CN110790829A - Application of antibody prepared by using pHLIP extracellular segment as antigen in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses an application of an antibody prepared by using a pHLIP extracellular segment as an antigen in preparation of an anti-tumor drug. The research of the invention finds that the extracellular segment of the improved low-pH insertion peptide can be used as an antigen to prepare an antibody, and the antibody can be used for treating tumors.

Description

Application of antibody prepared by using pHLIP extracellular segment as antigen in preparation of antitumor drugs

This application is a divisional application of the following patent applications: application No. 2018114594499, filing date 2018.11.30, entitled "an improved low pH insertion peptide".

Technical Field

The invention belongs to the field of biomedicine, and relates to application of an antibody prepared by using a pHLIP extracellular segment as an antigen in preparation of an antitumor drug.

Background

Chemotherapy has been of great importance in the treatment of tumors over the last 10 years, and has also received much attention. However, the conventional antitumor drugs still have many limitations, such as that they cannot distinguish normal tissues from tumor tissues, so that the treatment efficiency is very low, and even more, they cause fatal adverse reactions. Therefore, the improvement of selectivity becomes the key for the research and development of the antitumor drugs. The targeted drug delivery system can specifically deliver the antitumor drug to tumor tissues, can reduce the uptake of the antitumor drug in normal tissues, can reduce the adverse reaction and improve the clinical treatment effect. Currently, targeted drug delivery systems are widely varied and some have been used in clinical therapy. However, the same receptors are expressed in normal tissues and they also recognize the targeting ligand, only at a low level, which significantly limits its targeting efficiency and therapeutic efficacy.

The greatest difference between tumor tissue and normal tissue is that the extracellular environment is acidic. In recent years, anti-tumor drugs targeting the acidic microenvironment of tumor tissues have been rapidly developed. Because of the high uptake of glucose by tumor cells, glucose is glycolyzed into lactic acid under the anaerobic condition to form an acidic environment; on the other hand, abnormal blood vessels of the tumor cause insufficient oxygen supply of the tumor, and the growth of transformation of tumor cells is out of control to cause oxygen deficiency and metabolic disorder so as to increase anaerobic metabolism; the tumor cells adapt to a hypoxic environment and an acidic environment after lactic acid is generated by corresponding glycolysis by up-regulating a hypoxia inducible factor, and finally the pH value of a tumor tissue microenvironment is 5.7-7.0, which is obviously lower than the pH value of a normal tissue 7.4. The acidic microenvironment is a very effective target for improving the selectivity of the antitumor drugs.

pHLIP is a water-soluble polypeptide capable of being inserted into a cell bilayer lipid membrane to form a stable transmembrane α helix.peptide folding and membrane insertion are driven by a neutral or basic (pH >7.4) pH down to weak acidity (pH 7.0-6.5 or lower). pHLIP has three main morphologies, no structural water soluble form I at neutral pH, no structural water soluble form II at neutral pH and bound to the surface of the cell membrane, state II at neutral pH and inserted at acidic pH and passed through the cell membrane at α helix III. the poor property of membrane peptides due to the ease of aggregation is the property of membrane peptides, pHLIP also has a tendency to aggregate as membrane peptides, especially at high and/or low pH, in aqueous solutions at neutral pH, the concentration of monomers present is less than that of membrane peptides which are found in the aqueous solution, and the protein molecules are found to be able to form a stable protein, and the protein molecule which is not found to be able to enter the cell cytoplasm as a membrane, and the protein molecule is a protein capable of being able to form a stable protein which is a protein capable of transporting a protein to the protein.

With the progress of research on pHLIP, it was found that the application of wild-type pHLIP is limited by some key factors, such as slow clearance in vivo and influence of charges on the membrane insertion process by the carboxyl end of the wild-type pHLIP, researchers have attempted to design pHLIP derivatives with better performance by adjusting the amino acid sequence of pHLIP.at present, pHLIP sequence adjustment mainly involves ① excision or inversion of the membrane insertion end of the wild-type pHLIP sequence, ② replacement of part or all of the aspartic acid residues with glutamic acid residues, positively charged lysine residues, or protonated non-standard amino acid residues (γ -carboxyl acid, α -aminooxalic acid). the pHLIP derivatives produced by sequence adjustment, such as pHLIP variant 3 (excision of the membrane insertion end), can reduce the charges carried by pHLIP, accelerate the entrance of pHLIP into the membrane forming helix of cells, and improve the tumor targeting property thereof.pHLIP variant 7, while maintaining good targeting property, accelerates the elimination rate in blood, and facilitates the in vivo delivery of drugs.

Disclosure of Invention

An object of the present invention is to provide an improved low pH insertion peptide and a method for preparing the same.

It is a further object of the present invention to provide compositions comprising the improved low pH insertion peptides described above for use in the treatment, diagnosis or identification of diseases.

It is a further object of the present invention to provide an antibody prepared from the above improved low pH insertion peptide as an antigen.

The fourth purpose of the invention is to provide the application of the antibody in preparing anti-tumor drugs.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an improved low-pH insertion peptide, the sequence of which contains the following sequence: the extracellular segment of the WT low pH insertion peptide or a variant thereof is repeated once, twice or more.

Preferably, the variant of the WT low pH insertion peptide comprises Var1-Var 16.

