CN114522246A - Pharmaceutical composition containing VEGF nano antibody and application thereof - Google Patents

Pharmaceutical composition containing VEGF nano antibody and application thereof Download PDF

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Publication number
CN114522246A
CN114522246A CN202011325582.2A CN202011325582A CN114522246A CN 114522246 A CN114522246 A CN 114522246A CN 202011325582 A CN202011325582 A CN 202011325582A CN 114522246 A CN114522246 A CN 114522246A
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vegf
pharmaceutical composition
cell
seq
penetrating peptide
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万亚坤
朱敏
盖军伟
李光辉
沈晓宁
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Shanghai Novamab Biopharmaceuticals Co Ltd
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Shanghai Novamab Biopharmaceuticals Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Abstract

The invention provides a pharmaceutical composition containing a VEGF nano antibody and application thereof. Specifically, the invention provides a pharmaceutical composition, which contains a VEGF nano antibody, a cell-penetrating peptide and a pharmaceutically acceptable carrier. The pharmaceutical composition can effectively penetrate cell membranes, and the contained cell-penetrating peptide can remarkably promote the cell membrane penetrability of the VEGF nano antibody. The pharmaceutical composition can effectively permeate cancer cells and corneal cells, the uptake effect of the VEGF nano antibody on the cells is obviously improved along with the increase of the concentration of the cell-penetrating peptide, and the pharmaceutical composition has only slight toxicity at high dose and very good safety.

Description

Pharmaceutical composition containing VEGF nano antibody and application thereof
Technical Field
The invention relates to the field of biomedicine or biopharmaceutical, in particular to a pharmaceutical composition containing a VEGF nano antibody and application thereof.
Background
Cell-penetrating Peptides (CPPs) are a class of short-chain polypeptides that can penetrate the Cell membrane. It has been found that CPPs are capable of binding to a variety of drug molecules and delivering the drug into cells. Many experiments and reports have explained the transmembrane and cellular uptake mechanisms of CPPs, but the exact pathway of CPPs into cells remains to be investigated further. At present, researches find that artificially set conditions can influence experimental results, different CPPs enter different cells to have different uptake ways, and cargos carried by the CPPs can also influence the internalization mechanism of the CPPs. Notably, the internalization pathway of CPPs is not constant, and many CPPs can accomplish internalization in multiple ways. Moreover, the internalization mechanism of CPPs varies depending on their concentration, cell organization, and cargo carried. The membrane-penetrating peptides currently studied more include: TAT, Pennetratin, Protamine, Transpotan, MPG, and the like. The Pennetratin is derived from a homologous structure domain of drosophila antennapedia protein, is an amphiphilic membrane-penetrating peptide, and is used as a potential drug carrier for various purposes due to a good membrane-penetrating effect. The cationic amino acids arginine and lysine in the Penetratin sequence are irreplaceable sites for membrane penetration activity, and if the amino acids are replaced or modified, the membrane penetration effect is reduced, while tryptophan, phenylalanine and isoleucine in the sequence are hydrophobic amino acids, and the interaction of the peptide and the lipid of the cell membrane can be promoted. Pentratin derivatives RQIKIWFQWRRMKWKK are disclosed in the documents Sgolstra F, Deronde B M, Sarapas J M, et al, design mixtures of Membrane Active Proteins [ J ]. Accounts of Chemical Research,2013,46(12):2977-87, indicating that hydrophobic residues have an important role in cellular uptake. Chinese invention patent CN101355970A discloses Penetratin derivative RQIKIWFWNRRMKWKK. Chinese invention patent CN108976288A discloses sequences of various pennetratin derivatives. The literature indicates that the introduction of hydrophobic amino acids into the pennetrin molecule can enhance the penetration capacity of pennetrin derivatives into cells.
