CN107739410B - CD3 single-chain antibody-iRGD fusion protein, preparation and application thereof as antitumor drug - Google Patents

CD3 single-chain antibody-iRGD fusion protein, preparation and application thereof as antitumor drug Download PDF

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Publication number
CN107739410B
CN107739410B CN201710978345.8A CN201710978345A CN107739410B CN 107739410 B CN107739410 B CN 107739410B CN 201710978345 A CN201710978345 A CN 201710978345A CN 107739410 B CN107739410 B CN 107739410B
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irgd
fusion protein
chain antibody
tumor
cells
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CN107739410A (en
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刘宝瑞
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Zhongheng Guoxin (Chongqing) Big Health Research Institute Co.,Ltd.
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Nanjing Drum Tower Hospital
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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Abstract

The invention discloses a CD3 single-chain antibody-iRGD fusion protein, preparation and application thereof as an anti-tumor medicament. The invention takes the penetrating peptide iRGD component as a molecule for realizing the active targeting and high penetrability of the tumor, and connects the single-chain antibody of CD3 with the penetrating peptide iRGD component to form a fusion protein which can activate T cells and effectively target and kill tumor cells. Repeated experiments after successful construction prove that the invention has definite tumor targeting property and high penetrability, can practically enhance the anti-tumor treatment effect of immune cells, and is applied to anti-tumor drugs.