The sequence of the WT low pH insertion peptide or variant thereof is as follows:

WT:ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT(SEQ ID NO.2)；

Var1:ACEDQNPYWARYADWLFTTPLLLLDLALLVDG(SEQ ID NO.3)；

Var2:ACEDQNPYWRAYADLFTPLTLLDLLALWDG(SEQ ID NO.4)；

Var3:ACDDQNPWRAYLDLLFPTDTLLLDLLW(SEQ ID NO.5)；

Var4:ACEEQNPWRAYLELLFPTETLLLELLW(SEQ ID NO.6)；

Var5:ACDDQNPWARYLDWLFPTDTLLLDL(SEQ ID NO.7)；

Var6:CDNNNPWRAYLDLLFPTDTLLLDW(SEQ ID NO.8)；

Var7:ACEEQNPWARYLEWLFPTETLLLEL(SEQ ID NO.9)；

Var8:CEEQQPWAQYLELLFPTETLLLEW(SEQ ID NO.10)；

Var9:CEEQQPWRAYLELLFPTETLLLEW(SEQ ID NO.11)；

Var10:ACEDQNPWARYADWLFPTTLLLLD(SEQ ID NO.12)；

Var11:ACEEQNPWARYAEWLFPTTLLLLE(SEQ ID NO.13)；

Var12:ACEDQNPWARYADLLFPTTLAW(SEQ ID NO.14)；

Var13:ACEEQNPWARYAELLFPTTLAW(SEQ ID NO.15)；

Var14:TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA(SEQ ID NO.16)；

Var15:CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET(SEQ ID NO.17)；

Var16:CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET(SEQ ID NO.18)；

the part of the sequence marked with a transverse line is the extracellular segment sequence of the low pH insertion peptide. Var1-Var16 are all variants of WT.

The polypeptide sequence obtained by repeating the extracellular segment of the low-pH insertion peptide with the sequence of SEQ ID NO.2-18 once, twice or more than twice comprises:

(extracellular region)_n+ Linker + SEQ ID No.2-18, wherein n ═ 1, 2, 3, 4.

Linker sequences useful in the present invention may be (GGGS) m, where m ═ 1, 2, 3, 4.

In a specific embodiment of the present invention, the sequence of the modified low pH insertion peptide is obtained by repeating once the extracellular segment of Var7 having the sequence of SEQ id No.9, the sequence is:

ACEEQNPGGGSACEEQNPWARYLEWLFPTETLLLEL(SEQ ID NO.1)。

in the specific embodiment of the present invention, although it is demonstrated that the sequence obtained after repeating the extracellular domain of the low pH insertion peptide once has more beneficial effect than the original sequence in the case of Var7, the skilled person would directly and meaninglessly conclude that the sequence obtained after repeating the extracellular domain once can also have more beneficial effect than the original sequence for other WT variants according to the research results of the present invention, and because the experimental results of the present invention show the common advantages of the extracellular domain of the low pH insertion peptide, the above WT, and the WT variants including Var1-Var16, are included in the protection scope of the present invention.

The present invention provides a composition comprising the improved low pH insertion peptide described above.

Further, the composition also comprises a functional body, wherein the functional body comprises a therapeutic agent, a diagnostic agent and a marker molecule.

The functional entity may be linked to the modified low pH insertion peptide as described above either at the N-terminus or at the C-terminus. Specifically, if the therapeutic agent exerts a therapeutic effect via a molecule on the cell surface, the therapeutic agent needs to be linked to the N-terminus of the low-pH insertion peptide, and if the therapeutic agent exerts a therapeutic effect via a molecule inside the cell, the therapeutic agent needs to be linked to the C-terminus of the low-pH insertion peptide; the diagnostic agent is used for displaying the existence of pathological states of diseases, and can be connected to the N end to be displayed on the cell surface or connected to the C end to be displayed in cytoplasm; the marker molecule is used to increase the expression of the marker molecule in cells that do not contain the marker molecule on the cell membrane surface, and thus, in general, the marker molecule is linked to the N-terminus of the low pH insertion peptide.

Further, the therapeutic agents include, but are not limited to, antibody drugs, small molecule drugs, antibiotics, polypeptides, peptide nucleic acids, nanoparticles, liposomes.

The antibody drug may be one directed against any tumor molecule, as long as it is capable of treating the tumor. Antibody drugs include: molecule-targeted monoclonal antibody drugs, targeted antibody conjugate drugs, bispecific antibody drugs, targeted immune checkpoint drugs, and the like. Examples of such antibody drugs include, but are not limited to: rituximab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, dinomumab, ipilimumab, bentuximab, pertuzumab, ado-trastuzumab, atrozumab, ramucirumab, pembrolizumab, bonatuzumab, nivolumab, darumamab, dinumuzumab, rituximab, elotuzumab, alemtuzumab, avizumab, denosumab, Necitumumab, Atezolizumab, and Atezolizumab.

Further, the antibiotics include antitumor antibiotics, which are chemical substances produced by metabolism of microorganisms and having antitumor activity. Antitumor antibiotics that may be used in the present invention include C1027, mitomycin, doxorubicin, CC-1065, adozelesin, ducarmycins, gilvus mycin, tetracyclines, cinnamamides, MMI-166, batimastat, green tea polyphenols, salvianolic acid A, C3368-A, C3368-B, emodin, tricyclic pyrones, gel danamycins, 17AAG, paclitaxel, epothilone A, epothilone B, calicheamicin, lidamycin.

Further, the small molecule drugs are usually signal transduction inhibitors, which can specifically block signal transduction pathways necessary for tumor growth and proliferation, and thus achieve therapeutic purposes, and examples of the small molecule drugs include but are not limited to: imatinib, nilotinib, dasatinib, everolimus, erlotinib, sunitinib, ibrutinib, sorafenib, crizotinib, pazopanib, gefitinib, carfilzomib, tofacitinib, acitinib, regorafenib, vemurafenib, sirolimus, ponatinib, lenvatinib, olapanapanib, aflibercept, ceritinib, romidepsin, elotinib, belinostat, bosutinib, vandetanib, cabozantinib, panobinostat, afatinib, palifermin, trametinib, daraflavimus, lapatinib, vorinostat, venococclaxax, gleevec, iressa.

