0625ng/ml, 9.765625ng/ml, 2.44140625ng/ml, 0) at 100 ul/hole and 4 ℃ overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100ul of each trispecific antibody with the concentration of 1ug/ml into each well, and incubating at 37 ℃ for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 6.
Example 5
FACS detection of binding Capacity of trispecific antibody to SK-BR-3 cells
50ul of SK-BR-3 cells (cell number 50000/well) were added to a 96-well round bottom plate, the antibody was diluted in a gradient with FACS buffer (sterile PBS, 0.2% BSA), 50 ul/well was added to the 96-well round bottom plate, and incubation was carried out at 4 ℃ for 1 hour. After centrifugation at 2000rpm for 3min, the supernatant was discarded, washed 2 with FACS buffer, 100 ul/well fluorescent secondary antibody (Southern Biotech, 2040-09) was added to a final concentration of 1ug/ml, incubated at 4 ℃ for 1h, centrifuged 3min at 2000rpm, discarded, washed 2 times with FACS buffer, resuspended in 100 ul/well FACS buffer, and examined by flow cytometry. The results are shown in FIG. 7.
Example 6
FACS detection of binding Capacity of trispecific antibody to SK-OV-3 cells
50ul of SK-OV-3 cells (cell number 50000/well) were added to a 96-well round bottom plate, the antibody was diluted in a gradient with FACS buffer (sterile PBS, 0.2% BSA), 50 ul/well was added to the 96-well round bottom plate, and incubation was carried out at 4 ℃ for 1 hour. After centrifugation at 2000rpm for 3min, the supernatant was discarded, washed 2 with FACS buffer, 100 ul/well fluorescent secondary antibody (Southern Biotech, 2040-09) was added to a final concentration of 1ug/ml, incubated at 4 ℃ for 1h, centrifuged 3min at 2000rpm, discarded, washed 2 times with FACS buffer, resuspended at 100 ul/well FACS buffer, and examined by flow cytometry. The results are shown in FIG. 8.
It should be noted that the above-mentioned contents only illustrate the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and it is obvious to those skilled in the art that several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations fall within the protection scope of the claims of the present invention.
Sequence listing
<110> Nanjing Jisheng Australia Ma biological medicine Co., Ltd
<120> design, preparation and use of tri-specific antibody
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 2
<211> 329
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Lys
325
<210> 3
<211> 329
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Lys
325
<210> 4
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile
245 250 255
Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Lys
<210> 5
<211> 449
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile
245 250 255
Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Lys
<210> 6
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 7
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 8
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 9
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys
115
<210> 10
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Arg Ala Lys
115
<210> 11
<211> 244
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45
Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80
Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Ile Asn Pro Ala Tyr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
130 135 140
Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln
145 150 155 160
Ser Val Asp Phe Asp Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys
165 170 175
Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu
180 185 190
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
210 215 220
Cys Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys
<210> 12
<211> 246
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu
115 120 125
Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Gln Pro Val Leu
130 135 140
Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile
145 150 155 160
Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln
165 170 175
Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys
180 185 190
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn
195 200 205
Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp
210 215 220
Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly
225 230 235 240
Thr Lys Leu Thr Val Leu
245
<210> 13
<211> 246
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu
130 135 140
Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln Thr Ala Thr Ile
145 150 155 160
Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln
165 170 175
Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Asn Lys
180 185 190
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn
195 200 205
Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp
210 215 220
Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu Phe Gly Gly Gly
225 230 235 240
Thr Lys Leu Thr Val Leu
245
<210> 14
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 15
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Ser Ala Ser Ala Pro Thr Leu Lys Leu Glu Glu Gly Glu Phe Ser
1 5 10 15
Glu Ala Arg Val
20
Claims (27)
1. Trispecific antibody comprising (a) the anti-Her2 monoclonal antibody Herceptin, (b) the anti-CD16a single chain antibody scFv, (c) the SIRPa D1 protein, (D) a flexible linker.
2. The trispecific antibody of claim 1, wherein Herceptin is used as the basic structure of the trispecific antibody, and the N-terminal or C-terminal of Herceptin heavy chain is connected with anti-CD16a single-chain antibody scFv or SIRPa D1 protein through flexible linker, and the N-terminal or C-terminal of Herceptin light chain is connected with SIRPa D1 protein or anti-CD16a single-chain antibody scFv through flexible linker.