Description

CD3 single-chain antibody-iRGD fusion protein, preparation and application thereof as antitumor drug
Technical Field
The invention belongs to the technical field of biology and new medicines, and particularly relates to a CD3 single-chain antibody-iRGD fusion protein, preparation and application thereof as an anti-tumor medicine.
Background
In the field of immunotherapy, which is becoming more and more important, the progress in the treatment of solid tumors is slow, although tumor targeting can be achieved similarly to chimeric antigen receptor T cell therapy, BiTE, and the like. The difficulty of efficient entry of immune cells into tumor tissue is one of the important reasons. Therefore, in immune cell therapy, the key to further improve the anti-tumor therapeutic effect is to improve the active targeting property and penetrability of immune cells.
Disclosure of Invention
The invention aims to overcome the defects and provide a CD3 single-chain antibody-iRGD fusion protein (named as anti-CD 3-iRGD) and a preparation method and application thereof as an anti-tumor medicament.
The invention uses the substance containing the targeting penetrating peptide iRGD as the molecule for realizing the active targeting and high penetrability of the tumor, and connects the single-chain antibody of CD3 with the molecule to form a fusion protein which can activate T cells and effectively target and kill tumor cells. Repeated experiments after successful construction prove that the invention has definite tumor targeting property and high penetrability, can practically enhance the anti-tumor treatment effect of immune cells, and is applied to anti-tumor drugs.
The technical scheme provided by the invention is as follows:
the invention provides a CD3 single-chain antibody-iRGD fusion protein, which is formed by connecting a targeted human CD3 single-chain antibody and a tumor penetrating peptide iRGD through a connecting peptide (such as GGGGSGGGGSGGS).
Further, the fusion protein includes, but is not limited to, CD3 single chain antibody, single domain antibody, monoclonal antibody, and CD3 domain capable of activating T cells or other molecules capable of activating T cells, and the tumor targeting penetrating peptide includes, but is not limited to, iRGD, and ignr, as well as fusion peptides, fusion proteins, or other modified components containing the aforementioned targeting penetrating peptide structure.
Further, the fusion protein has the following amino acid sequence:
(1) a protein consisting of an amino acid sequence shown in SEQ ID No. 1; or
(2) An amino acid sequence which has 80 to 100 percent of homology with the amino acid sequence shown in the sequence SEQ ID No.1 and encodes the protein with the same function; or
(3) And (2) the protein which is derived from the protein (1) and has the same activity by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.
The specific preparation method for preparing the CD3 single-chain antibody-iRGD fusion protein comprises the following steps:
(1) construction of expression vector pET28a-anti-CD 3-iRGD:
the expression vector is synthesized by Nanjing Kinshire company, and the verification of the plasmid is completed, including nucleic acid electrophoresis and DNA sequencing;
(2) the expression vector pET28a-anti-CD3-iRGD is transformed into an expression strain BL21 DE 3:
transforming a confirmed 100 ng/muL expression vector pET28a-anti-CD3-iRGD 0.1 muL-5 muL into an expression strain BL21 DE3 competent cell, coating the competent cell on an LB plate with specific resistance, and selecting positive clone to store the strain by glycerol in a low-temperature refrigerator;
(3) induced expression, denaturation and renaturation of recombinant anti-CD3-iRGD, and purification:
1) the recombinant anti-CD3-iRGD isopropyl-beta-D-thiogalactoside is induced to express:
inducing the recombinant anti-CD3-iRGD with 1mM isopropyl-beta-D-thiogalactoside for expression, and performing polyacrylamide gel whole protein electrophoresis after inducing for 4 hours;
2) carrying out renaturation on recombinant anti-CD 3-iRGD;
collecting 1000mL of BL21 thallus subjected to isopropyl-beta-D-thiogalactoside induced expression, resuspending the thallus by PBS and 5mM imidazole, carrying out ultrasonic disruption at 350W power, centrifuging at 12000 r.min < -1 >, collecting precipitate, dissolving the precipitate by 8M urea denaturant, and dialyzing and renaturing in 6M, 4M, 2M and 0M urea solutions in sequence;
3) recombinant anti-CD3-iRGD passes through AKTA Purifier 900 FPLC system and nickel ion affinity layer
And (3) column chromatography purification:
(4) inducing the recombinant anti-CD3-iRGD with 1mM isopropyl-beta-D-thiogalactoside (IPTG) for 4 hours, collecting bacterial liquid, carrying out ultrasonication, centrifuging, collecting precipitate, dissolving the precipitate with 8M urea denaturant, dialyzing and renaturing in 6M, 4M, 2M and 0M urea solutions in sequence, purifying the recombinant anti-CD3-iRGD with an AKTA Purifier 900 FPLC system and a nickel ion affinity chromatography column: the target protein was detected by SDS-PAGE.
The invention has the beneficial effects that:
the single-chain antibody part of the fusion protein CD3 is combined with T cells to promote the polyclonal proliferation, activation (including phenotype change and cytokine secretion increase) and killing effect on tumor cells, and the iRGD part of the fusion protein is combined with the T cells through integrin alphaνβ3/β5 The receptor pathway targets the tumor locally and further increases T cell penetration in tumor vasculature and parenchyma.
Drawings
FIG. 1 shows the nucleic acid electrophoresis of target gene expression vector pET28a-anti-CD 3-iRGD.
Lane 1: complete plasmid electrophoresis results; lane 2: carrying out enzyme digestion on the plasmid by Nco I and Hind III nucleases and then carrying out electrophoresis; lane 3: and (5) DNA marker.
FIG. 2 is a schematic representation of the sequencing of the sequence between the Nco I and Hind III sites in the expression vector pET28a-anti-CD 3-iRGD.
FIG. 3 shows the expression and purification of anti-CD3-iRGD protein: (left panel) lane 1: marker; lane 2: not inducing; lane 3: after induction; lane 4: supernatant fluid; lane 5: precipitating; lane 6: after renaturation. (right panel) lane 1: marker; lane 2: flowing through the protein sample; lane 3: 1% imidazole eluting protein; lane 4: % imidazole eluting protein; lane 5: 25% -100% imidazole linear elution protein, i.e. after purification.
FIG. 4 is a diagram of a nickel column affinity chromatography purification of a protein of interest (arrows indicate the protein of interest).
FIG. 5 shows that the fusion protein anti-CD3-iRGD can bind to receptors on the surface of MKN45 cells and T cells at different concentrations. (half an hour later the binding of the cell surface to the protein was detected by flow, the left panel shows the binding of the fusion protein to MKN45 cell surface iRGD receptor, and the right panel shows the binding of the fusion protein to T cell surface CD3 molecule).
FIG. 6 is the effect of the fusion protein anti-CD3-iRGD on the T cell surface CD107a phenotype. (24 h later flow-detection of cell surface binding to proteins, CD107a is a marker of T cell killing function).
FIG. 7 is the effect of the fusion protein anti-CD3-iRGD on the T cell surface CD27 phenotype (CD 27 is a marker for T cell activation).
FIG. 8 shows that the fusion protein promotes secretion of IFN-. gamma.by T cells. (the secretion of IFN-. gamma.by T cells was measured by ELISPOT after 24 hours, and the right graph shows the quantitative results).
FIG. 9 shows that the fusion protein anti-CD3-iRGD promotes the killing effect of T cells on MKN 45. (killing of T cells was tested by LDH release assay after 24h and 48 h).
FIG. 10 shows that the fusion protein anti-CD3-iRGD promotes T cell penetration in 3D tumor cell spheres in vitro. (penetration of T cells in tumor spheres was observed under confocal microscopy after 6h and 24 h).
FIG. 11 shows that the fusion protein anti-CD3-iRGD promotes the killing effect of T cells on 3D tumor cell balls in vitro. (24 h and 48h later the size and morphology of the tumor spheres was observed under an inverted light microscope).
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the accompanying drawings and the detailed description, but do not limit the scope of the claims of the present invention.
Example 1
A CD3 single-chain antibody-iRGD fusion protein is composed of a targeting human CD3 single-chain antibody and a tumor penetrating peptide iRGD which are connected through a connecting peptide (such as GGGGSGGGGSGGS).