Further, examples of the polypeptide include, but are not limited to, toxins, cyclic peptides, microtubule inhibitors, protease activated receptors. Examples of toxins are amanitines, examples of cyclic peptides are phalloidin, examples of microtubule inhibiting factors are monomethyl auristatin e (mmae), examples of protease activated receptors are P1 AP.

Further, the peptide nucleic acid includes an anti-miR (antisense nucleic acid) oligonucleotide peptide.

Further, the nanoparticles comprise chitosan targeting nanoparticles, long-circulating nanoparticles, polylactic acid nanoparticles, solid lipid nanoparticles, gold nanoparticles, doxorubicin-loaded mesoporous silicon nanoparticles and superparamagnetic iron oxide nanoparticles.

Further, the liposome comprises phospholipid and cholesterol.

The phospholipids described herein include, but are not limited to, soybean lecithin (SPC), polyethylene glycol 1000 vitamin E succinate (TPGS), dimyristoyl lecithin (DMPC), Dilauroyl Lecithin (DLPC), distearoyl lecithin (DPPC), dipalmitoyl lecithin (DPPC), distearoyl lecithin (DSPC), 1-myristoyl-2-palmitoyl lecithin (MPPC), 1-palmitoyl-2-myristoyl lecithin (PMPC), 1-palmitoyl-2-stearoyl lecithin (PSPC), 1-stearoyl-2-palmitoyl lecithin (SPPC), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), dioleoyl lecithin (DOPC), Dioleoylphosphatidylethanolamine (DOPE), dilauroyl phosphatidylglycerol (DLPG), dipalmitoyl glycerol (DPPG), Distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidyl ethanolamine (DMPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphatidyl serine (DMPS), dipalmitoyl phosphatidyl serine (DPPS), brain Phosphatidyl Serine (PS), Brain Sphingomyelin (BSP), dipalmitoyl sphingomyelin (DPSP), distearoyl sphingomyelin (DSSP), distearoyl phosphatidyl ethanolamine (DSPE), with preference given to any one or a mixture of any of: soybean lecithin (SPC), distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phosphatidylethanolamine (DOPE).

Further, the diagnostic agent includes a fluorescent dye. Fluorescent dyes include, but are not limited to, Alexa750, Alexa546, Alexa647, Cy5.5, DyLight 680, DyLight 6804 PEG-conjugate (DyP680) and,680RD(IR680)、

800CW (IR800), indocyanine green ICG, PE, Percy-Cy5.5, FITC, APC, Cy7, FITC, GFP, Alexa Fluar488, Bidipy, Fluo-3, Propidium Iodid (PI), PerCP, PE-Cy5, PE-TesesRed, 7-AAD, PE-Cy7, PE-Alexa Fluur 750, Alexa Fluor660, Alexa Fluor700, APC-Cy7, APC-Alexa Fluur 750, Hoechsr33342-Blue, DAPI, EChsr 33Red, aric Blue, Cascade Blue 342, Hoexa Fluur 405, paridic orange.

Further, the marker molecule comprises a tumor surface antigen or a functional domain thereof, and the tumor surface antigen generally refers to an antigen substance newly appeared or overexpressed on the cell surface during the process of tumor occurrence and development.

Examples of tumor surface antigens include, but are not limited to, ER, PR, P53, EGFR, IGFR, Her2, CD20, CD25, CD117, CD34, CD138, CD33, VEGFR, BCMA, Mesothelin, CEA, PSCA, MUC1, EpCAM, S100, CD22, CD19, CD70, CD30, ALK, RANK, GPC2, GPC3, Her3, EGFRvIII, GD2, PD-L1, PD-L2.

The present invention also provides a novel antigen, the novel antigen sequence comprises the extracellular segment sequence of the improved low pH insertion peptide or the variant sequence thereof.

In a particular embodiment of the invention, the neoantigen sequence is shown in SEQ ID NO. 19.

The novel antigen of the invention has the following functions: (1) antigenicity; (2) after being linked with carrier protein, the carrier protein can be used as immunogen to stimulate animals to produce specific antibody.

The preparation method of the novel antigen can use a chemical synthesis method: antigen was synthesized by a solid phase method using an automatic polypeptide synthesizer.

The invention also provides a nucleic acid molecule encoding the novel antigen as described above.

The present invention also provides a recombinant vector comprising an empty vector and a target gene inserted into the empty vector, wherein the target gene is the nucleic acid molecule as described above.

In the present invention, the "empty vector" (or "vector") may be any vector known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses, and the like. The empty vector may include a variety of commonly used detection markers (e.g., fluorescent markers, antibiotic markers, etc. reporter genes) and enzyme cleavage sites. The construction of the recombinant vector can adopt various endonucleases of multiple cloning sites of the empty vector to carry out enzyme digestion to obtain linear plasmids, and the linear plasmids are connected with gene segments cut by the same endonucleases to obtain the recombinant plasmids.

The present invention also provides a recombinant host cell comprising the recombinant vector as described above.

The recombinant vector can be transformed, transduced or transfected into a host cell by methods conventional in the art, such as calcium chloride chemical transformation, high voltage shock transformation, preferably shock transformation; the host cell can be prokaryotic cell or eukaryotic cell, preferably Escherichia coli, Bacillus subtilis, yeast (such as Pichia pastoris) or various animal and plant cells, more preferably the host cell is a genetically engineered bacterium commonly used in the art, such as Escherichia coli, Bacillus subtilis or Pichia pastoris.

