CN114514040A - Targeting functional molecule modified antibody compound, composition and application thereof - Google Patents
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- CN114514040A CN114514040A CN202080069530.5A CN202080069530A CN114514040A CN 114514040 A CN114514040 A CN 114514040A CN 202080069530 A CN202080069530 A CN 202080069530A CN 114514040 A CN114514040 A CN 114514040A
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Abstract
The invention belongs to the technical field of pharmacy, and relates to an antibody compound modified by a targeting functional molecule, wherein the targeting functional molecule and an antibody are connected in a certain way to form the compound, so that the compound is used for improving the problem that the curative effect of the antibody is influenced when the antibody is used for treating brain diseases, such as brain tumors (blood-brain barrier and blood-brain tumor barrier), Alzheimer disease and Parkinson disease (blood-brain barrier) because the antibody cannot cross related biological barriers, and the test result shows that: the antibody compound modified by the targeting functional molecules does not influence the activity of the antibody, can overcome the limitation of biological barriers, promotes the antibody to enter a focus part across the biological barriers, releases the antibody under the micro-environmental response of the focus part, obviously improves the treatment effect of the antibody on brain diseases, and has good clinical application prospect.
Description
The invention belongs to the technical field of pharmacy, and relates to an antibody compound modified by a targeting functional molecule. The compound utilizes the targeting functional molecules to enable the antibody to cross a biological barrier when treating brain diseases, enhance the delivery of the antibody to a focus part, and release the antibody in the microenvironment of the focus, thereby achieving the purpose of enhancing the drug effect.
With the rapid development of biomedical technology, biomacromolecule drugs are widely applied to prevention, treatment and diagnosis of diseases, and have good drug research and development prospects. The antibody drug, as a biological macromolecule drug, takes an important role in diagnosis and treatment of various diseases such as tumors, neurodegenerative diseases (such as Alzheimer's Disease (AD) and Parkinson's Disease (PD)), autoimmune diseases and the like due to the advantages of high specificity, effectiveness and safety. For example, in the antitumor field, pembrolizumab and nivolumab (PD-1 antibody) approved by the FDA for melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, etc., and Atezolizumab (PD-L1 antibody) for bladder cancer and non-small cell lung cancer have a very good antitumor effect. The immune checkpoint antibody prevents checkpoint recognition of T lymphocytes and tumor cells by binding PD-1 on the surface of the T lymphocytes or PD-L1 highly expressed on the surface of the tumor cells, thereby avoiding immune escape of the tumor cells. However, the therapeutic effect of the immune checkpoint antibody on the brain tumor with the same high expression of PD-L1 is not optimistic, and the clinical test results show that the response rate of the immune checkpoint antibody on the treatment of the brain tumor is low, and the survival of the brain tumor is not significantly improved by the treatment of Atezolizumab (PD-L1 antibody). This is because the brain tumor has biological barriers (including blood-brain barrier (BBB), blood-brain tumor barrier (BBTB)), and the antibody, as a biomacromolecule drug, has a large molecular size and is difficult to cross the biological barrier into the brain tumor site, so that the immune checkpoint antibody does not achieve the expected effect when used for brain tumor therapy. In addition to brain tumors, other brain diseases such as neurodegenerative diseases also have biological barrier problems, which results in the ineffectiveness of antibody drugs for the related diseases due to failure to cross the barrier.
Based on the current state of the prior art, the inventor intends to construct an antibody compound, mediates the delivery of an antibody into the brain across biological barriers such as a blood-brain barrier, a blood-tumor barrier and the like in a mode of connecting a targeting functional molecule and the antibody, releases the antibody at a focus part, improves the treatment effect of the antibody on the intracerebral diseases and reduces the toxic and side effects of the antibody.
Disclosure of Invention
The invention aims to construct an antibody compound based on the current state of the prior art, and particularly relates to an antibody compound modified by a targeting functional molecule, which mediates the delivery of an antibody into the brain by crossing biological barriers such as a blood-brain barrier, a blood-tumor barrier and the like in a mode of connecting the targeting functional molecule and the antibody, releases the antibody at a focus part, improves the treatment effect of the antibody on intracerebral diseases and reduces the toxic and side effects of the antibody.
The first aspect of the invention provides an antibody complex modified by a targeting functional molecule, wherein the antibody complex is formed by covalently and/or non-covalently linking the targeting functional molecule and an antibody;
preferably, the targeting functional molecule is a targeting molecule or a functional molecule consisting of the targeting molecule and a specific sensitive structure; and/or
The antibody is an anti-tumor antibody directed against an immune checkpoint or a related antigen, or an antibody for the treatment of alzheimer's disease, or an antibody for the treatment of parkinson's disease, or an engineered form of the above antibodies.
The antibody complex modified by the targeting functional molecule according to the first aspect of the present invention, wherein the targeting functional molecule is a small molecule, a polypeptide molecule or a protein molecule having a cross-blood-brain barrier and/or a cross-blood-brain tumor barrier.
Preferably, the small molecule having a cross-blood-brain barrier and/or a cross-blood-brain tumor barrier is selected from the group consisting of p-hydroxybenzoic acid and derivatives thereof and/or fatty acids;
more preferably, the fatty acid is myristic acid.