The VEGF nano antibody has the characteristics of high stability, good water solubility, simple humanization, high targeting property, strong penetrability and the like, and plays an extremely imaginable huge function in immune experiments, diagnosis and treatment. If the VEGF nano antibody is used together with the cell-penetrating peptide, the utilization rate of the VEGF nano antibody medicament can be improved, and further, a VEGF nano antibody anti-tumor medicament preparation with high medicament effect and a VEGF nano antibody ophthalmic preparation form which is administrated in an eye-drop mode can be developed. At present, no pharmaceutical composition combining the VEGF nano antibody with high drug effect and low toxicity and the cell-penetrating peptide is disclosed.
Therefore, the VEGF nano antibody pharmaceutical composition is developed, the uptake effect of the VEGF nano antibody on cells is improved, the clinical application prospect is good, and a good foundation is laid for preparing the VEGF nano antibody pharmaceutical preparation with high drug effect and low toxicity, especially for developing eye-drop type pharmaceutical preparations.
Disclosure of Invention
The invention aims to provide a pharmaceutical composition containing a VEGF nano antibody and application thereof.
In a first aspect of the present invention, there is provided a pharmaceutical composition comprising:
(a) anti-VEGF nanobodies;
(b) a cell-penetrating peptide; and
(c) a pharmaceutically acceptable carrier.
In another preferred example, the Complementarity Determining Regions (CDRs) of the anti-VEGF nanobody are CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO: 3.
In another preferred example, the amino acid sequence of the anti-VEGF nanobody is shown in SEQ ID NO. 4.
In another preferred embodiment, the membrane-penetrating peptide comprises Penetratin and derivatives thereof.
In another preferred embodiment, the amino acid sequence of the Penetratin is shown in SEQ ID NO. 5.
In another preferred embodiment, said Penetratin derivative is a derivative polypeptide having at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified and/or substituted based on the amino acid sequence shown in SEQ ID NO. 5 and retaining transmembrane activity.
In another preferred embodiment, the derived polypeptide retains ≥ 80%, preferably ≥ 90%; more preferably more than or equal to 95% of the transmembrane activity of the polypeptide of SEQ ID NO. 5.
In another preferred embodiment, the derived polypeptide has an identity of 80% or more, preferably 90% or more, to SEQ ID NO 5; more preferably not less than 95%.
In another preferred embodiment, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8.
In another preferred embodiment, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID NO. 7.
In another preferred embodiment, the pharmaceutical composition comprises:
(a) the VEGF resisting nano antibody with the amino acid sequence shown as SEQ ID NO. 4;
(b) the amino acid sequence is shown as the penetrating peptide of SEQ ID NO. 7; and
(c) a pharmaceutically acceptable carrier.
In another preferred embodiment, the molar ratio of the anti-VEGF nanobody to the cell-penetrating peptide is 1:20 to 10:1, preferably 1:10 to 2:1, more preferably 1:5 to 1: 1.
In another preferred embodiment, the molar ratio of the anti-VEGF nanobody to the cell-penetrating peptide is 1:3 to 1: 1.
In another preferred embodiment, the total content of the anti-VEGF nanobody and the cell-penetrating peptide is 1 to 99 wt%, and more preferably 5 to 90 wt% of the composition.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a freeze-dried preparation and a liquid preparation.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises an injection (such as a periocular and intraocular injection), an eye drop, an ophthalmic gel or an ophthalmic ointment.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a sustained release dosage form or a non-sustained release dosage form.
In another preferred embodiment, the pharmaceutical composition may further comprise other pharmaceutically active ingredients, preferably active ingredients for treating cancer or ocular diseases.
In a second aspect of the invention, there is provided a use of a pharmaceutical composition according to the first aspect of the invention for the preparation of a medicament for the treatment of a VEGF-related disease or disorder.
In another preferred embodiment, the VEGF-related disease or disorder comprises cancer or an ocular disease.
In another preferred embodiment, the cancer includes, but is not limited to: breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, nasopharyngeal cancer, or a combination thereof.
In another preferred embodiment, the ocular disease is a neovascularization-associated ocular disease.
In another preferred embodiment, the ocular diseases include, but are not limited to: age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, or a combination thereof.