The novel antigens of the present invention can be isolated and purified from recombinant host cells using methods commonly used in the art. For example, the culture medium and recombinant host cells are centrifuged, cells are disrupted by high pressure homogenization, cell debris is removed by centrifugation, and the neoantigen is purified by affinity chromatography. For the isolation and purification of the resulting neoantigen product, purity identification can be performed using methods commonly used in the art. For example, Coomassie blue method, Kjeldahl method, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, antigen-antibody method, electrophoresis (for example, sodium dodecyl sulfate polyacrylamide gel electrophoresis), sedimentation analysis, diffusion analysis, isotachy method, protein mass spectrometry, and the like.

The invention also provides a fusion protein comprising the novel antigen as described above and a protein or polypeptide linked to the novel antigen.

Further, the fusion protein comprises the novel antigen as described above and a carrier protein coupled to the novel antigen.

Carrier proteins useful in the present invention include, but are not limited to, KLH (keyhole limpet hemocyanin), Bovine Serum Albumin (BSA), ovalbumin OVA, and the like. KLH (keyhole limpet hemocyanin) is preferred because it has high immunogenicity, many binding sites, good immunological effects, is distant from the immunized animal, and is less likely to cause cross-reactions when used as a carrier protein.

The fusion protein of the present invention is immunogenic and specific, and is an immunogen that can be used to immunize animals to produce antibodies specific for the novel antigens described above.

The invention also provides a novel antibody which is prepared from the novel antigen or the fusion protein.

Preferably, the above novel antibody of the present invention is a monoclonal antibody.

The present invention also provides a pharmaceutical combination comprising the novel antibody as described above.

Further, the pharmaceutical combination also comprises the modified low pH insertion peptide.

In a specific embodiment of the invention, the novel antibody is present independently of the modified low pH insertion peptide, not linked to each other, and when administered to act simultaneously, the antibody binds to the extracellular domain of the modified low pH insertion peptide.

The monoclonal antibodies of the invention can be prepared using conventional techniques in the art, and methods commonly used in the art for preparing antibodies include:

(1) mouse/rabbit based hybridoma technology.

The method comprises the following basic steps: animal immunization, cell fusion, screening and monoclonal antibody detection of hybridoma cells, cloning of hybridoma cells, identification and preparation of monoclonal antibodies and the like.

(2) Antibody screening techniques based on phage antibody display libraries.

① separating B lymphocyte from peripheral blood or spleen and lymph node, extracting mRNA and reverse transcribing to cDNA, ② applying antibody light chain and heavy chain primer to amplify different Ig gene segments via PCR, ③ constructing phage vector, ④ expressing vector to transform bacteria to construct complete antibody library, and finally screening out antigen specific antibody clone.

(3) Screening techniques based on monoclonal antibody libraries.

The low pH insertion peptides of the invention, as described above, may be prepared using conventional techniques in the art, such synthetic techniques including: solid phase synthesis and liquid phase synthesis.

The principle of solid phase synthesis consists in: the carboxyl end of amino acid is fixed on insoluble resin through proper connecting molecule, and then amino acid is condensed on the resin in sequence through removing amino protecting group, and the peptide chain is prolonged until the required polypeptide is obtained. Finally, the side chain protecting groups are removed with an appropriate reagent and the product cleaved from the resin. Compared with a liquid phase, the solid phase synthesis of the polypeptide has the advantages that: (1) the purification purpose can be achieved only by simply filtering and washing the resin in each step of reaction, the difficulty that the product in each step of a classical liquid phase synthesis method needs to be purified is overcome, and the operation is time-saving and labor-saving; (2) the soluble reagent can be excessive to complete the reaction and obtain high yield, and the excessive reagent can be simply washed by a solvent and filtered to remove; (3) all reactions can be carried out in one vessel, thus avoiding procedures and losses of transfer of reaction intermediates; (4) if proper connecting molecules and cracking conditions are selected, the high molecular resin can be recycled.

The strategies for solid-phase synthesis of the polypeptide comprise a Boc solid-phase method and an Fmoc solid-phase method. In a specific embodiment of the present invention, the present invention uses the Fmoc solid phase method.

The invention provides the use of an improved low pH insertion peptide as hereinbefore described for the preparation of a novel antigen as hereinbefore described.

The invention provides the use of an improved low pH insertion peptide as hereinbefore described for the preparation of a fusion protein as hereinbefore described.

The invention provides the use of an improved low pH insertion peptide as hereinbefore described in the preparation of a composition or novel antibody as hereinbefore described. Specifically, the method comprises the following steps:

the invention provides application of the improved low-pH insertion peptide in preparing a tumor drug targeted delivery system. The tumor therapeutic agent is connected with the low-pH insertion peptide, and the tumor therapeutic agent is delivered to the tumor tissue in a targeted mode and is specifically killed and killed by depending on the targeting of the low-pH insertion peptide to the micro-acid environment.

The invention provides the application of the improved low-pH insertion peptide in preparing a tumor diagnosis tool. The tumor diagnostic agent used in the above is linked with the low pH insertion peptide, and the targeting of the low pH insertion peptide to the microacid environment is relied on to deliver the tumor diagnostic agent to the tumor tissue in a targeted manner and to mark the presence of the tumor tissue, thereby judging whether the subject has the tumor.