Preferably, the targeting functional molecule is a polypeptide molecule and/or a protein molecule with a cross blood-brain barrier and/or a cross blood-brain tumor barrier;
more preferably, the polypeptide molecule is selected from one or more of: VAP polypeptide, cVAP polypeptide,SVAP polypeptides,DVAP polypeptide, pHA-SVAP polypeptides and pHA-DVAP polypeptide, MC-SVAP polypeptides and MC-DVAP polypeptides, D8 polypeptides, D8-VAP polypeptides, D8-SVAP polypeptides and D8-DVAP polypeptide, WSW polypeptide,DWSW polypeptides, WSW-VAP polypeptides,DWSW- SVAP polypeptides andDWSW- DVAP polypeptide, TGN polypeptide,DTGN polypeptide, TGN-VAP polypeptide,DTGN- SVAP polypeptides andDTGN- Da VAP polypeptide; A7R polypeptide, cA7R polypeptide,DA7R polypeptide, pHA-A7R polypeptide and pHA-DA7R polypeptide, MC-A7R polypeptide and MC-DA7R polypeptide, D8-A7R polypeptide and D8-DA7R, WSW-A7R polypeptide andDWSW- DA7R polypeptide, TGN-A7R polypeptide andDTGN- DA7R polypeptide; RGD polypeptide, staged-RGD polypeptide, pHA-RGD polypeptide, MC-RGD polypeptide, D8-RGD polypeptide, WSW-RGD polypeptide, TGN-RGD polypeptide; RW polypeptide, mn polypeptide, pHA-RW polypeptide and pHA-mn polypeptide, MC-RW polypeptide and MC-mn polypeptide, D8-RW polypeptide and D8-mn polypeptide, WSW-RW polypeptide andDWSW-mn polypeptide, TGN-RW polypeptide andDa TGN-mn polypeptide; t7 polypeptide andDa T7 polypeptide; RAP12 polypeptide andDa RAP12 polypeptide; and/or
The protein molecule is selected from transferrin and/or lactoferrin.
The antibody complex modified by the targeting functional molecule according to the first aspect of the present invention, wherein the specific sensitive structure is a domain or a chemical bond that dissociates in response to the microenvironment of the disease lesion;
preferably, the specific sensitive structure is an enzyme sensitive polypeptide or a pH sensitive chemical bond.
More preferably, the enzyme-sensitive polypeptide is a polypeptide substrate for a matrix metalloproteinase.
The antibody complex modified by targeting functional molecules according to the first aspect of the invention, wherein the antibody is an anti-tumor antibody directed against an immune checkpoint or a related antigen, or an antibody for the treatment of alzheimer's disease, or an antibody for the treatment of parkinson's disease, or an engineered form of the above-mentioned antibodies.
Preferably, the anti-tumor antibody is an antibody acting on PD-1, PD-L1, CTLA-4, LAG-3, TIM-3 immune checkpoints, or an antibody acting on HER-2, VEGFR, EGFR, GD2, PDGF-R alpha, gp100, MAGE-1 tumor associated antigens.
Preferably, the antibody for treating alzheimer's disease is an antibody against a β and Tau.
Preferably, the antibody for the treatment of Parkinson's disease is an antibody acting on leucine-rich repeat kinase 2(LRRK2), alpha-synuclein (alpha-synuclein), DJ-1, RAB8A and RAB 10.
Preferably, the antibody is modified into Fab fragment, single domain antibody, Fv fragment, single chain antibody, bivalent small molecule antibody, micro antibody or nano antibody by using genetic engineering technology.
According to the antibody compound modified by the targeting functional molecule, the covalent connection is that the targeting functional molecule is directly connected with the antibody through a chemical reaction mode between the targeting functional molecule and the antibody, or the targeting functional molecule is directly fused with the antibody through genetic engineering for expression.
The targeting functional molecule modified antibody complex according to the first aspect of the present invention, wherein the non-covalent attachment is an indirect attachment of the targeting functional molecule to the antibody by means of affinity coupling of avidin to biotin.
A second aspect of the present invention provides a pharmaceutical composition for treating an intracerebral disease, the pharmaceutical composition comprising:
a targeting functional molecule modified antibody complex of the first aspect; and
pharmaceutically acceptable adjuvants;
preferably, the intracerebral disease is selected from one or more of: brain tumor, Alzheimer's disease, and Parkinson's disease.
A third aspect of the invention provides a method of treatment of an intracerebral disease, the method comprising: administering to a subject in need thereof a targeting function molecule modified antibody complex of the first aspect or a pharmaceutical composition of the second aspect;
preferably, the intracerebral disease is selected from one or more of: brain tumor, Alzheimer's disease, and Parkinson's disease.
The fourth aspect of the invention provides the use of the functional targeting molecule modified antibody complex of the first aspect in the preparation of a medicament for the treatment of an intracerebral disease;
preferably, the intracerebral disease is selected from one or more of: brain tumor, Alzheimer's disease, and Parkinson's disease.
The invention forms the target functional molecule modified antibody compound with the antibody through a certain connection mode.
In the present invention, the specific connection mode is covalent connection and/or non-covalent connection.
Wherein, the covalent connection is the connection through the chemical reaction between the targeting functional molecule and the antibody, or the fusion expression of the targeting functional molecule and the antibody through genetic engineering; the non-covalent connection is to indirectly connect the targeting functional molecule and the antibody by the affinity coupling mode of avidin and biotin.
In the invention, the targeting functional molecule comprises a targeting molecule and a specific sensitive structure.
In the present invention, the targeting molecule is a molecule having a cross-blood-brain barrier and/or a blood-brain tumor barrier.
Preferably, the targeting molecule is a small molecule compound, such as: p-hydroxybenzoic acid (pHA) and its derivatives, fatty acids such as myristic acid (MC).