In a third aspect of the present invention, there is provided a process for preparing a pharmaceutical composition according to the first aspect of the present invention, comprising the steps of:
(i) respectively providing an anti-VEGF nano antibody and a cell-penetrating peptide,
(ii) physically mixing the anti-VEGF nanobody with a cell-penetrating peptide to obtain a mixture, an
(iii) Mixing said mixture with a pharmaceutically acceptable carrier under conditions suitable for the preparation of a medicament, thereby producing said pharmaceutical composition.
In another preferred embodiment, in step (ii), the anti-VEGF nanobody may be pre-mixed with any pharmaceutically acceptable carrier to obtain a first pre-mixture, and the first pre-mixture may be physically mixed with the cell-penetrating peptide to obtain the mixture.
In another preferred embodiment, in step (ii), the cell-penetrating peptide and any pharmaceutically acceptable carrier can be pre-mixed to obtain a second pre-mixture, and the second pre-mixture is physically mixed with the anti-VEGF nanobody to obtain the mixture.
In another preferred embodiment, in step (ii), the anti-VEGF nanobody and the cell-penetrating peptide may be pre-mixed with any pharmaceutically acceptable carrier to obtain a first pre-mixture and a second pre-mixture, and the first pre-mixture and the second pre-mixture may be physically mixed to obtain the mixture.
In a fourth aspect of the invention, there is provided a kit comprising:
(i) a first container, and an active ingredient (a) anti-VEGF nanobody, or a drug containing the active ingredient (a), contained in the first container;
(ii) a second container, and an active ingredient (b) a cell-penetrating peptide or a drug containing the active ingredient (b) contained in the second container; and
(iii) instructions for the combined administration of active ingredient (a) and active ingredient (b) for the treatment of a VEGF-related disease or disorder are described.
In another preferred embodiment, the medicament in the first container and the second container is a single preparation containing the active ingredient (a) and a single preparation containing the active ingredient (b).
In a fifth aspect of the invention, there is provided a method of treating a VEGF-related disease or disorder, said method comprising the steps of:
(i) administering to a subject in need thereof an active ingredient (a) an anti-VEGF nanobody and an active ingredient (b) a cell-penetrating peptide, or a pharmaceutical composition according to the first aspect of the present invention.
In another preferred embodiment, said administering comprises simultaneous administration or sequential administration.
In another preferred embodiment, the subject includes human and non-human mammals.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the transmembrane effect of the transmembrane peptide Pennetratin and its mutants in binding VEGF nanobodies. The results show that: the Pentratin-Mut 2 can effectively penetrate cell membranes after being mixed with the VEGF nano antibody, brings the VEGF nano antibody into cells, and has a membrane penetrating effect remarkably superior to that of wild Pentratin-WT and mutant Pentratin-Mut 1.
Fig. 2A and 2B show the permeation effect of different VEGF antibodies and a mixture of cell-penetrating peptides in different types of cells. The results show that: after the VEGF nano antibody and the Penetrat-Mut 2 are mixed, the infiltration and transmembrane effect on various cells is obviously better than that of a mixture of commercially available monoclonal antibody Eylea and Penetrat-Mut 2; and with the increase of the concentration of the Pennetratin-Mut 2, the uptake effect of the VEGF nano antibody on various types of cells is obviously improved.
Fig. 3A and 3B show the toxicity of VEGF nanobodies and a mixture of cell-penetrating peptides on different types of cells. The results show that: the low-dose group (9 mu M) of VEGF nanobody, Pentratin-Mut 2, Pentratin-Mut 3, VEGF nanobody/Pentratin-Mut 2 mixture (3:1) and VEGF nanobody/Pentratin-Mut 3 mixture (3:1) has no toxic or side effect on five types of cells basically; and the mixture of VEGF nano antibody/Penetrat-Mut 2 in the high-dose group (90 mu M) shows slight toxicity on cells HT29 and MCF7 of the colon adenocarcinoma cells, and the mixture of VEGF nano antibody/Penetrat-Mut 3 in the high-dose group (90 mu M) shows obvious toxicity on cells HT29 and MCF7 of the colon adenocarcinoma cells. Wherein, the dosage (9 μ M or 90 μ M) of the mixture of the VEGF nano antibody and the cell-penetrating peptide is the content of the nano antibody in the mixture.
