CN117616053A - Design, preparation and application of trispecific antibody - Google Patents
Design, preparation and application of trispecific antibody Download PDFInfo
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- CN117616053A CN117616053A CN202380012703.3A CN202380012703A CN117616053A CN 117616053 A CN117616053 A CN 117616053A CN 202380012703 A CN202380012703 A CN 202380012703A CN 117616053 A CN117616053 A CN 117616053A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- C07K—PEPTIDES
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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- C07K2317/622—Single chain antibody (scFv)
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Abstract
The invention provides a design of a trispecific antibody, a preparation method and application thereof, wherein the trispecific antibody comprises (a) an anti-Her2 monoclonal antibody Herceptin, (b) an anti-CD16a (FcgRIIIa) single-chain antibody scFv, (c) SIRPa D1 protein, and (D) a flexible linker; the C end of the antibody Herceptin heavy chain is connected with the anti-CD16a single-chain antibody through a linker, and the C end of the antibody Herceptin light chain is connected with SIRPa D1 through a linker; modifying the Fc fragment of the antibody to alter its binding capacity to the receptor while extending the half-life 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 to activate macrophages to phagocytize tumor cells; binding specifically to NK (Nature Killing) cells by anti-CD16 a; thus recruiting macrophages and NK cells of the tumor microenvironment to kill tumor cells via SIRPa and anti-CD16 a; the invention ensures the specificity of anti-CD16a, and does not cause side effects (Neutropenia) caused by binding to CD16b on the surface of neutrophils.
Description
Cross Reference to Related Applications
The present application claims the benefit of chinese patent application No. 202210521832.2 filed on 5.13 of 2022, which is incorporated herein by reference in its entirety.
Sequence listing
The present application contains a sequence listing and is incorporated by reference in its entirety.
The invention belongs to the technical field of biological medicine, and particularly relates to design, preparation and application of a trispecific antibody.
Along with the continuous development and penetration of oncology and immunology, tumor immunotherapy has become the most advanced treatment means in the field of anti-tumor. At presentThe main research direction of tumor immunotherapy at home and abroad is immune checkpoint inhibitor. CD47 has been regarded by the industry as the most important target in the tumor immunity field following PD-1/PD-L1, and numerous candidate drugs targeting CD47 are currently available worldwide in preclinical and clinical development stages, but no approved anti-CD 47 therapies exist at present [1] 。
CD47 is widely expressed on the surface of many cancer cells, but also on the surface of erythrocytes to protect itself from phagocytosis. This means that the targeted CD47 drug inevitably damages red blood cells while killing tumor cells, thereby causing the reduction of the number of red blood cells and platelets to cause serious anemia reaction [2] 。
The present 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 proven to be a target of significant clinical efficacy; CD16a is expressed on NK cell surfaces. The mechanism of action of the present invention includes, but is not limited to, the following three: (1) Can pull Her2 and CD47 double positive tumor together with NK cells, and form channels after NK cells release Perforin to damage tumor cell membrane to make NK cells massive Granzyme B (Granzyme B) enter tumor cells to kill tumor cells directly [3] The method comprises the steps of carrying out a first treatment on the surface of the (2) Meanwhile, because the CD47 of the tumor cells is combined and shielded by SIRPa D1 of the third antibody, macrophages in the tumor microenvironment do not receive an inhibition signal of ' do't eat me ', so that the macrophages are activated to phagocytose and kill the tumor cells; (3) The anti-Her 2 end can also directly inhibit Her2 polymer formation so as to inhibit proliferation of Her2 positive tumor cells [4] . Figure 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, belonging to the First-In-Class.
Since human CD16 protein has two subtypes, CD16a and CD16b, CD16a is mainly expressed in NK cells, and also in monocytes and macrophages, belonging to the activated receptor; CD16b is expressed on neutrophils, both subtypes are highly homologous, and thus, antibodies against CD16a typically bind CD16b as wellTo cause neutropenia [5] . The anti-CD16a in this invention specifically binds to the CD16a receptor and does not bind 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, and not to combine with CD16b on the surface of neutrophil, thereby not causing some side effects of anti-CD16a antibodies such as neutropenia, and simultaneously maintaining the high specificity and high affinity of Herceptin antibodies and SIRPa D1 proteins, and minimizing side effects caused by non-specific combination.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
it is an object of the present invention to provide a trispecific antibody or trispecific antigen-binding fragment thereof having specificity for three antigens, said 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.
In one embodiment, herceptin is used as the basic structure of the trispecific antibody, the N end or the C end of the Herceptin heavy chain is connected with the anti-CD16a single-chain antibody scFv or the SIRPa D1 protein through a flexible linker, and the N end or the C end of the Herceptin light chain is connected with the SIRPa D1 protein or the anti-CD16a single-chain antibody scFv through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C end of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the C end of the Herceptin heavy chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N end of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the C end of the Herceptin heavy chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C end of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the N end of the Herceptin heavy chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N end of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the N end of the Herceptin heavy chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N end of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C end of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the C end of the Herceptin light chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C end of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C end of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the N end of the Herceptin heavy chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N end of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the C end of the Herceptin light chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the C end of the Herceptin light chain through a flexible linker, and the anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N end of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the C end of the Herceptin heavy chain through a flexible linker.
In a specific embodiment, herceptin is used as the basic structure of a trispecific antibody, the SIRPa D1 protein is connected to the N end of the Herceptin heavy chain through a flexible linker, and the anti-CD16a scFv is connected to the C end of the Herceptin light chain through a flexible linker.
In one embodiment, the heavy chain variable region of the anti-Her2 monoclonal antibody Herceptin comprises: CDR1 as shown in SEQ ID NO. 16, CDR2 as shown in SEQ ID NO. 17 and CDR3 as shown in SEQ ID NO. 18; the light chain variable region comprises: CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 21.
In a further embodiment, the heavy chain variable region of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID No. 1 and the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 6.
In a further embodiment, the anti-Her2 monoclonal antibody Herceptin heavy chain constant region comprises the amino acid sequence shown in SEQ ID No. 2 or 3, and the light chain constant region comprises the amino acid sequence shown in SEQ ID No. 7.
In a further embodiment, the heavy chain of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID No. 4 or 5, and the light chain comprises the amino acid sequence shown in SEQ ID No. 8.
In one embodiment, the heavy chain variable region of the anti-CD16a single chain antibody scFv comprises: CDR1 as shown in SEQ ID NO. 22, CDR2 as shown in SEQ ID NO. 23 and CDR3 as shown in SEQ ID NO. 24; the light chain variable region comprises: CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 27.
In a further embodiment, the heavy chain variable region of the anti-CD16a single chain antibody scFv comprises the amino acid sequence shown in SEQ ID NO. 28 or 29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 30 or 31.
In a further embodiment, the anti-CD16a single chain antibody scFv comprises the amino acid sequence shown in SEQ ID NO. 11, 12 or 13.