Preferably, the targeting molecule is a polypeptide molecule or a protein molecule; wherein the polypeptide molecule is: VAP polypeptide, cVAP polypeptide,SVAP polypeptides,DVAP polypeptide, pHA-SVAP polypeptides and pHA-DVAP polypeptide, MC-SVAP polypeptide and MC-DVAP polypeptides, D8 polypeptides, D8-VAP polypeptides, D8-SVAP polypeptides and D8-DVAP polypeptide, WSW polypeptide,DWSW polypeptides, WSW-VAP polypeptides,DWSW- SVAP polypeptides andDWSW- DVAP polypeptide, TGN polypeptide,DTGN polypeptide, TGN-VAP polypeptide,DTGN- SVAP polypeptides andDTGN- Da VAP polypeptide; A7R polypeptide, cA7R polypeptide,DA7R polypeptide, pHA-A7R polypeptide and pHA-DA7R polypeptide, MC-A7R polypeptide and MC-DA7R polypeptide, D8-A7R polypeptide and D8-DA7R polypeptide, WSW-A7R polypeptide andDWSW- DA7R polypeptide, TGN-A7R polypeptide andDTGN- DA7R polypeptide; RGD polypeptide, staged-RGD polypeptide, pHA-RGD polypeptide, MC-RGD polypeptide, D8-RGD polypeptide, WSW-RGD polypeptide, and TGN-RGD polypeptide; RW polypeptide, mn polypeptide, pHA-RW polypeptide and pHA-mn polypeptide, MC-RW polypeptide and MC-mn polypeptide, D8-RW polypeptide andd8-mn polypeptide, WSW-RW polypeptide andDWSW-mn polypeptide, TGN-RW polypeptide andDa TGN-mn polypeptide; t7 polypeptide andDa T7 polypeptide; RAP12 polypeptide andDa RAP12 polypeptide; the protein molecules are transferrin, lactoferrin and the like, and the polypeptide molecules or the protein molecules can be used in one or any combination.
In the invention, the amino acid sequence of the polypeptide is written from an amino terminal to a carboxyl terminal; wherein, the VAP polypeptide is a ligand of GRP78 protein; the D8 polypeptide is a cell-penetrating peptide; the WSW polypeptide is quorum sensing polypeptide; the TGN polypeptide is brain-targeted polypeptide obtained by phage display; the A7R polypeptide is a ligand for VEGFR2 and NRP-1 receptors; RGD polypeptide, RW polypeptide and mn polypeptide are ligands of integrin; the T7 polypeptide is a ligand of transferrin receptor; RAP12 polypeptide is a high binding activity polypeptide of low density lipoprotein receptor-related protein-1.
TABLE 1
In the invention, the specific sensitive structure can respond to the microenvironment of a disease focus part and dissociate to release the antibody. Preferably, the specific sensitive structure is an enzyme sensitive polypeptide or a pH sensitive chemical bond, more preferably, the enzyme sensitive polypeptide is a polypeptide substrate of a matrix metalloproteinase.
In the invention, the antibody is an antibody acting on immune check points such as PD-1, PD-L1, CTLA-4, LAG-3, TIM-3 and the like, or an anti-tumor antibody acting on tumor-related antigens such as HER-2, VEGFR, EGFR, GD2, PDGF-R alpha, gp100, MAGE-1 and the like; or an antibody acting on Abeta, Tau or the like for use in the treatment of Alzheimer's disease; or an antibody acting on leucine-rich repeat kinase 2(LRRK2), alpha-synuclein (alpha-synuclein, DJ-1), RAB8A, RAB10 and the like for treating Parkinson's disease; or the antibody fragment combination of the above antibodies, which is modified by genetic engineering means, comprises Fab fragments, single domain antibodies, Fv fragments, single-chain antibodies, bivalent small molecule antibodies, minibodies, nanobodies and the like.
In order to achieve the above object, the technical scheme adopted by the present invention comprises a random site modification method and a site-specific site modification method: wherein,
random site modification: activating free amino in the antibody into a maleimide group, and then covalently connecting the maleimide group with a targeting functional molecule with sulfydryl to obtain an antibody compound modified by the targeting functional molecule;
site-directed modification: the target functional molecules are connected with the antibody through affinity coupling, specifically, Streptavidin (SA) is expressed on an Fc fragment of the antibody by using a biological engineering technology, and then the SA-antibody and the biotinylated target functional molecules are mixed to obtain the target functional molecule modified antibody compound.
The invention provides an antibody compound modified by targeting functional molecules and a preparation method thereof, and biological activity, in vivo and in vitro targeting and in vivo and in vitro efficacy evaluation are carried out, and results show that the modification of the targeting functional molecules of the antibody compound modified by the targeting functional molecules has small influence on the biological activity of the antibody, so that the antibody can overcome biological barriers such as blood-brain barriers and the like, the drug delivery of the antibody to lesions in the brain can be increased, the antibody is released under the specific microenvironment condition of the lesions, and the treatment effect of the antibody is remarkably enhanced.
The experimental result shows that the antibody compound modified by the targeting functional molecule has the advantage of crossing biological barriers, can improve the treatment effect of the antibody on brain diseases, has important significance for expanding the clinical application range of the antibody, and has good application prospect.
Brief description of the drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the HPLC and mass spectrum of pHA, where
The chromatographic method comprises the following steps: chromatography column (YMC, C18): 150X 4.6 mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution procedure: 0-45min, 5% B-65% B; flow rate: 0.7 mL/min; column temperature: 40 ℃; and (3) detection: UV 214nm, retention time: 8.89min, ESI-MS: 415.2Da, corresponding to the theoretical molecular weight.
Fig. 2 shows HPLC and mass spectra of mRAP, wherein,
the chromatographic method comprises the following steps: chromatography column (YMC, C18): 150X 4.6 mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution procedure: 0-45min, 5% B-65% B; flow rate: 0.7 mL/min; column temperature: 40 ℃; and (3) detection: UV 214nm, retention time: 14.63min, ESI-MS: 2321.8Da, corresponding to the theoretical molecular weight.
Figure 3 shows HPLC and mass spectra of mCDX, wherein,
the chromatographic method comprises the following steps: chromatography column (YMC, C18): 150X 4.6 mm; mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); elution procedure: 0-45min, 5% B-65% B; flow rate: 0.7 mL/min; column temperature: 40 ℃; and (3) detection: UV 214nm, retention time: 17.49min, ESI-MS: 2775.0Da, corresponding to the theoretical molecular weight.