The invention provides the application of the improved low-pH insertion peptide in the preparation of a tumor identification system. The tumor surface antigen is connected with the low pH insertion peptide, and the tumor surface antigen is delivered in a targeted manner and stays on the surface of tumor cells depending on the targeting property of the low pH insertion peptide to the micro-acid environment, so that the tumor cells are marked by the tumor surface antigen, and the tumor drug aiming at the specific antigen can kill the tumor. Taking HER2 as an example, trastuzumab only has a therapeutic effect on HER2 positive breast cancer patients, and HER2 connected by low-pH insertion peptide is targeted and positioned on the surface of breast cancer cells of HER2 negative breast cancer patients, so that trastuzumab can also play a therapeutic effect on HER2 negative breast cancer patients, and the application range of trastuzumab is expanded.

The invention also provides the use of the improved low pH insertion peptide as described above for the preparation of a CAR-T sequence. The antibody obtained by using the improved low-pH insertion peptide as an antigen screening can be used as a brand-new Scfv sequence to design a CAR-T sequence.

The invention also provides the use of the novel antigen as described above for the preparation of the fusion protein as described above, or the novel antibody as described above.

The invention also provides the application of the novel antibody in preparing anti-tumor drugs.

The invention also provides the use of the novel antibodies described above for the preparation of CAR-T sequences.

The invention also provides the use of the novel antibodies described hereinbefore in the preparation of a composition as described hereinbefore.

The invention also provides the use of the novel antibodies described above for the preparation of the pharmaceutical combinations described above.

The term "CAR-T" is used herein to refer collectively to the Chimeric Antigen Receptor T-Cell Immunotherapy. Based on the characteristics of the tumor microenvironment, scientists have optimized a series of CART sequences with completely different affinities for antigen at different pH values, thereby activating at different pH values.

The term "targeted antibody conjugated drug" or immunoconjugate is used herein. The immune conjugate molecule consists of a monoclonal antibody and a warhead drug. There are three main classes of substances that can be used as "warheads", namely radionuclides, drugs and toxins; and the monoclonal antibody is connected with the monoclonal antibody to respectively form a radioimmunoconjugate, a chemoimmunoconjugate and an immunotoxin.

The term "bispecific antibody drug" as used herein refers to an antibody that binds to two epitopes simultaneously, and diabodies can be divided into two types, i.e., T cell recruiting, comprising a tumor cell target-T cell recruiting site, which accounts for a majority of the proportion of diabodies, wherein T cell recruiting site refers to CD3(T cells), CD16 target (NK cells), and target is normally located on tumor cells; in addition, double antibodies may bind to double target sites (such as VEGF-PDGF, VEGF-Ang2) and inhibit 2 signaling pathways, thereby reducing the possibility of drug resistance.

The term "peptide nucleic acid" (PNA) as used herein is an artificially synthesized DNA or RNA analog having a backbone composed of repeating N-2 (aminoethyl) -glycine (N (2-aminoethyl) glycine) units, and a base and the backbone are linked by methylene carbonyl bonds. Because the PNA has no phosphate group on DNA or RNA, the PNA has neutral charge, no electric repulsion between the PNA and DNA, strong base pairing specificity, high thermal stability and difficult hydrolysis by protease or nuclease, and can form a stable complex when the PNA is combined with the DNA and the RNA, so the PNA has wide application prospect in the fields of biological research and clinical medicine.

The term "monoclonal antibody" is used herein to refer to a highly homogeneous antibody, produced by a single B cell clone, directed against only a particular epitope of an antigen, and is referred to as a monoclonal antibody.

The polypeptide sequences of the present invention are listed in order from N-terminus to C-terminus.

The invention has the advantages and beneficial effects that:

the invention carries out sequence improvement on the basis of known low-pH insertion peptide, and the improved polypeptide has stronger selectivity in an acidic tumor tissue microenvironment and is maintained in vivo for a longer time.

Drawings

FIG. 1 shows the fluorescence map of the localization of the low pH insertion peptide var7 on cells;

FIG. 2 shows the fluorescence map of the localization of the modified low pH insertion peptide p-var7 on cells;

figure 3 shows a graph demonstrating the localization of low pH insertion peptides in animals using in vivo imaging techniques, wherein a: 24 h; b: 48 h; c: 72 h; d: 72h, dissecting tumor tissues obtained from the mice;

FIG. 4 shows a graph of the effect of 1G12 on tumor growth;

FIG. 5 shows a statistical plot of the effect of 1G12 on tumor weight;

FIG. 6 shows a statistical plot of the effect of 1G12 on mouse body weight;

FIG. 7 shows a liver pathology staining map;

FIG. 8 shows a staining pattern of kidney pathology;

FIG. 9 shows a pathological staining pattern of the lung;

FIG. 10 shows a pathological staining pattern of a large intestine;

FIG. 11 shows pathological spleen staining patterns.

Detailed Description

The invention will be more readily understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to limit the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 Synthesis of modified Low pH insertion peptides

According to the sequences of SEQ ID NO.9 and SEQ ID NO.1, the synthesis is performed in sequence from carboxyl terminal to amino terminal.

(1) Attachment of the first amino acid to the resin

2-Chlorotryl Chloride Resinl g was placed in a dry clean peptide synthesis column, 8ml DCM was added and the mixture was swollen for 5min and the solvent was removed in vacuo. 2mmol Fmoc amino acid and 5mmol DIEA, respectively, were dissolved in 8ml DCM and added to the resin and reacted for 60min with gentle shaking at room temperature. The solvent was discarded in vacuo. The resin was washed 2 times with 10ml DMF for 2min each time. 10ml DCM/MeOH/DIEA (80: 15: 5) were added, the reaction was shaken gently for 10min, and the solvent was removed in vacuo. And repeating the steps once. The resin was washed 3 times with 10ml DMF for 2min each time. Vacuum stripping of the solvent, N₂And (5) drying.