In one embodiment, the SIRPa D1 protein comprises an amino acid sequence shown in SEQ ID NO 9 or 10.
In a specific embodiment, the amino acid sequence of one chain of the trispecific antibody is:
or is:
or is:
or is:
or is:
the amino acid sequence of the other strand is:
or is:
in one aspect, the present invention prepares a trispecific antibody that specifically binds Her2, CD16a and CD47, and the resulting antibody is designated as TriAB01, triAB02, triAB03 or TriAB04. In another aspect, the invention provides bispecific antibodies that specifically bind Her2 and CD47, the resulting antibodies being designated as TriAB-C. The amino acid sequence of one chain of the trispecific antibody TriAB01 is SEQ ID NO. 32, and the amino acid sequence of the other chain is SEQ ID NO. 37. The amino acid sequence of one chain of the trispecific antibody TriAB02 is SEQ ID NO. 33, and the amino acid sequence of the other chain is SEQ ID NO. 37. The amino acid sequence of one strand of the trispecific antibody TriAB03 is SEQ ID NO. 34, and the amino acid sequence of the other strand is SEQ ID NO. 37. The amino acid sequence of one strand of the trispecific antibody TriAB04 is SEQ ID NO. 35, and the amino acid sequence of the other strand is SEQ ID NO. 37. The amino acid sequence of one chain of the bispecific antibody TriAB-C is SEQ ID NO. 36, and the amino acid sequence of the other chain is SEQ ID NO. 37. In some embodiments, the trispecific antibody that specifically binds Her2, CD16a and CD47 is preferably TriAB04.
In some embodiments, the Fc fragment of the monoclonal antibody Herceptin introduces the L234A or L235A mutation, while introducing the M252Y, S254T or T256E mutation at the Fc fragment.
In some embodiments, the Fc fragment of the monoclonal antibody Herceptin is wild-type, while the M252Y, S254T or T256E mutation is introduced in the Fc fragment.
In some embodiments, the invention provides an isolated nucleic acid molecule encoding a trispecific antibody or trispecific antigen-binding fragment thereof described herein.
In some embodiments, the invention provides a vector comprising a nucleic acid sequence encoding a trispecific antibody or trispecific antigen-binding fragment thereof described herein.
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 shown in figure 1 and is a symmetrical structure. The anti-CD16a single-chain antibody scFv or SIRPa D1 protein is connected at the N end or the C end of the antibody Herceptin heavy chain through a flexible linker. 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 order of the domains may be VH-linker-VL or VL-linker-VH. The N end or the C end of the light chain of the Herceptin antibody is connected with SIRPa D1 or anti-CD16a single-chain antibody scFv through a flexible linker. Introducing an L234A/L235A mutation in the Fc segment of the antibody Herceptin to reduce the binding capacity to the receptors FcgRIII and FcgRIIA, and introducing an M252Y/S254T/T256E mutation in the Fc segment to prolong the half-life of the protein;
(2) Cloning the DNA fragment obtained in step (1) into a pcDNA series vector or other vector for a mammalian cell expression system by conventional molecular biological means, cloning the DNA fragment obtained in step (1) into another pcDNA series vector or other vector for an expression system including, but not limited to, mammalian cells, the vector of the expression system including a fusion DNA sequence having appropriate transcriptional and translational regulatory sequences attached thereto;
(3) And (3) transfecting the recombinant vector obtained in the step (2) into mammalian cells to express and purify the fusion protein, thereby obtaining the trispecific antibody. The transfection may be chemical transfection or electroporation transfection, and the mammalian cells may be HEK293 cells or CHO (Chinese hamster ovary ) cells or derived cells or other expression systems of the above cells (e.g.E.coli and yeast). The resulting antibody was designated as TriAB01/TriAB02/TriAB03/TriAB04/TriAB-C.
In certain embodiments, in the step (1), the method specifically comprises the following steps:
(a) Synthesizing the DNA sequences of the heavy chain and the light chain of the Herceptin antibody and the scFv of the anti-CD16a single-chain antibody;
(b) Designing a primer, and respectively amplifying the heavy chain of the Herceptin antibody and the anti-CD16a single-chain antibody scFv by taking the plasmid in the step (a) as a template;
(c) Using the DNA fragment in (b) as a template, and performing overlay PCR by using an upstream primer for amplifying a heavy chain DNA sequence of the 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 a mutation point as a limit, taking the fragment in (c) as a template, 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 the upstream mutation primer and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv;
(e) Using the two fragments obtained in the step (d) as templates, and amplifying by using an upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv to obtain mutated fragments;
(f) Obtaining another DNA fragment in the same manner;
(g) Ligating 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, and preparing plasmids after picking up the monoclonal;
(h) Transfecting the plasmid prepared in (g) into HEK293 cells at 37℃with 8% CO 2 Culture in 125rpm shaker, supernatant after 7 days of transient expression was purified by protein A affinity chromatography to obtain recombinant antibodies, and antibody concentration was determined by UV280 binding theoretical extinction coefficient.
It is also an object of the present invention to provide the use of the above-described trispecific antibodies in the preparation of an antitumor drug. The trispecific antibody can be used for any tumor therapy that expresses Her2 and CD47 simultaneously, or expresses Her2 or CD47 singly, or expresses Her2 under or Her2 negative, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, biliary tract cancer, ovarian cancer, and the like. Uses also include monotherapy for treating tumors or in combination with other methods of tumor treatment.
It is also an object of the present invention to provide the use of a trispecific antibody prepared according to the above method for the preparation of a bispecific, trispecific or multispecific antibody medicament. Bispecific, trispecific or multispecific antibodies may be used in any tumor therapy that expresses Her2 and CD47 simultaneously or that expresses Her2 or CD47 singly, or that expresses Her2 low or Her2 negative, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, biliary tract cancer, ovarian cancer, and the like. Uses also include monotherapy for treating tumors or in combination with other methods of tumor treatment.
The invention is characterized in that:
1. in order to solve the defect of weak specificity of targeted CD16a, the anti-CD16a scFv specifically binds to CD16a but not to CD16b, thereby avoiding neutropenia caused by the combination of neutrophils and antibodies.
2. NK cells are recruited by anti-CD 16a, which also kills tumor cell function when Fc function is insufficient. When Fc function is sufficient, the function of killing tumor cells is more powerful.
3. Tumor cells expressing CD47 can be linked by SIRPa D1 and CD47 ligand binding; meanwhile, the signal of ' Don't eat me ' can be blocked to activate macrophages to phagocytose tumor cells;
4. the binding capacity of the Fc segment of the antibody and the receptor FcgRIIIa is reduced by mutation means, so that the neutropenia caused by the combination of the neutrophil and the antibody is avoided.
5. At the same time, M252Y/S254T/T256E mutation is introduced in the Fc segment to prolong the half life of the protein.
6. The antibody structure is similar to the traditional IgG, adopts symmetrical structure design, has no knob-into-hole mutation, and avoids the generation of homologous isomers. Has remarkable advantages in terms of development and production cost of the purification process.