FIG. 4 shows the mass spectrometry characterization of targeting functional molecule-PDL 1 antibody complex,
the molecular weight of the targeting functional molecule-PDL 1 antibody compound is increased compared with that of an unmodified antibody, the average molecular weight is increased by about 5-7 targeting functional molecules, and the successful preparation of the compound is proved; and mRAP-alpha PDL1 and mCDX-alpha PDL1 can be reduced into unmodified antibody molecular weight after being subjected to enzymolysis by MMP, which shows that the target functional molecules can be removed by enzymolysis.
FIG. 5 shows the results of gel electrophoresis characterization of targeting functional molecule-PDL 1 antibody complex,
the molecular weights of the light chain and the heavy chain of the targeting functional molecule-PDL 1 antibody compound are increased compared with the molecular weight of an unmodified antibody, which indicates that the targeting functional molecule is modified on the light chain and the heavy chain of the antibody, and further confirms that the compound is successfully prepared; and mRAP-alpha PDL1 and mCDX-alpha PDL1 can be degraded by MMP, and the molecular weight is reduced, which shows that the target functional molecules can be removed by enzymolysis.
FIG. 6 shows functional binding activity characterization of targeting functional molecule-PDL 1 antibody complex, wherein,
the binding activity of the targeting functional molecule-PDL 1 antibody complex and PDL1 protein is verified in vitro by an Elisa method, and the binding activity of the targeting functional molecule-PDL 1 antibody complex and PDL1 protein is verified by an SPR technology, so that the effect of the targeting molecule on the functional binding activity of the targeting functional molecule-PDL 1 antibody complex is small.
FIG. 7 shows the binding effect of PDL1 antibody complex as a targeting functional molecule to PDL1 on the surface of tumor cells, wherein,
the graph A is a fluorescent photograph that the targeting functional molecule-PDL 1 antibody compound can block the combination of PDL1 protein on the surface of a tumor cell and other PDL1 antibodies, and the graph B is a flow-type quantitative result for further quantitatively verifying the blocking effect of the targeting functional molecule-PDL 1 antibody compound on PDL1 protein, and the result shows that the targeting functional molecule-PDL 1 antibody compound can also block the combination of PDL1 protein on the surface of the tumor cell with an unmodified antibody.
FIG. 8 shows that targeting of functional molecule-PDL 1 antibody complex inhibits PD pathway activation vs CD3+Killing of T cells, wherein,
panel A shows that targeting of functional molecule-PDL 1 antibody complex inhibits CD3 induced by PD pathway activation+Flow chart of T cell apoptosis, panel B is the quantitative result (n is 3), and the result shows that pHA-alpha PDL1 has comparable PD pathway inhibition ability to alpha PDL1, mRAP-alpha PDL1 and mCDX-Alpha PDL1 also inhibits T cell apoptosis.
FIG. 9 shows the evaluation of the in vitro cross-blood-brain barrier capacity of targeting functional molecule-PDL 1 antibody complex,
the targeting functional molecule-PDL 1 antibody complex was shown to be evaluated in vitro across the blood-brain barrier (n ═ 3), and the targeting molecule was able to mediate antibody crossing the in vitro blood-brain barrier model barrier, with significant differences compared to the unmodified antibody group.
FIG. 10 shows the evaluation of targeting in vivo in normal mice of the targeting functional molecule-PDL 1 antibody complex, wherein, the graph A shows that when the targeting functional molecule-PDL 1 antibody complex is injected into the tail vein for 8h, the targeting functional molecule-PDL 1 antibody complex is distributed in the brain of a normal C57BL/6 mouse (n is 3), the graph B shows the distribution of the targeting functional molecule-PDL 1 antibody complex in the main organs of normal mice when the tail vein is injected for 8h, panel C shows distribution of the targeting molecule-PDL 1 antibody complex in the brain of normal C57BL/6 mice when injected into tail vein for 24h (n-3), and the graph D shows that the distribution of the targeting functional molecule-PDL 1 antibody complex in the main organs of normal mice is increased in comparison with that of an unmodified antibody when the targeting functional molecule-PDL 1 antibody complex is injected into tail vein for 24h, and the PDL1 antibody can cross the complete blood brain barrier under the mediation of the targeting molecule.
FIG. 11 shows the evaluation of targeting molecule-PDL 1 antibody complex in vivo in mice bearing orthotopic brain tumors, wherein,
panel A shows that when target functional molecule-PDL 1 antibody complex is injected into tail vein for 8h, the complex is distributed in the brain of model mouse with orthotopic brain tumor C57BL/6 (n is 3), panel B shows that when the target functional molecule-PDL 1 antibody complex is injected into tail vein for 8h, when the compound is distributed in the main organs of a Homophore brain tumor C57BL/6 model mouse, and a graph C shows that when a target functional molecule-PDL 1 antibody compound is injected into the tail vein for 24 hours, it is distributed in the brain of a Homophore brain tumor C57BL/6 model mouse (n is 3), panel D shows that when the target functional molecule-PDL 1 antibody complex is injected into tail vein for 24h, the distribution of the antibody in main organs of an in situ-loaded brain tumor C57BL/6 model mouse shows that the distribution of a targeting functional molecule-PDL 1 antibody compound in a brain tumor is increased compared with that of an unmodified antibody, and PDL1 antibody can be more accumulated in the brain tumor part under the mediation of the targeting molecule.
FIG. 12 shows the results of pharmacokinetic studies of targeting functional molecule-PDL 1 antibody complex in normal mice,
the pharmacokinetic profile and parameters (n is 4) of the targeting functional molecule-PDL 1 antibody complex in normal mice after tail vein injection are shown, and the results show that the pharmacokinetics behaviors of pHA-alpha PDL1, mRAP-alpha PDL1 and unmodified alpha PDL1 are not obviously different, and the blood clearance of mCDX-alpha PDL1 is faster.