(2) Determination of the coupling ratio of the first amino acid to the resin

2mg of dried Fmoc amino acid-resin was weighed accurately and placed in a cuvette, 3ml of 20% piperidine/DMF was added, the reaction was gently shaken for 10min, zeroed using 20% piperidine/DMF as an air self control, and the light absorption at 290nm of the sample was measured with an ultraviolet spectrophotometer. The measurement was repeated 2 times, and the average value was taken. The coupling ratio was calculated by the following formula:

coupling ratio (mmol/g) ═ (Abs samples)/(sample weight mg × 1.75)

(3) Deprotection of Fmoc group

To the resin was added l 0ml Deprotection (DEBLOCK) reagent, mixed well and reacted for 5min with gentle shaking at room temperature. The solvent was discarded and the resin was washed 3 times with 10ml DMF for 2min each time. The resin was washed 3 times with 6ml of isopropanol each time for 5 min. The resin was washed 3 times with 6ml of hexane each time for 5 min. The solvent was discarded in vacuo. A small amount of resin sample is taken, and the content of free amino groups on the resin is rapidly determined by a ninhydrin color method (Kaiser method): washing resin 2ml with ethanol for 3 times, adding 2 drops of 5% ninhydrin, 80% phenol and KCN (2ml 0.001M KCN: 98ml piperidine), mixing well, and heating at 120 deg.C for 4-6 min. Judging the degree of deprotection reaction of the Fmoc group.

(4) Coupling reaction of the second amino acid

The second amino acid is connected by an in-situ activation method, 2mmol of Fmoc amino acid, 4.0mmol of TBTU and 4.0mmol of HOBT are taken, a minimum amount of DMF is added for dissolution, 5mmol of DIEA is added, and the mixture is added into the Fmoc group-removed resin after being fully and uniformly mixed. The reaction was gently shaken at room temperature for 60 min. The solvent was discarded in vacuo. The resin was washed 3 times with 5ml of methanol each time for 5 min. The resin was washed 3 times with 10ml DMF for 2min each time. The solvent was discarded in vacuo. A small sample of the resin was taken for ninhydrin color analysis. The coupling ratio was measured.

(5) Extension reaction of peptide chain

The Fmoc protecting group at the N-terminus of the last amino acid was removed with 10ml of DEBLOCK reagent, the resin was washed 3 times with 10ml of DMF and the solvent was removed by vacuum suction. A small sample of the resin was taken for ninhydrin color analysis. The next amino acid was coupled according to (3). And repeating the deprotection of Fmoc protecting group and the amino acid coupling reaction until the desired polypeptide chain is obtained by coupling.

(6) Peptide chain N-terminal marker Alexa647

The resin with all amino acid sequences is synthesized, the Fmoc protecting group at the N end of the amino acid is removed, and the resin is washed 3 times with 10ml of isopropanol for 5min each time. 1.38g of Alexa647, 1.6g of TBTU53 and 0.76ml of DIEA were mixed and added to the peptide-resin and reacted for 60min at room temperature with gentle shaking. The solvent was discarded in vacuo. The resin was washed 3 times with 5ml of methanol each time for 5 min. The resin was washed 3 times with 10ml DMF for 2min each time. The solvent was discarded in vacuo.

(7) Side chain deprotection of peptide chains and cleavage from resin

The resin with the synthesized complete amino acid sequence was washed with 10ml DMF and then with 6ml isopropanol 3 times for 5min each. The resin was washed 3 times with 6ml of hexane each time for 5 min. After vacuum suction of the solvent N₂Blow-drying and putting into a cracking container. Adding 25ml of cutting reagent into 1g of resin, performing cutting reaction at room temperature for 2h, shaking occasionally and mixing uniformly, filtering the resin by using a glass filter for the mixed solution after the reaction, collecting the mixed solution of the cutting reaction, and washing the resin 3 times by using TFA. The reaction mixture was transferred to a round bottom flask, washed 4 times with an equal volume of precooled ether and the precipitate collected. Drying to obtain the crude product of the synthetic polypeptide.

(8) Desalting of synthetic polypeptides

Dissolving the crude polypeptide product in distilled water. Weighing 15G of Amersham G-25 gel, swelling, then loading the gel on a column, balancing the loaded column with 50ml of distilled water, loading 3-5ml of the gel each time after balancing, eluting the gel with distilled water, detecting ultraviolet absorption at 220nm by using an ultraviolet spectrophotometer, and collecting the polypeptide according to peaks.

(9) HPLC purification of polypeptides

The polypeptide was isolated and purified by HPLC high performance chromatography using Waters Delta Prep 4000 from Waters. The column was a radial pressurized column (25X 100, 15 μm, DELTA PAKC18 packing) and the elution system was: solution A: 5% acetonitrile (0.1% TFA); and B, liquid B: 95% acetonitrile (0.08% TFA). Manually injecting samples, wherein the flow rate is 4ml/min, the linear gradient is adopted, the B liquid is increased from 5% to 50% within 45min, and then is increased to 95% B liquid within 5min for final elution, wherein the sample injection amount is 1ml each time. And detecting ultraviolet absorption at 215nrn, collecting components according to peaks, and using the components for mass spectrum detection. Collecting the components with correct molecular weight detection, and vacuum freeze-drying to obtain the required pure product for later use.