Herceptin itself has the function of directly inhibiting tumor growth [6] 。
8. Under normal conditions, the main toxic and side effect of the expression of Her2 by myocardial cells is the damage to the myocardial cells, and the long-term use can lead the left ventricular ejection fraction to be reduced (4-18%) or serious heart failure (4%) [7] . CD47 is expressed less in cardiomyocytes and therefore the binding and damage probability of trispecific antibodies to the myocardium is lower. Notably, CD47 is elevated during myocarditis and myocardial ischemia reperfusion, and several documents show that antagonism of CD47 reduces myocarditis, reduces damage from myocardial ischemia reperfusion, and reduces atherosclerosis [8] . Therefore, antagonism of CD47 contained in the three antibodies of the invention can help to reduce the damage of Her2 targeted therapy and reduce toxic and side effects.
9. In most cases, only tumor cells express Her2 and CD47 simultaneously (biscationity); normal cells typically express only Her2 or CD47. At a certain concentration, the trispecific antibody binds only to tumor cells and not to normal cells expressing Her2 or CD47, thereby reducing damage to normal cells.
10. Since the trispecific antibody simultaneously mobilizes macrophages and NK cells as effector cells, the trispecific antibody also has a killing effect on Her2 low-expression or non-expression tumors, and is similar to the side killing effect of Enhertu [9] . The toxic and side effects of the trispecific antibody are obviously lower than those of Enheretu with toxin because of no small molecular toxin.
FIG. 1 is a schematic structural diagram 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 invention;
FIG. 3 is an SDS-PAGE electrophoresis of a trispecific antibody of the present invention;
FIGS. 4-5 are graphs showing the binding capacity of the trispecific antibodies of the present invention to Her2 protein;
FIGS. 6-7 are graphs showing the binding capacity of the trispecific antibodies of the present invention to the CD47 protein;
FIGS. 8-9 are graphs showing the binding capacity of the trispecific antibodies of the present invention to the CD16a protein;
FIGS. 10-11 are graphs showing the binding capacity of the trispecific antibodies of the present invention to the CD16b protein;
FIG. 12 is a graph showing the binding capacity of the trispecific antibody of the present invention to SK-BR-3 cells;
FIG. 13 is a graph showing the binding capacity of the trispecific antibodies of the present invention to SK-OV-3 cells.
FIG. 14 is a graph of the phagocytic capacity of the recruited macrophages with a trispecific antibody of the invention;
FIG. 15 is a graph showing the detection of coagulation by a trispecific antibody of the present invention;
FIG. 16 is a graph showing the detection of Her2 internalization by a trispecific antibody of the invention;
FIG. 17 is a graph showing the affinity detection of a trispecific antibody of the invention with FcRn protein;
FIG. 18 is a graph showing ELISA detection of binding capacity of a trispecific antibody of the invention to FcRn protein;
FIG. 19 shows ADCC activity of a trispecific antibody of the invention against NCI-N87 cells;
FIG. 20 shows ADCC activity of a trispecific antibody of the invention against OE19 cells;
FIG. 21 shows ADCC activity of a trispecific antibody of the invention against MDA-MB-231 cells;
FIG. 22 shows ADCC activity of a trispecific antibody of the invention against SK-OV-3 cells;
FIG. 23 shows ADCC activity of a trispecific antibody of the invention against BT-474 cells;
FIG. 24 is an anti-tumor activity of a trispecific antibody of the invention in NCI-N87 xenograft models;
FIG. 25 is an evaluation of the efficacy of a trispecific antibody of the invention in the ExVivo organoid system.
Detailed Description
Unless defined otherwise herein, all scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques associated with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly employed in the art.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description, and from the claims.
For a better understanding of the present invention, definitions and explanations of related terms are provided below.
The term "antibody" as used herein is used in its broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., trispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
The trispecific antibodies of the present disclosure may be isolated and/or purified. The term "isolated" as used herein means having been removed from its natural environment. The term "purified" as used herein means an improvement in purity, where "purity" is a relative term and is not necessarily to be construed as absolute purity. In exemplary embodiments, the trispecific antibody is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% or about 100% pure. Bispecific or trispecific antibodies of the present disclosure may be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. In one embodiment, affinity chromatography is used to purify bispecific or trispecific antibodies of the disclosure.
The term "bispecific antibody" as used herein is an antibody having two different antigen binding specificities. For example, bispecific antibodies of the present disclosure are capable of binding Her2 and CD47 with sufficient affinity.
The terms "trispecific antibody", "trispecific antibody specifically binding Her2, CD47 and CD16 a", "trispecific antibody against Her2, CD47 and CD16 a" are used interchangeably and refer to a trispecific antibody capable of binding Her2, CD47 and CD16a with sufficient affinity such that the trispecific antibody can be used as a therapeutic/diagnostic agent targeting Her2 and/or CD47 or CD16a expressing cells. The trispecific antibodies of the present disclosure do not bind or bind with very low affinity to CD16b, thereby reducing or lowering side effects, such as neutropenia, caused by binding to CD16b on the surface of neutrophils. In addition, the trispecific antibodies of the present disclosure maintain high specificity and high affinity of Herceptin antibodies and SIRPa D1 proteins, minimizing side effects due to non-specific binding.
In some embodiments, the trispecific antibodies have high affinity for Her2 antigen, EC 50 The value is 0.1-0.3nM, as detected by ELISA or FACS. In some casesIn embodiments, the trispecific antibodies have high affinity for the CD47 antigen, EC 50 The value was 0.5-3.5nM, as detected by ELISA or FACS. In some embodiments, the trispecific antibodies have high affinity for the CD16a antigen, EC 50 Values of 5-30nM, as detected by ELISA or FACS.
The term "flexible linker", "flexible peptide linker" or "linker" as used herein refers to a peptide linker, preferably a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5-100, more preferably 10-50, most preferably 10-20 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein g=glycine, s=serine, and (x=3, n=3, 4,5 or 6 and m=0, 1,2 or 3) or (x=4 and n=2, 3,4 or 5 and m=0, 1,2 or 3), preferably x=4 and n=2 or 3, more preferably x=4 and n=3. In a preferred embodiment, the amino acid sequence of the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 14). In a preferred embodiment, the amino acid sequence of the peptide linker is GSASAPTLKLEEGEFSEARV (SEQ ID NO: 15).
The term "amino acid mutation" as used herein encompasses amino acid substitutions, deletions, insertions and modifications. Substitutions, deletions, insertions and modifications may be made to the trispecific antibody to achieve the desired function. Genetic or chemical methods known in the art may be used to generate amino acid mutations. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. Various names may be used herein to indicate amino acid mutations. For example, a mutation from leucine to alanine at position 234 of the Fc fragment may be indicated as L234A.