FIG. 13 shows the pharmacodynamic results of targeting functional molecule-PDL 1 antibody complex on GL261 carcinoma-bearing C57BL/6 model, in which,
panel a is the survival curve (n-8) of tumor-bearing C57BL/6 mice, demonstrating that the targeting functional molecule-PDL 1 antibody complex significantly prolonged survival compared to the unmodified antibody group and the PBS group; FIG. B, C is a photograph of brain tumor section ki67 and tunnel immunohistochemistry of tumor-bearing C57BL/6 mouse and its quantitative results, which shows that there are fewer malignant cells and more apoptosis in the target functional molecule-PDL 1 antibody complex group, consistent with the pharmacodynamic results.
FIG. 14 shows the immunomodulatory effect of targeting functional molecule-PDL 1 antibody complex on brain glioma in situ, wherein,
the immune effect of brain tumor sites of mice with tumor-bearing orthotopic brain glioma model (n is 3) is shown in fig. a-F, the immune effect of neck lymph nodes of the tumor-bearing mice is shown in fig. G-I, and the immune effect of spleen of the tumor-bearing mice is shown in fig. J-L, compared with the unmodified antibody and PBS group, the immunosuppressive cells of the target functional molecule-PDL 1 antibody complex group are obviously reduced, the effector cells are obviously increased, and the immunosuppressive environment of tumors and organisms can be improved.
Fig. 15 shows the toxic side effects of targeting functional molecule-PDL 1 antibody complex, wherein,
the result shows that the liver function index of the targeting functional molecule-PDL 1 antibody compound is close to that of the PBS group, the liver toxicity of the unmodified antibody group is higher, and meanwhile, the level of the inflammatory factor in the blood further confirms that the targeting functional molecule-PDL 1 antibody compound delivery system has lower toxic and side effects.
Best Mode for Carrying Out The Invention
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
Example 1 Synthesis and characterization of targeting molecules
Synthesis and characterization of the pHA targeting molecules: the polypeptide is prepared by adopting a solid phase synthesis technology, firstly, MBHA resin (the substitution degree is 0.54mmol/g) is weighed, N-dimethylformamide is added for swelling, 20% piperidine-DMF solution is added for removing Fmoc protecting groups on the resin, after washing, 8.8 times of Fmoc-amino acid (dissolved in DMF solution of HBTU/HOBT/DIEA) is added, and the mixture is placed in an air shaking table (37 ℃, 180rpm) for shaking for 45 min. After the reaction was complete, the resin was washed with fresh DMF and drained, and 20% piperidine-DMF solution was added to remove the Fmoc protecting group from the α -amino group. The above-described operation was repeated according to the sequence (pHA sequence: p-Hydroxybenzoic acid-SSC). After all amino acids are condensed, the resin is washed and drained, a polypeptide cleavage reagent (TFA/TIPS/H2O ═ 95/2.5/2.5, v/v) is added, the mixture is stirred for 2 hours, the TFA is removed by rotary evaporation, and the crude polypeptide is obtained by filtration and collection after precipitation by ethyl ether. The pHA polypeptide molecules were characterized by preparative liquid phase purification, lyophilization, HPLC and ESI-MS, and the results are shown in FIG. 1.
Synthesis and characterization of mRAP targeting molecules: mRAP was synthesized as described above and characterized by its amino acid sequence of CGPLGIAGQEAKIEKHNHYQK, HPLC and ESI-MS as shown in FIG. 2.
Synthesis and characterization of mCDX targeting molecules: the mCDX is synthesized by the method, the amino acid sequence of the mCDX is greirtgarewsekfGPLGIAGQC (the lower case is D-configuration amino acid and the upper case is L-configuration amino acid), and the HPLC and ESI-MS characterization results are shown in figure 3.
Example 2 preparation and characterization of targeting functional molecule-PDL 1 antibody complexes
Preparation of targeting functional molecule-PDL 1 antibody complex:
to the α PDL1 solution, sulfol-SMCC (1mg/mL) was added and the reaction was carried out at room temperature for 30min, and excess sulfol-SMCC was removed by desalting column. Adding 108 μ L of pHA PBS solution (2mg/mL), mixing, reacting at 4 deg.C for 12 hr, dialyzing in 14K Da dialysis bag with pure water for 2 times (30 min each time), and collecting the liquid in dialysis bag to obtain pHA modified PDL1 antibody complex (pHA- α PDL 1). mRAP-alpha PDL1 and mCDX-alpha PDL1 were prepared in the same manner.
And MMP enzymolysis verification:
MMP-2 was added to 1. mu.L of APMA (1M), diluted to 100. mu.L with the reaction solution, and incubated at 37 ℃ for 1 h. After activation, 50. mu.L of enzyme was mixed with 50. mu.L of mRAP- α PDL1 and mCDX- α PDL1, incubated at 37 ℃ for 2h, and dialyzed.
Characterization of targeting functional molecule-PDL 1 antibody complex:
the above products were characterized by MALDI-TOF and gel electrophoresis, respectively, the MALDI-TOF results are shown in FIG. 4, wherein the gel electrophoresis method is: preparing 12% separation gel, and mixing 0.35mg/mL pHA-alpha PDL1, mRAP-alpha PDL1, mCDX-alpha PDL1, mRAP-alpha PDL1+ MMP and
mCDX-alpha PDL1+ MMP solution 60. mu.L, DTT (1M) solution 0.6. mu.L was added to the solution to give a final concentration of 10mM DTT, and after standing overnight at 4 ℃, 15. mu.L of loading buffer was added, and the mixture was boiled in a water bath at 100 ℃ for 5min, cooled to room temperature, and loaded to 20. mu.L. Concentrating the sample at 80V, separating the sample at 120V, washing with pure water for 5min each time for 3 times; staining SimpleBlue, and shaking gently for 1h at room temperature; the sample was washed with pure water for 1 hour and 2 times, and photographed, and the results are shown in FIG. 5.