Further, the fusion protein includes, but is not limited to, CD3 single chain antibody, single domain antibody, monoclonal antibody, and CD3 domain capable of activating T cells or other molecules capable of activating T cells, and the tumor targeting penetrating peptide includes, but is not limited to, iRGD, and ignr, as well as fusion peptides, fusion proteins, or other modified components containing the aforementioned targeting penetrating peptide structure.
Further, the fusion protein has the following amino acid sequence:
(1) a protein consisting of an amino acid sequence shown in SEQ ID No. 1; or
(2) An amino acid sequence which has 80 to 100 percent of homology with the amino acid sequence shown in the sequence SEQ ID No.1 and encodes the protein with the same function; or
(3) And (2) the protein which is derived from the protein (1) and has the same activity by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.
The expression and purification process of the fusion protein with the anti-tumor effect is as follows:
1. construction of expression vector pET28a-anti-CD 3-iRGD:
the vector is synthesized by Nanjing Kinshire company, and the plasmid is subjected to nucleic acid electrophoresis after being digested by Nco I and Hind III nucleases, and the result shows that the size of a target gene fragment is about 800bp, as shown in figure 1: the fragment size is seen to be consistent with expectations; and the plasmid was subjected to sequencing verification. The nucleotide sequence of the sequenced gene and the deduced amino acid sequence were analyzed in comparison with the designed results. Comparison shows that the vector pET28a-anti-CD3-iRGD is successfully constructed, and the sequencing result is shown in FIG. 2.
2. The expression vector pET28a-anti-CD3-iRGD is transformed into an expression strain BL21 DE 3:
the confirmed 100 ng/. mu.L expression vector pET28a-anti-CD3-iRGD 0.1. mu.L-5. mu.L was transformed into expression strain BL21 DE3 competent cells prepared from calcium chloride and plated on specific kanamycin-resistant LB plate. And selecting positive clone and storing the strain in glycerol in a low-temperature refrigerator.
3. Induced expression and purification of recombinant anti-CD 3-iRGD:
1) the recombinant anti-CD3-iRGD IPTG induces expression:
the recombinant anti-CD3-iRGD is induced and expressed by 1mMIPTG, after 4 hours of induction, SDS-PAGE holoprotein electrophoresis can see clear specific protein bands at about 30KD, and the comparison of no IPTG induction and IPTG induction shows that the anti-CD3-iRGD protein is successfully expressed.
The whole protein IPTG induction expression comprises the following experimental steps:
a) adding 3 mu L of bacterial liquid (expression strain glycerol preservation bacteria) into 3mL of LB (kana resistance) culture medium, and shaking the bacteria at 220rpm and 37 ℃ overnight;
b) measuring OD600 to 0.5;
c) adding IPTG at a concentration of 1 mM;
d) shaking the bacteria at 220rpm and 37 ℃ for 4 hours;
e) taking out the bacterial liquid, centrifuging at 12000g for 10min, and removing the supernatant;
2) the recombinant anti-CD3-iRGD is denatured by 8M urea, renatured by dialysis, and purified by AKTA Purifier 900 FPLC system and nickel ion affinity chromatography column:
1000mL of the bacterial liquid BL21 DE3 subjected to IPTG induction expression is collected, and the bacterial liquid is centrifuged for 10min at 4200 r.min < -1 >, and the bacterial cells are collected. The pellet was resuspended in PBS (pH 7.4) and 80mL of 5mM imidazole for sonication under 350W, 3s working, 3s pause, and 45min total time. The ultrasonic lysate was centrifuged at 12000 rpm-1 for 20 min at 4 ℃. The precipitate was collected and dissolved thoroughly in a protein denaturing solution containing 8M urea and was renatured by dialysis sequentially in 6M, 4M, 2M, 0M urea solution and purified on an AKTA Purifier 900 FPLC system. The nickel column was equilibrated with 5 column volumes of PBS (pH 7.4) and 5 mmol/L-1 imidazole, and the sample was loaded, washed with PBS (pH 7.4) and 40 mmol/L-1 imidazole, and the target protein was eluted with PBS (pH 7.4) and 500 mmol/L-1 imidazole, as shown in FIG. 4. The eluted purified protein was dialyzed against PBS (pH 7.4).
4. Detection of the protein of interest by SDS-PAGE method:
selecting 1mL of expression strain BL21 DE3 pET28a-anti-CD3-iRGD bacterial liquid without induced expression, adding 1mL of bacterial liquid after IPTG induced expression for 4 hours, centrifuging for 1 min at 12000 r.min < -1 >, discarding supernatant, precipitating with 100 mu LddH2O resuspension as "uninduced" and "induced"; centrifuging 100 μ L of the bacteria solution after ultrasonic treatment at 12000 r min-1 for 1 min, transferring the supernatant to a new EP tube, and precipitating with 100 μ L ddH2O heavy suspension is respectively used as supernatant and sediment; the dialyzed renatured protein was used as "renatured" and subjected to SDS-PAGE, stained with Coomassie Brilliant blue and destained after electrophoresis, and photographed as shown in FIG. 3.
5. Respectively mixing MKN45 and PBMC with anti-CD3-iRGD (0, 0.1, 1, 10 ug/ml) at different concentrations
Incubate 30min on ice, wash 1ml PBS for 1 time, then add 3ul anti-His flow antibody separately, incubate 30min in dark place, wash 1ml PBS for 1 time, centrifuge 5min at 300g, leave 100ul volume and carry on flow detection MKN45, PBMC cell surface and anti-CD3-iRGD bind, as shown in figure 5.
6. MKN45 cells and PBMC cells were cultured as follows E: T = 20:1, adding anti-CD3-iRGD (0, 0.1, 1 and 10 ug/ml) with different concentrations, and performing flow detection on the expression of CD107a and CD27 on the surface of the T cell after 24 hours. The flow-through results show that the expression of CD107a (shown in figure 6) and CD27 (shown in figure 7) on the surface of T cells is obviously up-regulated when the protein concentration is 0.1ug/ml compared with the control group.
7. MKN45 cells and PBMC cells were cultured as follows E: T = 20:1, adding anti-CD3-iRGD (0, 0.1, 1 and 10 ug/ml) with different concentrations, and measuring ELISPOT after 24h, wherein the number of T cells secreting IFN-gamma is obviously increased when the protein concentration is 5ug/ml and 10ug/ml, as shown in figure 8.
8. LDH release experiments: MKN45 cells and PBMC cells were cultured as follows E: T = 20:1 or 40:1, taking 100ul of supernatant after 24h and 48h, adding 100ul of LDH reaction solution, and measuring the absorbance at A492nm after keeping away from light for 30min, as shown in figure 9.
9. MKN45 cells were added to the ultra low adsorption 96 well plate at a concentration of 1000 cells/ml, and after 2 days the cells pelleted, CFSE labeled PBMCs were expressed as E: T = 4: 1 into 96-well plate, adding 10ug/ml anti-CD3-iRGD protein, observing T cell penetration in tumor ball under confocal microscope after 6h and 24h (as shown in FIG. 10). Or unlabeled PBMCs as described E: T = 5: 1. 10: 1. 20:1 into 96-well plates, and 24h and 48h later, tumor spheres were observed under inverted light microscope for size and morphology (see FIG. 11). The results show that anti-CD3-iRGD can promote penetration and killing effects of T cells at the level of 3D tumor spheres.
Sequence listing
<110> Nanjing drum building hospital
<120> CD3 single-chain antibody-iRGD fusion protein, preparation and application thereof as antitumor drug
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 263
<212> PRT
<213> Artificial sequence ()
<400> 1
His His His His His His Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu
1 5 10 15
Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly
20 25 30
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly
35 40 45
Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
50 55 60
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
65 70 75 80
Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
85 90 95
Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly
115 120 125
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp
130 135 140
Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu
145 150 155 160
Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn
165 170 175
Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp
180 185 190
Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp
210 215 220
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe
225 230 235 240
Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Cys Arg
245 250 255
Gly Asp Lys Gly Pro Asp Cys
260