The multispecific antibodies of the present disclosure may comprise one or more amino acid substitutions, which may be non-conservative amino acid substitutions, i.e., one amino acid is substituted with another amino acid having a different structure and/or chemical property. The substitutions may also be conservative amino acid substitutions that do not adversely affect or alter the essential properties of the protein/polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted with another amino acid residue having a similar side chain, e.g., an amino acid residue is substituted with a physically or functionally similar residue (e.g., of similar size, charge, chemical nature including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions preferably the corresponding amino acid residue is substituted with another amino acid residue from the same side chain family.
Amino acid substitutions may be introduced into the multispecific antibodies of the present disclosure and screened for desired activity, retained/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC), and the like. Both conservative and non-conservative amino acid substitutions are contemplated for use in preparing antibody variants.
In one aspect of the invention, the antibody Herceptin Fc fragment may be mutated to reduce the binding capacity to the receptors fcgcriii and fcgcriia. In one embodiment, an L234A/L235A mutation is introduced in the Fc segment of the antibody Herceptin to reduce binding to the receptors FcgRIII and FcgRIIA. In another aspect of the invention, the antibody Herceptin Fc fragment may be modified to extend the half-life of the protein. In one embodiment, the M252Y/S254T/T256E mutation is introduced at the Fc segment of the antibody Herceptin to extend the half-life of the protein. In one embodiment, the L234A/L235A mutation is introduced in the Fc segment of the antibody Herceptin to reduce binding to the receptors FcgRIII and FcgRIIA, while the M252Y/S254T/T256E mutation is introduced in the Fc segment to extend the half-life of the protein.
The term "single chain variable fragment" or "scFv" as used herein refers to an antibody fragment comprising a light chain variable region and a heavy chain variable region, wherein the light chain and heavy chain variable regions are linked via a linker, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the anti-CD 16a binding domain is an scFv comprising the light and heavy chain variable regions of the amino acid sequences provided herein. The light chain variable region and the heavy chain variable region of the scFv may be, for example, any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
The heavy and light chain variable regions are responsible for binding to the target antigen, most of the sequence variability occurs in 6 Complementarity Determining Regions (CDRs), 3 CDRs per Variable Heavy (VH) and Variable Light (VL) domain. The complementarity determining regions form antigen binding sites. There are various schemes for identifying complementarity determining regions, for example, the Kabat numbering system defines complementarity determining regions based on sequence variability at antigen binding regions of VH and VL domains, the Chothia numbering system defines "hypervariable loops" based on the position of structural loop regions in VH and VL domains, and the IMGT numbering system is based on amino acid sequence alignment of germline V genes. For a given variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues make up a particular CDR. CDR regions may be numbered according to the kabat numbering system, the Chothia numbering system, or the IMGT numbering system, etc. One of ordinary skill in the art will be able to use such numbering system for any variable region sequence, independent of any experimental data outside of the sequence itself. Unless otherwise indicated, the VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 amino acid sequences described herein are numbered according to the IMGT numbering system.
The invention further provides isolated nucleic acid molecules encoding the trispecific antibodies described herein or trispecific antigen-binding fragments thereof. "isolated nucleic acid molecule encoding a trispecific antibody" or "isolated nucleic acid molecule encoding a trispecific antibody or trispecific antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding the heavy and/or light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more positions in a host cell. Nucleic acid molecules encoding the trispecific antibodies of the invention may be expressed as a single polynucleotide encoding the complete trispecific antigen-binding molecule or as a plurality (e.g., two or more) of co-expressed polynucleotides. The polypeptides encoded by the co-expressed polynucleotides may be joined via, for example, disulfide bonds or other means to form functional trispecific antibodies.
In some aspects, the invention relates to expression vectors comprising the isolated nucleic acid molecules encoding the trispecific antibodies of the invention, and prokaryotic or eukaryotic host cells comprising the isolated nucleic acid molecules or vectors encoding the trispecific antibodies of the invention. In one embodiment, the host cell comprises a vector (e.g., has been transformed or transfected with the vector) comprising a polynucleotide encoding a trispecific antibody (or trispecific antigen-binding fragment thereof) of the invention. The term "host cell" as used herein refers to any cellular system that can be engineered to express a trispecific antibody or trispecific antigen-binding fragment thereof of the invention. Host cells suitable for expressing the trispecific antibodies of the invention are well known in the art.
The invention also provides a method of producing a trispecific antibody comprising the steps of:
(1) Connecting anti-CD16a single-chain antibody scFv to the C end or N end of the heavy chain of the Herceptin antibody through a flexible connector; connecting SIRPa D1 protein to the C end or N end of a Herceptin antibody light chain through a flexible connector; introducing mutation in the Fc segment of Herceptin antibody;
(2) Cloning the first DNA fragment obtained in step (1) into a pcDNA series vector or other vectors for use in expression systems including, but not limited to, mammalian cells; cloning the second DNA fragment obtained in the step (1) into another pcDNA series vector or other vectors used in expression systems including but not limited to mammalian cells to obtain a recombinant vector;
(3) And (3) transfecting the recombinant vector obtained in the step (2) into a mammalian cell or other expression systems, and carrying out expression and purification of fusion proteins to obtain the trispecific antibody.
In one embodiment, the Fc fragment of the Herceptin antibody in step (1) is introduced with the L234A or L235A mutation, while the M252Y, S T or T256E mutation is introduced at the Fc fragment.
In one embodiment, the Fc fragment of the Herceptin antibody in step (1) is wild-type, while the M252Y, S T or T256E mutation is introduced in the Fc fragment.
In a specific embodiment, the step (1) specifically includes the following steps:
(a) Synthesizing heavy chain of Herceptin antibody and scFv DNA sequence of anti-CD16a single chain antibody;
(b) Designing a primer, and respectively amplifying the heavy chain of the Herceptin antibody and the anti-CD16a single-chain antibody scFv by taking the plasmid in the step (a) as a template;
(c) Using the DNA fragment in (b) as a template, and performing overlay PCR by using an upstream primer for amplifying the heavy chain DNA sequence of the antibody Herceptin and a downstream primer for amplifying the scFv DNA sequence of the anti-CD16a single-chain antibody to obtain a DNA sequence;
(d) Designing a mutation primer by taking a mutation point M252Y, S T or T256E as a limit, taking the fragment in (c) as a template, amplifying the fragment before the mutation point by using an upstream primer and a downstream mutation primer for amplifying the heavy chain DNA sequence of the Herceptin antibody, and amplifying the fragment after the mutation point by using the upstream mutation primer and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv;
(e) Using the two fragments obtained in the step (d) as templates, and amplifying by using an upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv to obtain a mutated DNA fragment;
(f) Obtaining a second DNA fragment in the same manner;
(g) Ligating 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, and preparing plasmids after picking up the monoclonal;
(h) Transfecting the plasmid prepared in (g) into HEK293 cells at 37℃with 8% CO 2 Culturing in 125rpm shaker, transiently expressing for 7 days, subjecting supernatant to protein A affinity chromatography, and purifying to obtain recombinant antibodyThe antibody concentration was determined by UV280 binding to the theoretical extinction coefficient.