Example 3 functional binding Activity characterization of Targeted functional molecule-PDL 1 antibody Complex
ELISA binding activity:
coating a 96-well ELISA plate with 100 mu L/well of murine PD-L1 protein, standing at room temperature for 2 hours, washing the plate for 2 times, adding BSA at room temperature for blocking for 1 hour, adding pHA-alpha PDL1, mRAP-alpha PDL1, mCDX-alpha PDL1 or unmodified alpha PDL1(600ng/mL, 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL, 2ng/mL) with a series of concentration gradients, placing the plate in a constant temperature shaker at 37 ℃ for incubation for 1 hour, after washing the plate, adding anti-rat Ig (H + L)/HRP for incubation for 1 hour, adding 100 mu L of color developing solution for standing at room temperature for 10min, adding 100 mu L of 1M sulfuric acid for terminating the reaction, measuring the absorbance value by a microplate reader, and detecting the wavelength of 450 nm. The results are shown in FIG. 6 (A).
SPR binding activity:
the binding activity of pHA-alpha PDL1, mRAP-alpha PDL1, mCDX-alpha PDL1 and PDL1 protein was examined by using the Surface Plasmon Resonance (SPR) technique. The pre-binding analysis was performed by the biacore system, and CM5 chips were selected. Recombinant murine PDL1 protein was coupled to a CM5 chip and RU values reached the target values. Unmodified α PDL1, pHA- α PDL1, mRAP- α PDL1, mCDX- α PDL1 were configured as sample solutions at concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, 25 and 50nM, respectively. Sample introduction is carried out in sequence from low to high according to concentration, the binding activity of the targeting functional molecule-PDL 1 antibody compound and PDL1 protein is analyzed by Biacore T200 Evaluation software, and K of the targeting functional molecule-PDL 1 antibody compound and the PDL1 protein is respectively calculatedDThe value is obtained. The results are shown in FIG. 6 (B-E).
PD pathway blockade:
the PD channel is blocked by utilizing a fluorescein labeled PDL1 antibody (PE-alpha PDL1) to compete for the binding of a PDL1 antibody complex modified by a targeting functional molecule and PDL1 protein, and the binding blocking capability of the targeting functional molecule PDL1 antibody complex and the PDL1 protein is examined. GL261 cells were digested and dispersed at 5X 10 per well3The density of each cell is inoculated on a laser confocal cell culture dish, cell culture solution containing IFN-gamma is added into each hole, and the cells are cultured for 48 hours in a cell culture box, so that the PDL1 protein on the surface is highly expressed. Discarding the culture solution, washing with PBS, fixing with 4% paraformaldehyde for 15min, staining with DAPI for 10min, washing with PBS again, loading and incubating by the above method, and observing the blocking effect of the polypeptide modified antibody on PDL1 protein on the cell surface by using a laser confocal microscope. The results are shown in FIG. 7.
Further, for quantitative examination of the blocking effect of the complex on the PD pathway, GL261 cells were stimulated with IFN-. gamma.for 48 hours to highly express PDL1 protein on the surface, GL261 cells highly expressing PDL1 protein were digested with pancreatin, the culture broth was neutralized with pancreatin, and then centrifuged and redispersed in PBS buffer. IgG, alpha PDL1, pHA-alpha PDL1, mRAP-alpha PDL1 and mCDX-alpha PDL1 were added to tumor cells highly expressing PDL1 at 4 ℃ and incubated for 5min, PE-alpha PDL1 was added and incubated for 15min, the cells were washed with PBS and resuspended in 200. mu.L of PBS buffer, the positive cells were counted by a flow cytometer, and the biological activity of the modified alpha PDL1 was examined, as shown in FIG. 7.
+Example 4 in vitro CD 3T cell killing experiment
Mouse CD3+Extraction of T cells:
c57BL/6 mice were inoculated ventrally and subcutaneously 106GL261 cells were inoculated on day 5 after tumor inoculation, spleen of mouse was ground into single cells, and CD3 was sorted out by magnetic beads+T cells are cultured on a U-shaped plate coated with anti-CD3 antibody, and then added with anti-CD28 antibody for co-stimulation and culture for 48 h.
T cell mediated cell killing experiment:
GL261 cells were stimulated with IFN-gamma for 48h to achieve high expression of surface PDL1 protein. Tumor cells highly expressing PDL1 protein were seeded in 96-well plates (5X 10)3One/well) with or without equimolar amounts of IgG, α PDL1, pHA- α PDL1, mRAP- α PDL1, mCDX- α PDL 1. Re-stimulated CD3+T cells (105/well) were co-cultured for 24 h. After staining with the apoptosis kit, flow cytometry was used for detection, and the results are shown in fig. 8.
Example 5 Targeted evaluation of targeting of functional molecule-PDL 1 antibody complexes
In vitro blood-brain barrier crossing capacity:
cutting head of 4-week-old SD rat, collecting brain, rapidly separating in ice-cold D-Hanks solution to obtain cerebral cortex, removing meninges and cerebral macrovascular, cutting, adding collagenase and DNase, digestingCentrifuging, transferring bottom capillary to culture solution, inoculating to 24-well transwell pre-plated with rat tail collagen, transferring into carbon dioxide incubator at 37 deg.C and 5% CO2Culturing for 24h under saturated humidity condition to allow the capillary vessel segment to adhere to the wall, changing into special endothelial culture solution containing puromycin, culturing for 72h, changing into special endothelial culture solution containing no puromycin, culturing for 72h, and measuring transmembrane resistance (TEER) over 250 Ω & cm2Thus, the in vitro blood-brain barrier modeling is successful.