Detailed Description
The inventor of the invention has extensively and deeply studied, and through a large amount of screening, the invention unexpectedly discovers a pharmaceutical composition formed by simply and physically mixing a VEGF nano antibody and a cell-penetrating peptide for the first time. Experiments show that the pharmaceutical composition can effectively penetrate cell membranes, and the cell-penetrating peptide can effectively bring the VEGF nano antibody into cells; after the VEGF nano antibody and the cell-penetrating peptide are mixed, the permeation and cell-penetrating effects on various cells are obviously better than those of a mixture of a commercially available monoclonal antibody Eylea (namely a VEGF conventional monoclonal antibody) and the cell-penetrating peptide; the VEGF nano antibody pharmaceutical composition has no cytotoxicity at low dose, has only slight toxicity at high dose, and has very high safety.
Specifically, the invention provides a VEGF nano antibody pharmaceutical composition which can effectively penetrate cell membranes and remarkably promote the cell membrane penetrability of VEGF nano antibodies; the VEGF nano antibody pharmaceutical composition can effectively permeate cancer cells and corneal cells, and the uptake effect of the VEGF nano antibody on the cells is remarkably improved along with the improvement of the concentration of the cell-penetrating peptide.
Term(s) for
As used herein, the terms "VEGF nanobody of the invention", "anti-VEGF nanobody", "VEGF nanobody" have the same meaning and are used interchangeably to refer to nanobody that specifically recognizes and binds to VEGF, including human VEGF. In a preferred embodiment, the amino acid sequence of the VEGF nanobody of the invention is shown in SEQ ID NO 4.
As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
As used herein, the terms "single domain", "VHH", "nanobody", "heavy chain antibody" (sdAb, or nanobody) have the same meaning and are used interchangeably to refer to cloning the variable region of an antibody heavy chain, constructing a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Nanobodies (VHHs) consisting of only one heavy chain variable region are typically constructed by first obtaining an antibody that naturally lacks light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially b-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a b-folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3, 2 or 1 amino acids are substituted with amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
Pharmaceutical composition
The present invention provides a pharmaceutical composition comprising (a) an anti-VEGF nanobody; (b) cell-penetrating peptides; and (c) a pharmaceutically acceptable carrier.
The active ingredients in the pharmaceutical composition refer to the anti-VEGF nano antibody and the cell-penetrating peptide, and the cell-penetrating peptide can effectively bring the VEGF nano antibody into cells and can be used for treating VEGF-related cancers and eye diseases.
"safe and effective amount" means: the amount of active ingredient is sufficient to significantly ameliorate the condition or symptom without causing serious side effects. When the pharmaceutical composition of the present invention is used, the total daily dose to be applied in most cases should be lower (or equal to or slightly larger than) the daily usual dose for each individual drug, and of course, the effective dose of the active ingredient to be used may vary depending on the mode of administration and the severity of the disease to be treated, etc.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. Such vectors include (but are not limited to): saline, buffers, dextrose, water, glycerol, ethanol, sugar alcohols, surfactants, powders, pH adjusters, bacteriostats, preservatives, thickeners, stabilizers, nutrients, and combinations thereof.
The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization.
In the present invention, the pharmaceutical composition may be administered to the eye by subretinal or intravitreal injection. One preferred dosage form is an injection.
The pharmaceutical compositions of the invention may also be used with other therapeutic agents, such as therapeutic agents for the treatment of cancer or ocular diseases.
Applications of
As described above, the nanobody pharmaceutical composition of the present invention has wide biological and clinical application values, and its applications relate to many fields such as diagnosis and treatment of VEGF-related diseases, basic medical research, biological research, etc. One preferred application is for clinical diagnosis and targeted therapy against VEGF, including VEGF-related cancers and ocular diseases.
