CN110713522A - Use of extracellular domain of low pH insertion peptide as antigen - Google Patents

Abstract

The invention discloses application of an extracellular segment of a low-pH insertion peptide as an antigen. The research of the invention finds that the extracellular segment of the low-pH insertion peptide has antigenicity, and the extracellular segment of the low-pH insertion peptide is used as an antigen to prepare the antibody which can be used for treating tumors. The research result of the invention provides a new candidate drug for clinical tumor treatment.

Description

Use of extracellular domain of low pH insertion peptide as antigen

Technical Field

The invention belongs to the field of biomedicine, relates to a low-pH insertion peptide, and also relates to an application of a low-pH insertion peptide extracellular segment as an antigen.

Background

The low pH insertion peptide (pHLIP), derived from the transmembrane helix protein C of bacteriorhodopsin, is a water-soluble polypeptide that can be inserted into the lipid bilayer membrane of a cell to form a stable transmembrane alpha helix. Peptide folding and membrane insertion are driven by a neutral or basic (pH >7.4) pH drop to weakly acidic (pH 7.0-6.5 or lower). pHLIP has three main forms: form I, in which no structure is soluble in water at neutral pH, state II, in which no structure is present and binds to the cell membrane surface, state III, in which insertion and alpha-helix cross the cell membrane occurs at acidic pH. Since the poor solubility due to the tendency to agglutinate is a property of the membrane peptide, pHLIP also tends to agglutinate as a membrane peptide, and particularly, pHLIP monomer is present at a concentration of less than 30 μ g/ml in an aqueous solution at neutral pH under high concentration and/or low pH conditions, and pHLIP peptides in states II and III are all present in monomer form under low pH conditions. Many studies have shown that the decrease in peptide solubility due to structural changes results in changes in the binding ability of the peptide to the membrane and the conformation of the entire peptide. The stability of the peptide in blood is a very important property, since proteases in blood degrade the peptide consisting of the L-form amino acid within minutes. Although polypeptides composed of D-amino acids are much more stable, they are not suitable for binding to specific receptors due to their variable chirality. Because there is no specific interaction between pHLIP and the lipid bilayer, it is not surprising that pHLIP consisting of either L-or D-form has the same biophysical and tumor localization properties, and there is increasing evidence that pHLIP localization does not require any specific molecular binding events to occur. Only one significant difference is that D-pHLIP forms a left-handed helix across the membrane, while L-pHLIP forms a right-handed helix across the membrane. In contrast to cell penetrating peptides, pHLIP remains in the cell membrane after insertion into the cell membrane, entering the cytoplasm on one end and the extracellular space on the other. Thus, the peptide has a dual delivery capacity: one is its ability to deliver cargo molecules to the cell surface and the other is its ability to inject or release membrane-impermeable cargo molecules into the cytoplasm. To achieve the first capability, cargo molecules can be attached to the N-terminus of pHLIP, such cargo molecules having a wide range of polarities and sizes, one example of an application is the delivery of imaging probes to acidic tissues and their stable tethering to the cell membrane surface. To achieve the second ability, the cargo molecule can be linked to the C-terminus of pHLIP through a cleavable linkage that can be cleaved in the cytoplasm, such as a cleavable disulfide bond, and an example of an application is the delivery of antitumor drugs to tumor tissues and their introduction into the tumor cytoplasm, such as fluorescent dyes, cyclic peptides, polar toxins, peptide nucleic acids, and the like.

Due to its special properties, pHLIP is widely used in the following fields: (1) performing fluorescent staining and fluorescent image navigation operation; (2) nuclear imaging, including PET and SPECT; (3) disease therapy, such as targeted delivery of toxins, targeted gene therapy; (4) and the nanotechnology is used for reinforcing the load transportation of the gold nanoparticles or the encapsulated liposome to tumor cells.

However, the prior art does not report the application of low pH insertion peptide having antigenic properties and utilizing the antigenic properties to prepare antibodies. The application fills the blank of the research direction and provides a new target for preparing the targeted tumor antibody medicine.

Disclosure of Invention

The invention provides an antigen comprising an extracellular segment of a low pH insertion peptide.

Further, the low pH insertion peptide comprises the following polypeptides:

WT:ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT(SEQ ID NO.1)；

Var-1:ACEDQNPYWARYADWLFTTPLLLLDLALLVDG(SEQ ID NO.2)；

Var-2:ACEDQNPYWRAYADLFTPLTLLDLLALWDG(SEQ ID NO.3)；

Var-3:ACDDQNPWRAYLDLLFPTDTLLLDLLW(SEQ ID NO.4)；

Var-4:ACEEQNPWRAYLELLFPTETLLLELLW(SEQ ID NO.5)；

Var-5:ACDDQNPWARYLDWLFPTDTLLLDL(SEQ ID NO.6)；

Var-6:CDNNNPWRAYLDLLFPTDTLLLDW(SEQ ID NO.7)；

Var-7:ACEEQNPWARYLEWLFPTETLLLEL(SEQ ID NO.8)；

Var-8:CEEQQPWAQYLELLFPTETLLLEW(SEQ ID NO.9)；

Var-9:CEEQQPWRAYLELLFPTETLLLEW(SEQ ID NO.10)；

Var-10:ACEDQNPWARYADWLFPTTLLLLD(SEQ ID NO.11)；

Var-11:ACEEQNPWARYAEWLFPTTLLLLE(SEQ ID NO.12)；

Var-12:ACEDQNPWARYADLLFPTTLAW(SEQ ID NO.13)；

Var-13:ACEEQNPWARYAELLFPTTLAW(SEQ ID NO.14)；

Var-14:TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA(SEQ ID NO.15)；

Var-15:CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET(SEQ ID NO.16)；

Var-16:CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEET(SEQ ID NO.17)；

the part of the sequence marked with a transverse line is the extracellular segment sequence of the low pH insertion peptide.