Claims (3)

1. A CD3 single-chain antibody-iRGD fusion protein, characterized in that: the fusion protein is formed by connecting a targeted human CD3 single-chain antibody and a tumor penetrating peptide iRGD through a connecting peptide, and has an amino acid sequence shown in SEQ ID No. 1.
2. A method of producing a CD3 single-chain antibody-iRGD fusion protein according to claim 1, comprising the steps of:
constructing an expression vector pET28a-anti-CD 3-iRGD;
transforming an expression vector pET28a-anti-CD3-iRGD into an expression strain BL21 DE 3;
induced expression and purification of recombinant anti-CD 3-iRGD;
inducing the recombinant anti-CD3-iRGD with 1mM isopropyl-beta-D-thiogalactoside (IPTG) for 4 hours, collecting bacterial liquid, carrying out ultrasonic crushing, centrifuging, collecting precipitate, dissolving the precipitate with 8M urea denaturant, dialyzing and renaturing in 6M, 4M, 2M and 0M urea solutions in sequence, purifying the recombinant anti-CD3-iRGD through an AKTA Purifier 900 FPLC system and a nickel ion affinity chromatography column; the target protein was detected by SDS-PAGE.
3. The use of the CD3 single-chain antibody-iRGD fusion protein in the preparation of anti-tumor drugs, wherein the tumors comprise breast cancer, gastric cancer, liver cancer, intestinal cancer, pancreatic cancer, lung cancer and cervical cancer.
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