The present invention also provides a method for treating a VEGF-related disease or disorder with the pharmaceutical composition of the present invention, comprising administering to a subject in need thereof an active ingredient (a) an anti-VEGF nanobody and an active ingredient (b) a membrane-penetrating peptide, or administering the pharmaceutical composition of the first aspect of the present invention.
When the two active ingredients of the present invention are used for the above-mentioned purpose, they may be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and may be orally administered in the form of: tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, from about 0.05 to 5% suspending agent), syrups (containing, for example, from about 10 to 50% sugar), and elixirs (containing, for example, from about 20 to 50% ethanol), or may be administered parenterally in the form of sterile injectable solutions or suspensions (containing from about 0.05 to 5% suspending agent in an isotonic medium). For example, these pharmaceutical preparations may contain from about 0.01% to about 99%, more preferably from about 0.1% to about 90%, by weight of the active ingredient in admixture with a carrier.
The two active ingredients or pharmaceutical compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration. Preferred routes of administration include oral, intraocular or intravenous administration.
The main advantages of the invention include:
(a) the VEGF nano antibody medicine composition can effectively penetrate cell membranes and can remarkably promote the cell membrane penetrability of the VEGF nano antibody.
(b) The VEGF nano antibody pharmaceutical composition can effectively permeate cancer cells and corneal cells, and the uptake effect of the VEGF nano antibody on the cells is remarkably improved along with the improvement of the concentration of the cell-penetrating peptide.
(c) The VEGF nano antibody pharmaceutical composition has no cytotoxicity at low dose and only slight toxicity at high dose.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
Universal material
The transmembrane peptide used in the examples was prepared from Sgosaprost F, Deronde B M, Sarapas J M, et al.design microorganisms of Membrane Active Proteins [ J ]. Accounts of Chemical Research,2013,46(12):2977-87, and mutant thereof, reference of Chinese patent No. 101355970A, Sgosaprost F, Deronde B M, Sarapas J M, et al.design microorganisms of Membrane Active Proteins [ J ]. Accounts of Chemical Research,2013,46(12):2977-87, and Chinese patent No. CN 108976288A.
The VEGF nano antibody is a nano antibody which is independently developed by the applicant and is combined with VEGF, and the amino acid sequence is shown as SEQ ID NO. 4. The monoclonal antibody Eylea used as a control in the examples is a conventional monoclonal antibody that binds VEGF in the art, purchased from Bayer (China) Inc.
Example 1 transmembrane effect of transmembrane peptide Pennetratin and its mutant binding VEGF Nanobody
Digesting malignant melanoma cells A375, centrifuging after complete medium neutralization to remove supernatant, resuspending the cells with fresh medium, inoculating to a 6-well plate, culturing for 24h, wherein 5E5 cells are in each well; mixing three cell-penetrating peptides, namely Penetrin-WT (the amino acid sequence is shown as SEQ ID NO: 5), Penetrin-Mut 1 (the amino acid sequence is shown as SEQ ID NO: 6) and Penetrin-Mut 2 (the amino acid sequence is shown as SEQ ID NO: 7) with a VEGF nano antibody (the amino acid sequence is shown as SEQ ID NO: 4) in advance, and standing for 1 day at room temperature; discarding cell culture supernatant on the next day, washing with DMEM blank medium for 2 times, adding DMEM medium containing cell-penetrating peptide/antibody mixture (the added DMEM medium contains 9uM nano antibody, and the molar ratio of each cell-penetrating peptide to the antibody is 3:1), and continuing to culture for 4 h; washing with 10mM PBS containing 0.02mg/ml heparin sodium for 2 times, discarding supernatant, adding pancreatin-digested cells, collecting cells, transferring to 96-well plate, adding fixative, and standing at room temperature for 20 min; then washing the cells with PBS, adding methanol and incubating for 10 min; washing cells for 2 times with PBS, adding diluted biotinylated goat anti-VEGF polyclonal antibody (diluted 1:500 for use), incubating at room temperature for 20min, washing cells once with PBS, and adding diluted SA-PE fluorescent antibody; after washing the cells with PBS, PE signal was detected with flow cytometry.