The bispecific, trispecific or multispecific antibodies of the present disclosure may be used, for example, to treat or diagnose cancer, such as to treat any tumor that expresses Her2 and CD47 simultaneously or Her2 or CD47 singly, or Her2 is low or Her2 negative, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, biliary tract cancer, ovarian cancer, and the like. Thus, in some embodiments, the disclosure relates to the use of trispecific antibodies in the preparation of antitumor drugs. In some embodiments, the disclosure relates to the use of a trispecific antibody produced using a method as described above in the preparation of an anti-tumor medicament. In certain embodiments, the invention provides methods of treating an individual having cancer, including but not limited to lung cancer, breast cancer, gastroesophageal cancer, esophageal cancer, biliary tract cancer, and ovarian cancer, using a trispecific antibody, comprising administering to the individual a therapeutically effective amount of the trispecific antibody. In further embodiments, the method further comprises administering to the individual an additional tumor treatment method. In the above embodiments, the individual is preferably a mammal, more preferably a human. The trispecific antibodies of the present invention may be administered to an individual in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal.
The term "treatment" as used herein refers to a therapeutic treatment with the aim of slowing (alleviating) an undesired physiological condition, disorder or disease, or obtaining a beneficial or desired clinical result. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; a reduction in the extent of a condition, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delayed onset or progression of the condition, disorder or disease is slowed; improvement of a condition, disorder or disease state. Treatment also includes extending survival compared to expected survival without treatment. In other embodiments, "treatment" refers to prophylactic measures, wherein the purpose is to delay the onset or reduce the severity of an undesired physiological condition, disorder or disease, for example, in a person susceptible to the disease (e.g., an individual carrying a genetic marker of a disease such as breast cancer).
The present invention is further illustrated in the following drawings and detailed description, which are to be understood as being merely illustrative of the invention and not limiting the scope of the invention.
Example 1
Molecular construction and production of trispecific antibodies
The antibody light and heavy chain genes were amplified by PCR using conventional molecular biology techniques and ligated to pcdna3.4 vectors by homologous recombination techniques. The positive clone after sequencing is transfected into an Expi293F cell after plasmid extraction, the culture is continued for 7 days in a shaking table of 37 ℃/5% CO2/125rpm, the supernatant is collected after the culture is finished and subjected to protein A affinity chromatography, the purified trispecific antibody is obtained, and the concentration of the antibody is determined through the UV280 binding theoretical extinction coefficient. SDS-PAGE electrophoresis results are shown in FIG. 3. The trispecific antibodies are successfully expressed and the purity of SDS-PAGE after one-step affinity chromatography reaches more than 80 percent.
Example 2
ELISA detection of binding of trispecific antibodies to Her2 protein
Coating 2ug/ml Her2 protein (10004-H08H 1, yiqiao Shenzhou), 100 ul/well at 4deg.C overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; three specific antibodies were added at different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) per well and incubated at 37℃for 1h for each 100ul of the other control samples; goat anti-human IgG/HRP was then added and incubated at 37℃for 1h, after development for 10min, OD450 was read on a microplate reader. The results are shown in FIG. 4.
Coating 2ug/ml Her2 protein, 100 ul/well at 4℃overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr, and cleaning with PBST for 3 times; different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) of trispecific antibody were added per well, 100ul of each of the other control samples were incubated at 37℃for 1h and then PBST was washed 3 times; then add 1:8000 diluted goat anti-human IgG-HRP 100ul, incubated at 37 ℃ for 1h, washed 6 times, added with 100ul TMB for 5min per well, added with 50ul stop solution, and read OD450 on an enzyme label instrument. The results are shown in FIG. 5. The trispecific antibodies have a high affinity with Her2 antigen.
Example 3
ELISA detection of binding of trispecific antibodies to CD47 protein
Coating 2ug/ml of CD47 protein (12283-H08H, yiqiao Shenzhou) at 100 ul/well 4℃overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) of trispecific antibody were added per well and incubated for 1h at 37 ℃; goat anti-human IgG/HRP was then added and incubated at 37℃for 1h, after development for 10min, OD450 was read on a microplate reader. The results are shown in FIG. 6.
Coating 2ug/ml of CD47 protein, 100 ul/well at 4 ℃ overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr, and cleaning with PBST for 3 times; different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) of trispecific antibody were added per well, 100ul of each of the other control samples were incubated at 37℃for 1h and then PBST was washed 3 times; then add 1:8000 diluted goat anti-human IgG-HRP 100ul, incubated at 37 ℃ for 1h, washed 6 times, added with 100ul TMB for 5min per well, added with 50ul stop solution, and read OD450 on an enzyme label instrument. The results are shown in FIG. 7. The trispecific antibodies have a high affinity for the CD47 antigen.
Example 4
ELISA detection of binding of trispecific antibodies to CD16a protein
Coating 2ug/ml of CD16a protein (10389-H08C, yiqiao Shenzhou), 100 ul/well at 4℃overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) of trispecific antibody were added per well and incubated for 1h at 37 ℃; goat anti-human IgG/HRP was then added and incubated at 37℃for 1h, after development for 10min, OD450 was read on a microplate reader. The results are shown in FIG. 8.
Coating 2ug/ml CD16a protein, 100 ul/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; three specific antibodies were added at different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) per well and incubated at 37℃for 1h for each 100ul of the other control samples; PBST is washed 3 times after incubation for 1h at 37 ℃; then add 1:8000 diluted goat anti-human IgG-HRP 100ul, incubated at 37 ℃ for 1h, washed 6 times, added with 100ul TMB for 5min per well, added with 50ul stop solution, and read OD450 on an enzyme label instrument. The results are shown in FIG. 9. The trispecific antibody may specifically bind CD16a.
Example 5
ELISA detection of binding of trispecific antibodies to CD16b protein
Coating 2ug/ml of CD16b protein (11046-H08H, yiqiao Shenzhou), 100 ul/well at 4℃overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) of trispecific antibody were added per well and incubated for 1h at 37 ℃; goat anti-human IgG/HRP was then added and incubated at 37℃for 1h, after development for 10min, OD450 was read on a microplate reader. The results are shown in FIG. 10.
Coating 2ug/ml CD16b protein, 100 ul/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; three specific antibodies were added at different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) per well and incubated at 37℃for 1h for each 100ul of the other control samples; PBST is washed 3 times after incubation for 1h at 37 ℃; then add 1:8000 diluted goat anti-human IgG-HRP 100ul, incubated at 37 ℃ for 1h, washed 6 times, added with 100ul TMB for 5min per well, added with 50ul stop solution, and read OD450 on an enzyme label instrument. The results are shown in FIG. 11. The trispecific antibody does not bind to CD16b, avoiding the toxic and side effects caused thereby.