3. A trispecific antibody according to any of claims 1-2, characterized in that the amino acid sequence of the flexible linker is GGGGSGGGGSGGGGS or GSASAPTLKLEEGEFSEARV.
4. The trispecific antibody of any one of claims 1-2, wherein the amino acid sequence of the heavy chain variable region of said Herceptin antibody is:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, respectively; the amino acid sequence of the light chain variable region is:
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK。
5. the trispecific antibody of any of claims 1-2, wherein the anti-CD16a single chain antibody scFv consists of a heavy chain variable region VH and a light chain variable region VL, which are linked together by a flexible peptide linker.
6. The trispecific antibody of any of claims 5, wherein the heavy chain variable region amino acid sequence of the anti-CD16a single chain antibody scFv is:
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAHIWWDDDKRYSPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARINPAYFAYWGQGTLVTVSS or is:
EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS；
the light chain variable region amino acid sequence is:
DIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK
or the following steps:
QPVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL
7. the trispecific antibody of claims 1-2, wherein the amino acid sequence of the SIRPa D1 protein is:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK
or the following steps:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK
8. the trispecific antibody of any one of claims 1-2, wherein the Herceptin antibody has an Fc stretch in which the L234A or L235A mutation is introduced, and in which the M252Y, S254T or T256E mutation is introduced.
9. The trispecific antibody of any one of claims 1-2, wherein the Fc fragment of said Herceptin antibody is wild type with introduction of M252Y, S254T or T256E mutations in the Fc fragment.
10. A method of producing a trispecific antibody according to any one of claims 1 to 9, comprising the steps of:
(1) connecting the anti-CD16a single-chain antibody scFv to the C end or the N end of the Herceptin antibody heavy chain through a flexible linker; connecting the SIRPa D1 protein to the C terminal or the N terminal of a Herceptin antibody light chain through a flexible linker; introducing mutation in the Fc segment of the Herceptin antibody;
(2) cloning the first DNA fragment obtained in the step (1) into pcDNA series vectors or other vectors for expression systems including but not limited to mammalian cells; cloning the second DNA fragment obtained in step (1) into another pcDNA series vector or other vectors for expression systems including but not limited to mammalian cells to obtain a recombinant vector;
(3) transfecting the recombinant vector obtained in the step (2) to a mammalian cell or other expression systems, and carrying out expression and purification of the fusion protein to obtain the tri-specific antibody.
11. The method of claim 10, wherein the mammalian cells in step (3) comprise HEK293 cells, CHO cells, or derived cells thereof.
12. The method for producing a trispecific antibody according to claim 10, wherein in step (1) the Fc region of the Herceptin antibody introduces L234A or L235A mutations, while the Fc region introduces M252Y, S254T or T256E mutations.
13. The method of claim 10, wherein the Fc region of the Herceptin antibody in step (1) is wild-type, and the M252Y, S254T or T256E mutation is introduced into the Fc region.
14. The method for producing trispecific antibody according to claim 10, wherein step (1) comprises the following steps:
(a) synthesizing heavy chains of Herceptin antibodies and scFv DNA sequences of anti-CD16a single-chain antibodies;
(b) designing primers, and respectively amplifying the heavy chain of the Herceptin antibody and the single-chain antibody scFv of anti-CD16a by using the plasmid in (a) as a template;
(c) taking the DNA fragment in the step (b) as a template, and carrying out Overlap PCR by using an upstream primer for amplifying a heavy chain DNA sequence of the antibody Herceptin and a downstream primer for amplifying an anti-CD16a single-chain antibody scFv DNA sequence to obtain a DNA sequence;
(d) designing a mutation primer by taking the mutation point M252Y, S254T or T256E as a boundary, taking the fragment in the step (c) as a template, amplifying the fragment before the mutation point by using an upstream primer and a downstream primer which are used for amplifying a heavy chain DNA sequence of the Herceptin antibody, and amplifying the fragment after the mutation point by using an upstream mutation primer and a downstream primer which are used for amplifying the anti-CD16a single-chain antibody scFv;
(e) amplifying by using the two fragments obtained in the step (d) as templates and using an upstream primer for amplifying a heavy chain DNA sequence of a Herceptin antibody and a downstream primer for amplifying an anti-CD16a single-chain antibody scFv to obtain a mutated DNA fragment;
(f) obtaining a second DNA fragment in the same manner;
(g) connecting the fragments prepared in (e) and (f) to pcDNA series vectors or other vectors for expression systems including but not limited to mammalian cells, transforming into E.coli competent cells DH5a or other competent cells, preparing plasmids after picking single clones;
(h) the plasmid prepared in (g) was transfected into HEK293 cells at 37 ℃ with 8% CO 2 Culturing in 125rpm shaker, performing protein A affinity chromatography on the supernatant after transient expression for 7 days, purifying to obtain recombinant antibody, and combining UV280 with theoretical extinction systemThe antibody concentration was determined.