α PDL1, pHA- α PDL1, mRAP- α PDL1 and mCDX- α PDL1 were quantified by BCA method and diluted to the same concentration (1.0mg/mL) with PBS, 10. mu.L of each well was added to the upper chamber of the transwell, 90. mu.L of EBM-2 culture medium containing culture factors was further added, and 600. mu.L of EBM-2 culture medium was added to the lower chamber. 60 μ L of the lower chamber solution was collected at 0.5h and 1h and 60 μ L of the solution was replenished. The CD274 coated ELISA plate measures the amount of antibody in the collected samples, and the results are shown in figure 9.
In vivo brain targeting verification:
60 μ L of α PDL1 antibody solution (0.5mg/mL) at a radiation dose of 0.78mCi125I, 20. mu.L PBS, in an Iodogen tube (80. mu.g) for 5min (about 34-37 ℃). After transferring, washing with water for 2 times, and purifying by Sephadex G-25 column to obtain125I-labeled α PDL1, with a measured radiation dose of 660 μ Ci, a labeling rate of about 81%, and a specific activity of 21.06 μ Ci/μ g. By using125I-labeled alpha PDL1 is prepared respectively125I-labeled pHA-alpha PDL1,125I-labeled mRAP-alpha PDL1,125I-labeled mCDX- α PDL 1.
12 normal mice were selected, three per group, with groups α PDL1, pHA- α PDL1, mRAP- α PDL1, mCDX- α PDL1, and a radiation dose of 15 μ Ci per mouse. Brains were taken at 8h, 24h, weighed and the radioactivity was measured to investigate the brain and major tissue distribution of the complex in normal mice, as shown in FIG. 10.
Meanwhile, male C57BL/6 with a weight of about 20g is selected, positioned in a cerebral stereotaxic apparatus to the striatum area with a fontanelle of 1.8mm right, 0.6mm forward and a depth of 3mm, and injected into the striatum area with a size of 1X 106Is arranged atIn logarithmic growth phase, GL261 cells (dispersed in 5. mu.L PBS buffer) in suspension were cultured in SPF environment after tumor inoculation, and the survival status of model mice was observed periodically. The mice inoculated with the in situ glioma were divided into 4 groups of 12 mice, as above. On day 7 after tumor inoculation, the tissue distribution experiment was performed in the same manner, and the radiation dose administered to each mouse was 15. mu. Ci. Brain tumors were harvested at 8h and 24h, weighed, and the radioactivity was measured, the results of which are shown in FIG. 11.
Example 6 pharmacokinetic evaluation of targeting functional molecule-PDL 1 antibody Complex in Normal mice
16 male C57BL/6 mice were randomly divided into 4 groups of 4 mice, and injected into the tail vein with α PDL1, pHA- α PDL1, mRAP- α PDL1, mCDX- α PDL1 at a dose equivalent to α PDL1 containing 5mg/kg, bled at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h of eye sockets, assayed for PDL1 antibody content by ELISA, plotted against the time of drug production and calculated for pharmacokinetic parameters, the results are shown in FIG. 12.
Example 7 pharmacodynamic evaluation of targeting functional molecule-PDL 1 antibody Complex pairs in orthotopic tumor mouse model
33 Male C57BL/6 mice were inoculated with in situ glioma cell GL261 (1X 10)6One/only),
they were randomized into 3 groups and tumor inoculation was day 0. On days 3, 5, 7, and 9, respectively, tail vein injection of PBS, α PDL1, and pHA- α PDL1 was performed at a dose equivalent to α PDL1 containing 5mg/kg, and the death time was observed and recorded to plot a survival curve. 24h after the last administration, that is, 10 days, brain tissues of 3 mice were paraffin-embedded in each group, and after sectioning, Ki67 and TUNEL immunohistochemical staining were performed to examine the proliferation ability and apoptosis of tumors in each administration group, and the results are shown in FIG. 13.
Example 8 immunomodulatory Effect of Targeted functional molecule-PDL 1 antibody Complex on brain glioma in situ
12 Male C57BL/6 mice were inoculated with in situ glioma cell GL261 (1X 10)6One/only) to randomly divide it into 3 groups eachGroup 4, tumor inoculation was day 0. On days 3, 5, 7, and 9, respectively, tail vein injection of PBS, α PDL1, and pHA- α PDL1 was performed at a dose of 5mg/kg of α PDL 1. Taking brain tumor, neck lymph node and spleen on day 10, processing into single cell suspension, adding flow cell surface antibody, staining with CD8, CD4, PD-1, CD62L, CD44 and CD25 fluorescence labeled antibody, and breaking cell nuclear membrane to stain Foxp 3; and the other part of cells are added into a Brefeldin A incubator to be incubated for 6 hours, and intracellular antibodies IFN-gamma and TNF-alpha are stained by breaking cell membranes after cell surface staining. Determine CD8 therein+T cell, CD4+T cells, IFN-. gamma.+T cells, TNF-alpha+、PD-1 +T cell and TregThe number of cells, and their ratio were varied, and the results are shown in FIG. 14.
Example 9 toxic side effects of targeting functional molecule-PDL 1 antibody complexes
12 Male C57BL/6 mice were inoculated with in situ glioma cell GL261 (1X 10)6One/one), it was randomly divided into 3 groups of 4 each, and the tumor was inoculated on day 0. On days 3, 5, 7 and 9, PBS, α PDL1 and pHA- α PDL1 were injected into the tail vein at a dose of 5mg/kg corresponding to α PDL 1. The body weight of each mouse was measured and recorded. Taking blood on the 10 th day, centrifuging at 4000rpm/min for 10 minutes, taking plasma, and measuring TNF-alpha and IFN-gamma factors in the blood by an ELISA method; and detecting ALT and AST liver function indexes, and evaluating the safety of the liver function indexes. The results are shown in FIG. 15.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Claims (15)
- The targeting functional molecule modified antibody complex is characterized in that the antibody complex is formed by covalently linking and/or non-covalently linking a targeting functional molecule and an antibody;preferably, the targeting functional molecule is a targeting molecule or a functional molecule consisting of the targeting molecule and a specific sensitive structure; and/or said antibody is an anti-tumor antibody directed against an immune checkpoint or a related antigen, or an antibody for the treatment of alzheimer's disease, or an antibody for the treatment of parkinson's disease, or an engineered form of the above-mentioned antibodies.