Example 6
FACS detection of binding Capacity of trispecific antibodies to cell SK-BR-3
50ul SK-BR-3 cells were added to the 96 well round bottom plate, the number of cells was 50000/well, antibody was diluted by FACS buffer (sterile PBS,0.2% BSA) gradient, 50 ul/well was added to the 96 well round bottom plate, and incubated at 4℃for 1h. The supernatant was centrifuged at 2000rpm for 3min, washed 2 with FACS buffer, 100 ul/Kong Yingguang secondary antibody (Southern Biotech, 2040-09) was added, the final concentration was 1ug/ml, incubated at 4℃for 1h, centrifuged at 2000rpm for 3min, and the supernatant was washed 2 times with FACS buffer and resuspended with 100 ul/well FACS buffer and examined by flow cytometry. The results are shown in FIG. 12.
Example 7
FACS detection of binding Capacity of trispecific antibodies to cell SK-OV-3
50ul SK-OV-3 cells were added to the 96-well round bottom plate at 50000 cells/well, antibody was diluted in FACS buffer (sterile PBS,0.2% BSA) gradient, added to the 96-well round bottom plate at 50 ul/well, and incubated for 1h at 4 ℃. The supernatant was centrifuged at 2000rpm for 3min, washed 2 with FACS buffer, 100 ul/Kong Yingguang secondary antibody (Southern Biotech, 2040-09) was added, the final concentration was 1ug/ml, incubated at 4℃for 1h, centrifuged at 2000rpm for 3min, and the supernatant was washed 2 times with FACS buffer and resuspended with 100 ul/well FACS buffer and examined by flow cytometry. The results are shown in FIG. 13.
Example 8
FACS detection of macrophage-induced phagocytosis assay
The mice macrophages RAW264.7 and OE-19 were cultured and expanded to the desired cell mass, target cells OE-19 were labeled with CFSE, washed 3 times with PBS, target cells were incubated with different concentrations of TriAB04 (2.5 nM, 5nM, 10nM, 25nM, 50nM, 100 nM) and TTI-622, incubated for 2 hours at 37℃and then washed 3 times with PBS, and target cell solutions containing the protein to be tested were transferred to plates containing RAW264.7 cells at an effective target ratio of 1:3, RAW264.7 cells were stimulated simultaneously with 1ug/mL LPS, and after 2 hours incubation, cells were collected for flow analysis to detect CFSE density in RAW264.7 cells [10] . The results are shown in FIG. 14. The trispecific antibodies activate a large number of tumor cells for phagocytosis.
Example 9
Detection of trispecific antibody Activity in hemagglutination experiments
Human RBC was centrifuged at 1500rpm for 5min, washed three times with DPBS, re-added to prepare 10% RBC suspension, antibody to be tested was prepared into samples of different concentrations (3 uM,1uM,0.33uM,0.11uM,0.037uM,0.012uM,0.004uM, 0) using PBS buffer, and mixed with human RBC 50ul+50ul volume for later useIn 96-well cell culture plate, standing at 37deg.C for 2 hr, and observing agglutination [11] . The results are shown in FIG. 15. Any concentration of trispecific antibodies does not cause clotting.
Example 10
Efficiency of internalization of trispecific antibody Her2 positive cells
Human ovarian cancer cells SK-OV-3 were 5X 10 per well 5 Target cells were incubated with 400nM TriAB04, positive control Herceptin for 1, 24, 48 and 72 hours respectively, cells were digested with pancreatin after incubation, washed 1 cell with 0.5% BSA buffer, triAB04 and Herceptin for 45 min on ice, PE anti-human IgG Fc Antibody secondary antibody for 45 min on ice, centrifuged and washed 1 cell with 0.5% BSA buffer, and cells were collected for flow analysis to detect FITC fluorescence intensity (MFI) in cells [12] . Internalization rate = (1-MFI Measurement value /MFI t＝1 )*100％
The results are shown in FIG. 16. The trispecific antibody was able to cause a level of her2 internalization comparable to Herceptin.
Example 11
Detection of binding Capacity of trispecific antibodies to FcRn protein
11.1 Fortebio affinity assay
The affinity of the test molecule to the antibodies was calculated using the ProA probe Load test antibody (concentration 4 ug/ml) and then binding and dissociating to FcRn proteins (4 ug/ml, 1ug/ml, 0.25ug/ml and 0) at different concentrations. The results are shown in FIG. 17. The affinity of the mutant Fc to FcRn was improved by a factor of 10 compared to wild type.
11.2 ELISA affinity assay
Coating 2ug/ml FcRn protein 100 ul/well at 4deg.C overnight; sealing with 3% skimmed milk powder at 37deg.C for 1 hr; three specific antibodies were added at different concentrations (10000 ng/ml, 2500ng/ml, 625ng/ml, 156.25ng/ml, 39.06ng/ml, 9.77ng/ml, 2.44ng/ml, 0) per well and incubated at 37℃for 1h for each 100ul of the other control samples; PBST is washed 3 times after incubation for 1h at 37 ℃; then add 1:8000 diluted goat anti-human IgG-HRP 100ul, incubated at 37 ℃ for 1h, washed 6 times, added with 100ul TMB for 5min per well, added with 50ul stop solution, and read OD450 on an enzyme label instrument. The results are shown in FIG. 18. The affinity of the mutated Fc for FcRn is significantly improved compared to wild type.
Example 12
ADCC Activity of trispecific antibodies
12.1 ADCC Activity against human gastric cancer cell NCI-N87
Expanded cells were cultured to the required amount, plated at 4000/well into 96-well plates, attached overnight, and fresh human PBMCs resuspended into 1640 medium according to an effective target ratio of 10:1 into 96-well plate, the gradient of the drug administration concentration is 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively, after further culturing for 5 days, PBS washes the plate 3 times, and the number of living cells is detected by CCK-8 reagent, and IC is calculated according to the drug effect measurement 50 . The results are shown in FIG. 19. The anti-tumor activity of the trispecific antibody is superior to Herceptin.
12.2 ADCC Activity against human esophageal cancer cell OE19
Expanded cells were cultured to the required amount, plated at 4000/well into 96-well plates, attached overnight, and fresh human PBMCs resuspended into 1640 medium according to an effective target ratio of 10:1 into 96-well plate, the gradient of the drug administration concentration is 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively, after further culturing for 5 days, PBS washes the plate 3 times, and the number of living cells is detected by CCK-8 reagent, and IC is calculated according to the drug effect measurement 50 . The results are shown in FIG. 20. The anti-tumor activity of the trispecific antibody is superior to Herceptin.
12.3 ADCC Activity against human breast cancer cells MDA-MB-231
Cells were expanded to the desired amount by plating cells into 96-well plates at 1500/well, overnight attachment, and fresh human PBMCs resuspended in 1640 medium according to an effective target ratio of 10:1 into 96-well plate, the gradient of the drug administration concentration is 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively, after further culturing for 5 days, PBS washes the plate 3 times, and the number of living cells is detected by CCK-8 reagent, and IC is calculated according to the drug effect measurement 50 . The results are shown in FIG. 21. The anti-tumor activity of the trispecific antibody is superior to that of the contrast agent Herceptin and the combination of Herceptin and TTI-622.