Although the present invention has been carried out with respect to the identification of antigenic properties of the extracellular domains of the low pH insertion peptides represented by SEQ ID NO.8 and SEQ ID NO.4, the experimental results of the present invention indicate that the extracellular domains of the low pH insertion peptides have a common antigenic property due to the similarity in the properties of the extracellular domains of the low pH insertion peptides.

The 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 antigen of the invention 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 an antigen as hereinbefore described.

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 antigens of the 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 antigen as described above and a protein or polypeptide linked to the antigen.

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

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 specific antibodies against the antigens described above.

The invention also provides an antibody, wherein the antibody is prepared from the antigen or the fusion protein.

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

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 also provides the application of the antigen in preparing the fusion protein, the antibody or the anti-tumor medicine.

The invention also provides the use of an antibody as hereinbefore described, including the use of any one of:

(1) use in the manufacture of a product for detecting an antigen as hereinbefore described;

(2) the application in preparing antitumor drugs;

(3) use in the preparation of a CAR-T sequence.

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 "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 discovers the antigenicity of the extracellular segment of the low-pH insertion peptide for the first time, and the antigen can be used for preparing the antibody for treating the tumor.

Drawings

FIG. 1 shows an SDS-PAGE electrophoresis of antibodies prepared according to the invention;

FIG. 2 is a graph showing the effect of antibodies immunized with an extracellular domain of a low pHLIP insertion peptide as an antigen on tumor cell growth, wherein A: var-7; b: var-3.

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 preparation of antibodies Using var-7 and var-3 Low pHLIP insertion extracellular domains as antigens

1. Experimental Material

MC38 cells, purchased from ATCC;

var-7 low pHLIP insertion peptide and Var-3 low pHLIP insertion peptide are synthesized by Beijing Huada protein research and development center Limited company, and dissolved in PBS with the concentration of 40 mu M;

female C57/BL6 mice 6-8 weeks old were purchased from Witongliwa.

2. Antibody preparation

(1) Extracellular segment linked KLH

The extracellular domain of Var-7 low pHLIP insertion peptide (Ala-Cys-Glu-Glu-Gln-Asn-Pro, SEQ ID NO.18) was linked to KLH (manufactured by Zhongzhou Middowkoku Co., Ltd.), and the extracellular domain of Var-3 low pHLIP insertion peptide (Ala-Cys-Asp-Asp-Gln-Asn-Pro, SEQ ID NO.19) was linked to KLH (manufactured by Zhongzhou Middowku Co., Ltd.).

(2) Animal immunization

KLH-linked low-pHLIP is inserted into an extracellular peptide segment to immunize a Balb/C mouse to prepare hybridoma, so that var-7 extracellular segment specific monoclonal antibody 3C1 and var-3 extracellular segment specific monoclonal antibody 4D3 are obtained, and the affinity and the antigen recognition specificity of the monoclonal antibody are verified. The results are shown in FIG. 1, confirming that a purer protein is obtained and that the light and heavy chains of the antibody are clearly observed after melting.

Example 2 evaluation of anti-tumor Effect of antibody

1. Experimental procedure

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: var-7/3 single injection group, 10; the var-7/var-3 combined with 3D1/4D3 injection group, 10; 3C1/4D3 single injection group, 10; saline N.S was injected into groups, 10. Tumor size was measured every 3 days.

The administration method comprises the following steps: var-7/3 administration: injecting each vein with 40 μ M/100 μ l, starting from the day after grouping, and injecting 1 time every 2 days; antibody administration: intraperitoneal injection, dose 5mg/kg, 2 times per week, until the end.

2. Results

As shown in FIG. 2, Var-7 in combination with 3C1 or Var-3 in combination with 4D3 significantly inhibited tumor growth, indicating that the low pHLIP inserted peptide extracellular domain was used as an antigen for immunizing antibodies against cancer.

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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Claims (10)

1. An antigen comprising an extracellular segment of a low pH insertion peptide.

2. The antigen according to claim 1, wherein the low pH insertion peptide comprises the low pH insertion peptide of sequence SEQ ID No.1 or a variant thereof; preferably, the variant comprises the polypeptides shown in SEQ ID NO.2-SEQ ID NO. 17.

3. A nucleic acid molecule encoding the antigen of claim 1 or 2.

4. 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 3.

5. A recombinant host cell comprising the recombinant vector of claim 4.

6. A fusion protein comprising the antigen of claim 1 or 2 and a protein or polypeptide linked to the antigen.

7. The fusion protein of claim 6, comprising the antigen of claim 1 or 2 and a carrier protein coupled to the antigen; preferably, the carrier protein comprises KLH, BSA, or OVA.

8. An antibody prepared from the antigen of claim 1 or 2, or the fusion protein of claim 6 or 7.

9. Use of the antigen of claim 1 or 2 for the preparation of the fusion protein of claim 6 or 7, or the antibody of claim 8, or an anti-tumor medicament.

10. The use of the antibody of claim 8, wherein said use comprises any one of the following:

(1) use in the manufacture of a product for detecting an antigen according to claim 1 or 2;

(2) the application in preparing antitumor drugs;

(3) use in the preparation of a CAR-T sequence.

Images