- The targeting molecule modified antibody complex of claim 1, wherein said targeting molecule is a small molecule, polypeptide molecule or protein molecule having a cross-blood-brain barrier and/or a cross-blood-brain tumor barrier.
- The antibody complex modified with targeting functional molecule according to claim 2, wherein said small molecule with a blood-brain barrier and/or a blood-brain tumor barrier is selected from p-hydroxybenzoic acid and derivatives thereof and/or fatty acids;preferably, the fatty acid is myristic acid.
- The targeting functional molecule modified antibody complex of claim 2, wherein said targeting functional molecule is a polypeptide molecule and/or a protein molecule having a cross-blood-brain barrier and/or a cross-blood-brain tumor barrier;preferably, the polypeptide molecule is selected from one or more of: VAP polypeptide, cVAP polypeptide,SVAP polypeptides,DVAP polypeptide, pHA-SVAP polypeptides and pHA-DVAP polypeptide, MC-SVAP polypeptides and MC-DVAP polypeptides, D8 polypeptides, D8-VAP polypeptides, D8-SVAP polypeptides and D8-DVAP polypeptide, WSW polypeptide,DWSW polypeptides, WSW-VAP polypeptides,DWSW- SVAP polypeptides andDWSW- DVAP polypeptide, TGN polypeptide,DTGN polypeptide, TGN-VAP polypeptide,DTGN- SVAP polypeptides andDTGN- Da VAP polypeptide; A7R polypeptide, cA7R polypeptide,DA7R polypeptide, pHA-A7R polypeptide and pHA-DA7R polypeptide, MC-A7R polypeptide and MC-DA7R polypeptide, D8-A7R polypeptide and D8-DA7R, WSW-A7R polypeptides andDWSW- DA7R polypeptide, TGN-A7R polypeptide andDTGN- DA7R polypeptide; RGD polypeptide, staged-RGD polypeptide, pHA-RGD polypeptide, MC-RGD polypeptide, D8-RGD polypeptide, WSW-RGD polypeptide, and TGN-RGD polypeptide; RW polypeptide, mn polypeptide, pHA-RW polypeptide and pHA-mn polypeptide, MC-RW polypeptide and MC-mn polypeptide, D8-RW polypeptide and D8-mn polypeptide, WSW-RW polypeptide andDWSW-mn polypeptide, TGN-RW polypeptide andDa TGN-mn polypeptide; t7 polypeptide andDa T7 polypeptide; RAP12 polypeptide andDa RAP12 polypeptide; and/orThe protein molecule is selected from transferrin and/or lactoferrin.
- The modified antibody complex of targeted functional molecules of claim 1, wherein said specific sensitive structure is a domain or chemical bond that dissociates in response to the microenvironment of the disease lesion;preferably, the specific sensitive structure is an enzyme sensitive polypeptide or a pH sensitive chemical bond.
- The functionally targeted molecule modified antibody complex of claim 5, wherein said enzyme-sensitive polypeptide is a polypeptide substrate for a matrix metalloproteinase.
- The modified antibody complex of targeted functional molecules of claim 1, wherein said anti-tumor antibody is an antibody acting on the immune checkpoint of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3 or an antibody acting on HER-2, VEGFR, EGFR, GD2, PDGF-ra, gp100, MAGE-1 tumor-associated antigens.
- The functional targeting molecule modified antibody complex of claim 1, wherein said antibody for alzheimer's disease treatment is an antibody against a β, Tau.
- The functional targeting molecule modified antibody complex of claim 1, wherein said antibody for PD treatment of parkinson's disease is an antibody directed against leucine rich repeat kinase 2(LRRK2), alpha-synuclein (alpha-synuclein), DJ-1, RAB8A and RAB 10.
- The antibody complex modified by targeting functional molecule according to claim 1, wherein the antibody is modified into Fab fragment, single domain antibody, Fv fragment, single chain antibody, bivalent small molecule antibody, micro-antibody or nano-antibody by genetic engineering technique.
- The targeting molecule modified antibody complex of claim 1, wherein said covalent linkage is achieved by directly linking the targeting molecule to the antibody by chemical reaction between the targeting molecule and the antibody, or by directly expressing the targeting molecule and the antibody by fusion by genetic engineering.
- The targeting molecule modified antibody complex of claim 1 wherein said non-covalent attachment is indirect attachment of said targeting molecule to said antibody by affinity coupling of avidin to biotin.
- A pharmaceutical composition for treating an intracerebral disease, the pharmaceutical composition comprising:the targeting functional molecule modified antibody complex of any one of claims 1 to 12; andpharmaceutically acceptable adjuvants;preferably, the intracerebral disease is selected from one or more of: brain tumor, Alzheimer's disease, and Parkinson's disease.
- A method of treating an intracerebral disorder, the method comprising: administering to a subject in need thereof a targeting function molecule modified antibody complex of any one of claims 1 to 12 or a pharmaceutical composition of claim 13;preferably, the intracerebral disease is selected from one or more of: brain tumor, Alzheimer's disease, and Parkinson's disease.
- Use of a targeted functional molecule modified antibody complex of any one of claims 1 to 12 in the manufacture of a medicament for the treatment of an intracerebral disease;preferably, the intracerebral disease is selected from one or more of: brain tumor, Alzheimer's disease, and Parkinson's disease.
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