12.4 ADCC Activity against human esophageal cancer cells SK-OV-3
Expanded cells were cultured to the required amount, plated at 4000/well into 96-well plates, attached overnight, and fresh human PBMCs resuspended into 1640 medium according to an effective target ratio of 10:1 into 96-well plate, the gradient of the drug administration concentration is 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively, after further culturing for 5 days, PBS washes the plate 3 times, and the number of living cells is detected by CCK-8 reagent, and IC is calculated according to the drug effect measurement 50 . The results are shown in FIG. 22. The anti-tumor activity of the trispecific antibody is superior to Herceptin.
12.5 ADCC Activity against human breast cancer cells BT-474
Expanded cells were cultured to the required amount, plated at 4000/well into 96-well plates, attached overnight, and fresh human PBMCs resuspended into 1640 medium according to an effective target ratio of 10:1 into 96-well plate, the gradient of the drug administration concentration is 500nM, 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0, respectively, after further culturing for 5 days, PBS washes the plate 3 times, and the number of living cells is detected by CCK-8 reagent, and IC is calculated according to the drug effect measurement 50 . The results are shown in FIG. 23. The trispecific antibodies have similar anti-tumor activity to Herceptin.
Example 13
Anti-tumor Activity of trispecific antibodies in NCI-N87 xenograft model
Human gastric cancer cell NCI-N87 in 1640 culture medium containing 10% FBS at 37deg.C and 5% CO 2 Culture, cell continuous culture, ensured inoculation of 28 SCID mice (24 for grouping, 4 for backup) ten passages ago, each of which was inoculated with approximately 8X 10 6 NCI-N87 cells were inoculated at a volume of 100uL and at the right back of the mice near the armpits. Mice were anesthetized with 3-4% isoflurane prior to inoculation.
When the tumor grows to an average of about 100-150mm 3 On the left and right, 24 tumor-bearing mice will be randomly divided into 4 groups of 6 based on tumor volume and body weight. The day of group dosing was defined as day 0. The grouping and dosing regimen is shown in the following table:
tumor volume: tumor volumes were measured twice weekly after grouping for 4 weeks. The calculation method of the tumor volume (V) is as follows: v= (length x width 2)/2. The calculation method of the Relative Tumor Volume (RTV) of each mouse is: RTV = Vt/V0, where Vt is the measured volume per day and V0 is the volume at the beginning of the treatment.
The results are shown in FIG. 24. In the xenograft model, the anti-tumor activity of the trispecific antibody is obviously superior to that of Herceptin.
Example 14
Drug efficacy study of trispecific antibodies in ExVivo organoid System
Collecting a chest water sample of a Her 22 + breast cancer patient, and separating cells in the sample, including tumor cells and immune cells. The candidate drug and the reference drug are incubated with the tumor cell immune cell complex in vitro. After 4-5 days of treatment, the medium and cells were collected for analysis, including tumor cells and Granzyme B. The results are shown in FIG. 25. In the ExVivo evaluation system of the sample, the anti-tumor activity of the trispecific antibody is obviously better than that of the combination of Herceptin and TTI-622.
It should be noted that the foregoing merely illustrates the technical idea of the present invention and is not intended to limit the scope of the present invention, and that a person skilled in the art may make several improvements and modifications without departing from the principles of the present invention, which fall within the scope of the claims of the present invention.
Reference is made to:
[1]Andre Veillete,Jun Chen.SIRPa–CD47 Immune Checkpoint Blockade in Anticancer Therapy.
[2]A.Neil Barclay.Signal regulatory protein alpha(SIRPa)/CD47 interaction and function.
[3]Song-Yang Wu,Tong Fu,Yi-Zhou Jiang,Zhi-Ming Shao.Natural killer cells in cancer biology and therapy.
[4]Marc Turini,Patrick Chames,Pierre Bruhns,Daniel Baty,Brigitte Kerfelec.A FcgRIII-engaging bispecific antibody expands the range of HER2-expressing breast tumors eligible to antibody therapy.
[5]Yawu Jing,Zhenya Ni,Jianming Wu,LeeAnn Higgins et al.Identification of an ADAM17 cleavage region in human CD16(FcgRIII)and the engineering of a non-cleavable version of the receptors in NK cells.
[6]Xuesai Zhang,Jianhe Chen,Zhibing Weng,Qingrou Li et al.A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action.
[7]Robert S.Copeland-Halperin,Jenifer E.Liu,Anthony F.Yu.Cardiotoxicity of HER2-targeted therapies.
[8]Quanli Cheng,Junlian Gu,Binay Kumar Adhikari et al.Is CD47 a potentially promising therapeutic target in cardiovascular diseases？–Role of CD47 in cardiovascular diseases.
[9]Yusuke Ogitani,Katsunobu Hagihara,Masataka Oitate,Hiroyuki Naito et al.Bystander killing effect of DS-8201a,a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate,in tumors with human epidermal growth factor receptor 2 heterogeneity.
[10]Treatment of CD47+disease cells with SIRP alpha-Fc fusions.
[11] CN110724672A hybridoma cell strain 105D11 antibody and application thereof.
[12]US20210380716A1.Recombinant bifunctional protein targeting CD47 and HER2.
Claims (23)
- A trispecific antibody or trispecific antigen-binding fragment thereof specific for three antigens, wherein said antibody comprises (a) the anti-Her2 monoclonal antibody Herceptin, (b) the anti-CD16a single chain antibody scFv, (c) a SIRPa D1 protein, (D) a flexible linker.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 1, wherein Herceptin is used as the basic structure of the trispecific antibody, the anti-CD16a single-chain antibody scFv or SIRPa D1 protein is connected to the N-terminus or C-terminus of Herceptin heavy chain through a flexible linker, and the SIRPa D1 protein or anti-CD16a single-chain antibody scFv is connected to the N-terminus or C-terminus of Herceptin light chain through a flexible linker.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region of the anti-Her2 monoclonal antibody Herceptin comprises: CDR1 as shown in SEQ ID NO. 16, CDR2 as shown in SEQ ID NO. 17 and CDR3 as shown in SEQ ID NO. 18; the light chain variable region comprises: CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 21.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID No. 1 and the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 6.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 4, wherein the anti-Her2 monoclonal antibody Herceptin heavy chain constant region comprises the amino acid sequence shown in SEQ ID No. 2 or 3 and the light chain constant region comprises the amino acid sequence shown in SEQ ID No. 7.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 5, wherein the heavy chain of the anti-Her2 monoclonal antibody Herceptin comprises the amino acid sequence shown in SEQ ID No. 4 or 5 and the light chain comprises the amino acid sequence shown in SEQ ID No. 8.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region of the anti-CD16a single chain antibody scFv comprises: CDR1 as shown in SEQ ID NO. 22, CDR2 as shown in SEQ ID NO. 23 and CDR3 as shown in SEQ ID NO. 24; the light chain variable region comprises: CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 27.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 7, wherein the heavy chain variable region of the anti-CD16a single chain antibody scFv comprises the amino acid sequence shown in SEQ ID No. 28 or 29 and the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 30 or 31.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 8, wherein the anti-CD16a single chain antibody scFv comprises the amino acid sequence shown in SEQ ID No. 11, 12 or 13.