Example 2 localization of Low pH insertion peptides on tumor cells cultured in vitro

1. Cell lines

Human colorectal cancer cell line SW480 (purchased at ADCC).

2. Reagent

RPMI 1640 medium (solarbio), fetal bovine serum (Yuan Hengjin Ma.), PBS (pH 7.4) (Gibco), hydrochloric acid, alexa 647-labeled var7(var7 is standard var7, sequence: Ala-Cys-Glu-Glu-Gln-Asn-Pro-Trp-Ala-Arg-Tyr-Leu-Glu-Trp-Leu-Phe-Pro-Thr-Glu-Thr-Leu-Leu-Leu-Glu-Leu (SEQ ID NO.9)) and alexa-labeled p-var7(p-var7 is a lengthened version of extra-cell 7 of var7 repeated once in extra-cell segment, sequence: Ala-Glu-Gln-Asn-Pro-Gly-Gly-Gly-Ser-Cys-Glu-Glu-Glu-Gln-Asn-Pro-Trp-Tyr-Ala-Leu-Trp-Glu-Glu-Pro-Trp-Tyr-Leu-Leu-Trp-Glu-Glu-Pro-Leu-Trp-Leu-Leu- -Leu-Phe-Pro-Thr-Glu-Thr-Leu-Leu-Leu-Glu-Leu (SEQ ID NO.1)), alexa647 is attached to Cys at position 2 of the N-terminus of the two polypeptides.

3. Instrument for measuring the position of a moving object

Clean bench (RONGFENG), carbon dioxide incubator (Thermo), centrifuge (Thermo), laser confocal cell culture dish (20mm) (Corning), electronic pH meter (Sertolis), optical microscope (Olympus), laser confocal microscope (Nikon).

4. Experimental methods

(1) Collecting SW480 cells in logarithmic phase, discarding culture solution, washing with physiological saline twice, adding appropriate amount of 0.25% pancreatin, and digesting until cells are obtainedAnd (3) not attaching to the wall, adding a proper amount of culture solution to stop digestion, transferring the mixture into a 10ml test tube, centrifuging the mixture at 1000rpm for 5min, sucking a supernatant, adding 1ml of RPMI 1640 culture medium containing 10% fetal calf serum, and re-suspending and uniformly mixing the cells. Taking 10 mul of cell suspension from the cell, adding the cell suspension into a cell counting plate for counting, taking a certain amount of cell suspension, adding the cell suspension into a laser confocal cell culture dish, and adjusting the cell suspension to 5 x10 by using a complete culture medium⁵1ml cell system, put into cell incubator to culture overnight.

(2) The culture solution was prepared using 1mol/L hydrochloric acid and a PBS buffer solution with pH 7.4. Hydrochloric acid was added dropwise to the PBS buffer, and finally the pH of the titration buffer was 6.3. The synthesized peptides ((p-var7 and var7) were added to PBS buffer solutions with pH 6.3 and 7.4, respectively, and mixed well, and diluted in proportion until the final peptide concentration was 2.5. mu. mol/L, which was the peptide-containing PBS culture solution.

(3) After overnight incubation, SW480 cells were attached, the culture supernatant was aspirated and washed twice with PBS buffer at pH 7.4.

(4) The PBS buffer was aspirated from the cell culture dishes, and 1ml of the previously prepared PBS/peptide mixture buffer at pH 6.3 and 7.4 was added to each of the two dishes, and 1ml of the peptide-free PBS medium at pH 7.4 was added to the other control dish, and both were incubated in a 37 ℃ cell incubator for 1 hour.

(5) After the completion of the incubation, the supernatant of the peptide-containing PBS culture solution was aspirated, washed three times with each set of peptide-free PBS buffer solutions having different pH values, and then PBS buffer solution having pH 7.4 was added.

(6) The prepared cell culture dish is placed under a laser confocal microscope (647mm excitation wavelength) to observe the fluorescence expression condition of the cell membrane surface.

3. The experimental results are as follows:

as a result, as shown in FIGS. 1 and 2, both var7 and p-var7 were able to insert efficiently into the surface of SW480, a human colon cancer cell in an acidic solution environment, but the membrane insertion ability of p-var7 was lost in a neutral solution environment (FIG. 2), while var7 retained this ability (FIG. 2), indicating that p-var7 has a greater selectivity in an acidic tumor tissue microenvironment.

Example 3 localization of Low pH insertion peptides in animals

1. The experimental steps are as follows:

mouse colon cancer cells CT26 Balb/c mice were inoculated subcutaneously with tumors growing to about 1cm, injected intravenously with saline (N.S.), alexa 647-labeled p-var7, alexa 647-labeled var7, at a dose of 60 μm/100 μ L N.S., 24, 48, 72 hr in vivo imaging (excitation with Cy5.5 wavelength), and photographed in supine position, respectively. Mice were sacrificed and tumors were imaged.

2. Results of the experiment

Results as shown in fig. 3, both var7 and p-var7 were able to label tumors, but p-var7 was more powerful to label and the fluorescence intensity of var7 decayed more significantly over time, with only a small amount of label being visible in tumor tissue by 72 hours, in contrast to the stronger fluorescence label still visible at 72 hours for p-var 7. This experiment demonstrates that p-var7 is able to target tumor tissue in vivo and remain for a longer period of time. The black arrows in panel A represent tumor tissue.