The result is shown in figure 1, the Pentratin-Mut 2 can effectively penetrate cell membranes after being mixed with the VEGF nano antibody, and the VEGF nano antibody is brought into cells, and the membrane penetrating effect of the VEGF nano antibody is obviously superior to that of the wild Pentratin-WT and the mutant Pentratin-Mut 1.
Example 2 permeation Effect of different VEGF antibodies with a mixture of cell-penetrating peptides in different cell types
Respectively digesting five cells of malignant melanoma cells A375, colon adenocarcinoma cells HT29, lung cancer cells H1299, breast cancer cells MCF7 and corneal epithelial cells HCEC, centrifuging after complete medium neutralization, removing supernatant, resuspending the cells with fresh medium, inoculating the cells to a 6-well plate, and culturing for 24H at 5E5/mL of each cell; mixing the Pennetratin-Mut 2 with the VEGF nano antibody and the commercially available monoclonal antibody Eylea according to different molar ratios in advance, and standing at room temperature for 1 day; discarding cell culture supernatant on the next day, washing with DMEM blank medium for 2 times, adding DMEM medium containing cell-penetrating peptide/antibody mixture (the added DMEM medium contains 9uM antibody, and the molar ratio of the cell-penetrating peptide to the antibody is 1:1, 2:1 or 3:1), and continuously culturing for 4 h; washing with 10mM PBS containing 0.02mg/ml heparin sodium for 2 times, removing supernatant, adding pancreatin digested cells, collecting cells, transferring to 96-well plate, adding fixative, and standing at room temperature for 20 min; then washing the cells with PBS, adding methanol and incubating for 10 min; washing cells with PBS for 2 times, adding diluted biotinylated goat anti-VEGF polyclonal antibody (diluted 1: 500) to the VEGF nano-antibody group, incubating at room temperature for 20min, washing cells with PBS once, and adding diluted SA-PE fluorescent antibody; for the commercially available monoclonal antibody Eylea group, add 1: 200 diluted Goat anti human IgG-PE antibody; after washing the cells with PBS, PE signal was detected by flow cytometry.
The results are shown in fig. 2A and fig. 2B, after the VEGF nanobody is mixed with the pennetrin-Mut 2, the infiltration and transmembrane effect on various cells is remarkably better than that of the mixture of the commercially available monoclonal antibody Eylea and the pennetrin-Mut 2; and with the increase of the concentration of the Pennetratin-Mut 2, the uptake effect of the VEGF nano antibody on various types of cells is obviously improved.
Example 3 toxicity of VEGF Nanobodies and transmembrane peptide mixtures on different types of cells
Respectively digesting five cells of malignant melanoma cell A375, colon adenocarcinoma cell HT29, lung cancer cell H1299, breast cancer cell MCF7 and corneal epithelial cell HCEC, completely neutralizing the culture medium, centrifuging to remove supernatant, suspending the cells with fresh culture medium, spreading the suspension in 96-well plate at 37 deg.C and 5% CO at 2E 4/well, and culturing2Culturing for 24 h; the supernatant was discarded, and 100 uL/well of each of the supernatant was added with DMEM medium containing VEGF nanobody, Pentratin-Mut 2, Pentratin-Mut 3 (amino acid sequence is shown in SEQ ID NO: 8), Pentratin-Mut 2/VEGF nanobody mixture (3:1), Pentratin-Mut 3/VEGF nanobody mixture (3:1) (the order of VEGF nanobody and transmembrane peptide was adjusted according to the data shown in FIG. 3, please re-nucleate), and the culture was continued for 4 hours at 37 ℃ with 5% CO2(ii) a Discarding the supernatant, washing the cells once with the complete culture medium, adding 100 uL/hole of new complete culture medium, and continuing to culture for 24 h; CCK 810 uL was added to each well and incubated at 37 ℃ for 4h, with OD450 being read by a microplate reader.