- The trispecific antibody or trispecific antigen-binding fragment thereof of claim 1 or 2, wherein the SIRPa D1 protein comprises the amino acid sequence shown in SEQ ID No. 9 or 10.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of one chain is SEQ ID No. 32 or SEQ ID No. 33 or SEQ ID No. 34 or SEQ ID No. 35 or SEQ ID No. 36 and the amino acid sequence of the other chain is SEQ ID No. 37 or SEQ ID No. 38.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 1 or 2, wherein the Fc-fragment of the Herceptin antibody introduces the L234A or L235A mutation, while introducing the M252Y, S T or T256E mutation at the Fc-fragment.
- The trispecific antibody or trispecific antigen-binding fragment thereof according to claim 1 or 2, wherein the Fc-fragment of the Herceptin antibody is wild-type, while introducing a M252Y, S254T or T256E mutation in the Fc-fragment.
- An isolated nucleic acid molecule encoding the trispecific antibody or trispecific antigen-binding fragment thereof of any one of claims 1 or 2.
- A vector comprising the nucleic acid sequence of claim 14.
- A cell comprising the nucleic acid sequence of claim 14 or the vector of claim 15.
- A method for preparing a trispecific antibody according to any one of claims 1 to 5, comprising the steps of:(1) Connecting anti-CD16a single-chain antibody scFv to the C end or N end of the heavy chain of the Herceptin antibody through a flexible connector; connecting SIRPa D1 protein to the C end or N end of a Herceptin antibody light chain through a flexible connector; introducing mutation in the Fc segment of Herceptin antibody;(2) Cloning the first DNA fragment obtained in step (1) into a pcDNA series vector or other vectors for use in expression systems including, but not limited to, mammalian cells; cloning the second DNA fragment obtained in the step (1) into another pcDNA series vector or other vectors used in expression systems including but not limited to mammalian cells to obtain a recombinant vector;(3) And (3) transfecting the recombinant vector obtained in the step (2) into a mammalian cell or other expression systems, and carrying out expression and purification of fusion proteins to obtain the trispecific antibody.
- The method of claim 17, wherein the mammalian cells in step (3) comprise HEK293 cells, CHO cells, or derivatives thereof.
- The method for preparing a trispecific antibody according to claim 17, wherein the Fc fragment of Herceptin antibody in step (1) is introduced with the L234A or L235A mutation, while the M252Y, S T or T256E mutation is introduced at the Fc fragment.
- The method for preparing a trispecific antibody according to claim 17, wherein the Fc fragment of Herceptin antibody in step (1) is wild-type while introducing M252Y, S T or T256E mutation in the Fc fragment.
- The method for preparing a trispecific antibody according to claim 17, wherein in the step (1), specifically comprising the steps of:(a) Synthesizing heavy chain of Herceptin antibody and scFv DNA sequence of anti-CD16a single chain antibody;(b) Designing a primer, and respectively amplifying the heavy chain of the Herceptin antibody and the anti-CD16a single-chain antibody scFv by taking the plasmid in the step (a) as a template;(c) Using the DNA fragment in (b) as a template, and performing overlay PCR by using an upstream primer for amplifying the heavy chain DNA sequence of the antibody Herceptin and a downstream primer for amplifying the scFv DNA sequence of the anti-CD16a single-chain antibody to obtain a DNA sequence;(d) Designing a mutation primer by taking a mutation point M252Y, S T or T256E as a limit, taking the fragment in (c) as a template, amplifying the fragment before the mutation point by using an upstream primer and a downstream mutation primer for amplifying the heavy chain DNA sequence of the Herceptin antibody, and amplifying the fragment after the mutation point by using the upstream mutation primer and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv;(e) Using the two fragments obtained in the step (d) as templates, and amplifying by using an upstream primer for amplifying the heavy chain DNA sequence of the Herceptin antibody and a downstream primer for amplifying the anti-CD16a single-chain antibody scFv to obtain a mutated DNA fragment;(f) Obtaining a second DNA fragment in the same manner;(g) Ligating 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, and preparing plasmids after picking up the monoclonal;(h) Transfecting the plasmid prepared in (g) into HEK293 cells at 37℃with 8% CO 2 Culture in 125rpm shaker, supernatant after 7 days of transient expression was purified by protein A affinity chromatography to obtain recombinant antibodies, and antibody concentration was determined by UV280 binding theoretical extinction coefficient.
- Use of the trispecific antibody of any one of claims 1-13 or the trispecific antibody prepared by the method of any one of claims 17-21 in the preparation of an anti-tumor medicament.
- The trispecific antibody of claim 2, wherein Herceptin is used as the basic structure of the trispecific antibody, and the SIRPa D1 protein and the anti-CD16a scFv are linked in the following manner:SIRPa D1 protein is connected to the C end of the Herceptin light chain through a flexible connector, and anti-CD16a scFv is connected to the C end of the Herceptin heavy chain through a flexible connector;SIRPa D1 protein is connected to the N end of the Herceptin light chain through a flexible connector, and anti-CD16a scFv is connected to the C end of the Herceptin heavy chain through a flexible connector;SIRPa D1 protein is connected to the C end of the Herceptin light chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin heavy chain through a flexible connector;SIRPa D1 protein is connected to the N end of the Herceptin light chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible connector;SIRPa D1 protein is connected to the N end of the Herceptin heavy chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible connector;SIRPa D1 protein is connected to the C end of the Herceptin heavy chain through a flexible connector, and anti-CD16a scFv is connected to the C end of the Herceptin light chain through a flexible connector;SIRPa D1 protein is connected to the C end of the Herceptin heavy chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible connector;SIRPa D1 protein is connected to the C end of the Herceptin heavy chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin heavy chain through a flexible connector;SIRPa D1 protein is connected to the N end of the Herceptin light chain through a flexible connector, and anti-CD16a scFv is connected to the C end of the Herceptin light chain through a flexible connector;SIRPa D1 protein is connected to the C end of the Herceptin light chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible connector;SIRPa D1 protein is connected to the N end of the Herceptin heavy chain through a flexible connector, and anti-CD16a scFv is connected to the N end of the Herceptin light chain through a flexible connector; or (b)SIRPa D1 protein is connected to N end of Herceptin heavy chain through flexible linker, and anti-CD16a scFv is connected to C end of Herceptin light chain through flexible linker.
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