Example 4 evaluation of antitumor Effect of antibody prepared with extracellular domain of p-var7 as antigen

Experimental materials: MC38 cells, purchased from ATCC; p-var7 polypeptide (molecular weight is 4095Da), which is synthesized by Beijing Huada protein research and development center, Inc., and dissolved in PBS with the concentration of 40 μ M; the extracellular segment of p-var7 (Ala-Cys-Glu-Glu-Gln-Asn-Pro-Gly-Gly-Gly-Ser-Ala-Cys-Glu-Glu-Gln-Asn-Pro, SEQ ID NO.19) was linked to KLH (synthesized by Beijing Kingsler Biotech Co., Ltd.); female C57/BL6 mice 6-8 weeks old were purchased from Witongliwa.

1. Preparing an antibody:

(1) the method comprises the following steps: and (3) immunizing Balb/c mice by using an extracellular section of p-var7 connected by KLH to prepare hybridoma, obtaining 2 monoclonal antibodies, and detecting the specific combination of the antibodies and the antigens and the antibody subtype by ELISA.

(2) As a result:

the results show that the monoclonal antibodies named 1G12 and 1G1 can specifically bind to the antigen, wherein 1G12 has higher affinity, 1G1 has lower affinity, OD values are 1.4423 and 0.4924 respectively, and the 2 strains are all IgG1 subtypes, and the prepared antibody concentration is about 0.7 mg/ml.

2. Evaluation of anti-tumor Effect of antibody

(1) Establishing a mouse colon cancer MC38 transplantation tumor model: MC38 is inoculated into C57/BL6 mouse, when the tumor diameter is 1cm, the tumor is aseptically stripped, sheared, homogenized, filtered to obtain single cell suspension, cultured and expanded in 1640 complete culture medium, and the cells are injected into the flank of C57/BL6 mouse at 2x10⁶And (3) removing oversize and undersize tumors from each cell/mouse when the diameter of the tumor is 0.8-1cm, and grouping mice with the tumors basically consistent in size. And (4) components in total: p-var7 injection group alone, 10; p-var7 combined with 1G12 injection group, 10; p-var7 combined with 1G1 injection group, 10; saline N.S was injected into groups, 10. Tumor size was measured every 3 days.

(2) The administration method comprises the following steps: p-var7 dosing: each intravenous injection was 40 μ M/100 μ l (approximately 16 μ g, referring to previous in vivo imaging results, the same dose of injection was able to observe the accumulation of fluorescence at the tumor site, but substantially subsided by the third day), starting the day after completion of the group, once a day, once every 2 days; antibody administration: intraperitoneal injection is carried out, the dosage is 5mg/kg (the molar ratio of the antibody to the p-var7 is about 1:5), the injection frequency is the same as that of the p-var7, and the injection time is 6-12 hours after the p-var7 administration until the end.

(3) As a result:

as shown in fig. 4 and 5, 1G12 was able to significantly inhibit tumor growth, the inhibition rate was 50% at 2 weeks, and 1G1 had no significant tumor-inhibiting effect; in the treatment process, the physical condition of the mouse is good, and symptoms such as activity reduction, diarrhea, weight reduction and the like do not appear, and the weight of the mouse is not changed as shown in figure 6; pathological results showed that no organic changes were observed in the organs of the liver, kidney, lung, spleen and intestine of the treated mice (FIGS. 7-11).

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Sequence listing

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Application of antibody prepared by using pHLIP extracellular segment as antigen in preparation of antitumor drugs

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Claims (10)

1. A neoantigen, wherein the neoantigen sequence is an extracellular segment sequence of an improved low pH insertion peptide; the improved low pH insertion peptide contains the following sequence: a polypeptide sequence obtained by repeating the extracellular segment of the low-pH insertion peptide or the variant thereof with the sequence of SEQ ID NO.2 once, twice or more;

preferably, the variant of the low pH insertion peptide with the sequence of SEQ ID NO.2 comprises a polypeptide with the sequence of SEQ ID NO.3-SEQ ID NO. 18.

2. The neoantigen of claim 1, wherein the sequence of the modified low pH insertion peptide from N-terminus to C-terminus is represented as follows: (extracellular domain) n + Linker + a low pH insertion peptide of SEQ ID No.2 or a variant thereof, wherein n ═ 1, 2, 3, 4 … … … …;

preferably, the Linker sequence is (GGGS) m, wherein m is 1, 2, 3, 4 … … … …;

more preferably, the Linker sequence is GGGS.

3. The neoantigen of claim 1 or 2, wherein the neoantigen has the sequence shown in SEQ ID No. 19.

4. A nucleic acid molecule encoding the neoantigen of any one of claims 1 to 3.

5. A recombinant vector comprising an empty vector and a target gene inserted into the empty vector, wherein the target gene is the nucleic acid molecule according to claim 5.

6. A recombinant host cell comprising the recombinant vector of claim 5 or the nucleic acid molecule of claim 4.

7. A fusion protein comprising the neoantigen of any one of claims 1-3 and a carrier protein coupled to the neoantigen;

preferably, the carrier protein comprises KLH, BSA, or OVA.

8. A novel antibody, which is produced from the novel antigen according to any one of claims 1 to 3 or the fusion protein according to claim 7.

9. A pharmaceutical combination comprising the novel antibody of claim 8; preferably, the pharmaceutical combination further comprises the improved low pH insertion peptide of claim 1 or 2.

10. Use of the neoantigen of any one of claims 1 to 3 or the neoantibody of claim 8, comprising the use of any one of:

(1) use of the neoantigen of any one of claims 1-3 in the preparation of the fusion protein of claim 7, or the neoantibody of claim 8;

(2) the use of the novel antibody of claim 8 for the preparation of an anti-tumor drug, or for the preparation of a CAR-T sequence;

(3) use of the novel antibodies of claim 8 for the preparation of a pharmaceutical combination according to claim 9.

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