15. Use of the trispecific antibody of any one of claims 1-9 in an anti-tumor medicament.
16. Use of a trispecific antibody produced according to any one of claims 10 to 14 in an anti-tumour medicament.
17. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin heavy chain through a flexible linker.
18. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin heavy chain through a flexible linker.
19. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin heavy chain through a flexible linker.
20. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin heavy chain through a flexible linker.
21. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin light chain through a flexible linker.
22. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin light chain through a flexible linker.
23. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin light chain through a flexible linker.
24. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin heavy chain through a flexible linker.
25. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin light chain through a flexible linker.
26. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the C-terminus of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is linked to the N-terminus of the Herceptin light chain through a flexible linker.
27. The trispecific antibody of claim 2, wherein the Herceptin is the basic structure of the trispecific antibody, the SIRPa D1 protein is linked to the N-terminus of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is linked to the C-terminus of the Herceptin heavy chain through a flexible linker.
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| PCT/CN2023/093454 WO2023217216A1 (en) | 2022-05-13 | 2023-05-11 | Design, prepration, and use of tri-specific antibody |
| CN202380012703.3A CN117616053A (en) | 2022-05-13 | 2023-05-11 | Design, preparation and application of trispecific antibody |
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| EP1513554B9 (en) * | 2002-05-30 | 2011-11-09 | Macrogenics, Inc. | Cd16a binding proteins and use for the treatment of immune disorders |
| CN100376599C (en) * | 2004-04-01 | 2008-03-26 | 北京安波特基因工程技术有限公司 | Recombining single chained three specific antibodies of anti CCA, anti CD 3, anti CD 28 through genetic engineering |
| GB0510790D0 (en) * | 2005-05-26 | 2005-06-29 | Syngenta Crop Protection Ag | Anti-CD16 binding molecules |
| US9611325B2 (en) * | 2014-07-21 | 2017-04-04 | Wuhan Yzy Biopharma Co., Ltd. | Construction and application of bispecific antibody HER2xCD3 |
| CN104892765B (en) * | 2015-06-29 | 2018-04-10 | 深圳市国创纳米抗体技术有限公司 | A kind of bispecific antibody of AntiCD3 McAb antigen and Her 2 antigen |
| CA3062479A1 (en) * | 2017-05-08 | 2018-11-15 | Shanghai Jmt-Bio Technology Co., Ltd. | Bispecific recombinant protein and use thereof |
| CN107325184A (en) * | 2017-08-08 | 2017-11-07 | 安徽大学 | Bispecific antibody targeting EGFR and HER2 and application thereof |
| US11066476B2 (en) * | 2018-09-14 | 2021-07-20 | Shanghai tongji hospital | Asymmetric bispecific antibody |
| EA202191763A1 (en) * | 2018-12-21 | 2022-03-10 | Озе Иммьюнотерапьютикс | BIFUNCTIONAL ANTI-PD-1/SIRP MOLECULE |
| CN113321735B (en) * | 2021-05-20 | 2023-01-06 | 盛禾(中国)生物制药有限公司 | Multifunctional fusion protein |
| CN114805592A (en) * | 2022-05-13 | 2022-07-29 | 南京吉盛澳玛生物医药有限公司 | Design, preparation and application of trispecific antibody |
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