As shown in FIGS. 3A and 3B, the low dose group (9. mu.M) of VEGF nanobody, Pentratin-Mut 2, Pentratin-Mut 3, Pentratin-Mut 2/VEGF nanobody mixture (3:1), Pentratin-Mut 3/VEGF nanobody mixture (3:1) had substantially no toxic side effects on five types of cells; while the VEGF nanobody/Pennetrat-Mut 2 mixture in the high dose group (90. mu.M) showed slight toxic effects on five cells, the VEGF nanobody/Pennetrat-Mut 3 mixture in the high dose group (90. mu.M) showed significant toxic effects on five cells.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Claims (10)

1. A pharmaceutical composition, comprising:
(a) anti-VEGF nanobodies;
(b) cell-penetrating peptides; and
(c) a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1, wherein the Complementarity Determining Regions (CDRs) of the anti-VEGF nanobody include CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3.
3. The pharmaceutical composition of claim 1, wherein the amino acid sequence of the anti-VEGF nanobody is shown in SEQ ID No. 4.
4. The pharmaceutical composition of claim 1, wherein the membrane-penetrating peptide comprises Penetratin and derivatives thereof.
5. The pharmaceutical composition of claim 1, wherein the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO 7, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 8.
6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises:
(a) the VEGF resisting nano antibody with the amino acid sequence shown as SEQ ID NO. 4;
(b) a cell-penetrating peptide with an amino acid sequence shown as SEQ ID NO. 7; and
(c) a pharmaceutically acceptable carrier.
7. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in a dosage form comprising an injection, an eye drop, an ophthalmic gel, or an ophthalmic ointment.
8. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of a VEGF-related disease or disorder.
9. A process for preparing a pharmaceutical composition according to claim 1, comprising the steps of:
(i) respectively providing an anti-VEGF nano antibody and a cell-penetrating peptide,
(ii) physically mixing the anti-VEGF nanobody with a cell-penetrating peptide to obtain a mixture, an
(iii) Mixing said mixture with a pharmaceutically acceptable carrier under conditions suitable for the preparation of a medicament, thereby producing said pharmaceutical composition.
10. A kit, comprising:
(i) a first container, and an active ingredient (a) anti-VEGF nanobody, or a drug containing the active ingredient (a), contained in the first container;
(ii) a second container, and an active ingredient (b) a cell-penetrating peptide or a drug containing the active ingredient (b) contained in the second container; and
(iii) instructions for the combined administration of active ingredient (a) and active ingredient (b) for the treatment of a VEGF-related disease or disorder are described.
CN202011325582.2A 2020-11-23 2020-11-23 Pharmaceutical composition containing VEGF nano antibody and application thereof Pending CN114522246A (en)

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CN110452297A (en) * 2019-09-03 2019-11-15 上海洛启生物医药技术有限公司 Anti-vegf single domain antibody and its application
CN110511273A (en) * 2019-07-12 2019-11-29 福州市皮肤病防治院 A kind of preparation method and applications of high efficiency cell transmembrane polypeptide
WO2020224475A1 (en) * 2019-05-05 2020-11-12 复旦大学 Drug delivery vector and pharmaceutical formulation using same

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Publication number Priority date Publication date Assignee Title
CN108976288A (en) * 2017-06-05 2018-12-11 复旦大学 Lipophilic derivatives based on wild type cell-penetrating peptide penetratin
WO2020224475A1 (en) * 2019-05-05 2020-11-12 复旦大学 Drug delivery vector and pharmaceutical formulation using same
CN110511273A (en) * 2019-07-12 2019-11-29 福州市皮肤病防治院 A kind of preparation method and applications of high efficiency cell transmembrane polypeptide
CN110452297A (en) * 2019-09-03 2019-11-15 上海洛启生物医药技术有限公司 Anti-vegf single domain antibody and its application

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