CN107739410A - CD3 single-chain antibody iRGD fusion proteins, preparation and its application as antineoplastic - Google Patents

CD3 single-chain antibody iRGD fusion proteins, preparation and its application as antineoplastic Download PDF

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Publication number
CN107739410A
CN107739410A CN201710978345.8A CN201710978345A CN107739410A CN 107739410 A CN107739410 A CN 107739410A CN 201710978345 A CN201710978345 A CN 201710978345A CN 107739410 A CN107739410 A CN 107739410A
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Prior art keywords
irgd
chain antibodies
fusion proteins
amino acid
acid sequence
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CN201710978345.8A
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CN107739410B (en
Inventor
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 kind of CD3 single-chain antibodies iRGD fusion proteins, preparation and its application as antineoplastic.CD3 single-chain antibody is connected thereto, forming one can be with activating T cell and the fusion protein of energy efficient targeting killing tumor cell by the present invention using penetrating peptide iRGD compositions as the molecule for realizing tumor-targeting, high-penetration.Success has clear and definite tumor-targeting and high-penetration, can effectively strengthen the antineoplaston effect of immunocyte, applied in antineoplastic after building through experimental verification repeatedly, the present invention.

Description

CD3 single-chain antibody-iRGD fusion proteins, preparation and its as antineoplastic Using
Technical field
The invention belongs to biology with new medical technology field, more particularly to a kind of CD3 single-chain antibodies-iRGD fusion proteins, Preparation and its application as antineoplastic.
Background technology
In the immunotherapy field increasingly attracted people's attention, treated despite the presence of similar to Chimeric antigen receptor T cell Method, BiTE etc. can realize the targeting of tumour, but be made slow progress in the treatment of solid tumor.Immunocyte is difficult to effectively enter It is the reason for one of them is important to enter tumor tissues.Therefore, in immune cell therapy, while the active target of immunocyte is improved Tropism is the further key for improving antineoplaston effect with penetrability.
The content of the invention
The purpose of the present invention, which is that, overcomes drawbacks described above, there is provided a kind of CD3 single-chain antibodies-iRGD fusion proteins（Name For anti-CD3-iRGD）There is provided the preparation method of above-mentioned fusion protein and its application as antineoplastic simultaneously.
The material that the present invention targets penetrating peptide iRGD compositions using containing is as realizing tumor-targeting, high-penetration Molecule, CD3 single-chain antibody is connected thereto, forming one can be with activating T cell and can efficient targeting killing tumor cell Fusion protein.Success has clear and definite tumor-targeting and high-penetration, can cut after building through experimental verification repeatedly, the present invention Strengthen the antineoplaston effect of immunocyte on the spot, applied in antineoplastic.
Technical scheme provided by the invention is：
The present invention provides a kind of CD3 single-chain antibodies-iRGD fusion proteins, the fusion protein by target people CD3 single-chain antibodies and Tumour penetrating peptide iRGD is via connection peptide（Such as：GGGGSGGGGSGGGGS）Connection composition.
Further, the fusion protein including but not limited to CD3 single-chain antibodies, single domain antibody, monoclonal antibody and Can activating T cell CD3 functional domains or other can activating T cells molecule, cancer target penetrating peptide including but not limited to iRGD, INGR, and fusogenic peptide, fusion protein or other ornamental equivalents containing foregoing targeting penetrating peptide structure.
Further, the fusion protein has following amino acid sequence：
（1）The protein being made up of the amino acid sequence shown in SEQ ID No.1；Or
（2）With the amino acid sequence homology shown in sequence SEQ ID No.1 in 80%-100% coding identical function protein Amino acid sequence；Or
（3）Amino acid sequence shown in SEQ ID No.1 has on an equal basis through increasing, lacking or replacing one or more amino acid Activity by（1）Derivative albumen.
The specific preparation method that the present invention prepares the CD3 single-chain antibodies-iRGD fusion proteins is as follows：
（1）Expression vector pET28a-anti-CD3-iRGD structure：
Expression vector is synthesized by Nanjing Jin Sirui companies, and has completed the checking to the plasmid, including nucleic acid electrophoresis and DNA are surveyed Sequence；
（2）Expression vector pET28a-anti-CD3-iRGD is converted to expression bacterial strain BL21 DE3：
The μ L-5 μ L of 100ng/ μ L expression vectors pET28a-anti-CD3-iRGD 0.1 having been acknowledged are taken to be transformed into expression bacterial strain In BL21 DE3 competent cells, it is applied on the LB flat boards of specific resistance, selects glycerine in positive colony low temperature refrigerator and preserve The strain；
（3）Recombinate anti-CD3-iRGD induced expression, denaturation and renaturation, and purifying：
1）Recombinate anti-CD3-iRGD isopropyl-β-D-thiogalactoside induced expressions：
To recombinating anti-CD3-iRGD 1mM isopropyl-β-D-thiogalactoside induced expressions, after induction 4 hours, Polyacrylamide gel holoprotein electrophoresis；
2）Recombinate anti-CD3-iRGD and carry out refolding strategy；
BL21 thalline of the 1000mL after isopropyl-β-D-thiogalactoside induced expression is collected, with PBS, 5mM imidazoles weight Outstanding thalline, 350W power carry out ultrasonication, the collected after centrifugation of 12 000 rmin 1 precipitation, with urea-denatured dose of dissolving of 8M Precipitation, and the dialysis renaturation in 6M, 4M, 2M, 0M urea liquid successively；
3）Anti-CD3-iRGD is recombinated through the FPLC systems of AKTA Purifier 900, nickel ion is affine layer
Analyse post purifying：
（4）To recombinating anti-CD3-iRGD 1mM isopropyl-β-D-thiogalactosides（IPTG）Induction 4 hours, collect Bacterium solution carries out ultrasonication, collected after centrifugation precipitation, is precipitated with urea-denatured dose of dissolving of 8M, and urinated successively in 6M, 4M, 2M, 0M Dialysis renaturation in plain solution, anti-CD3-iRGD is through the FPLC systems of AKTA Purifier 900, nickel ion affinity chromatograph for restructuring Post purifies：With SDS-PAGE method testing goal albumen.
The beneficial effects of the invention are as follows：
Fusion protein CD3 single chain antibody portions are combined with T cell, promote polyclonal propagation, the activation of T cell（Change including phenotype Become and cytokine secretion increases）With the lethal effect to tumour cell, fusion protein iRGD parts then pass through integrin alphaνβ3/ β5 Receptor pathway further increases penetrability of the T cell in tumor vessel and essence targeted to tumor by local.
Brief description of the drawings
Fig. 1 is destination gene expression carrier pET28a-anti-CD3-iRGD nucleic acid electrophoresis figures.
Swimming lane 1：Complete plasmid electrophoresis result；Swimming lane 2：Plasmid is through Nco I and the nuclease digestion rear electrophoresis results of Hind III； Swimming lane 3：DNA marker.
Fig. 2 is the sequencing result of sequence between Nco I and the sites of Hind III in expression vector pET28a-anti-CD3-iRGD Schematic diagram.
Fig. 3 is the expression and purification of anti-CD3-iRGD albumen：（Left figure）Swimming lane 1：Marker；Swimming lane 2：Do not induce；Swimming Road 3：After induction；Swimming lane 4：Supernatant；Swimming lane 5：Precipitation；Swimming lane 6：After renaturation.（Right figure）Swimming lane 1：Marker；Swimming lane 2：Albumen sample Product flow through；Swimming lane 3：1% imidazoles elutes albumen；Swimming lane 4：% imidazoles elutes albumen；Swimming lane 5：25%-100% imidazoles linear elution eggs In vain, i.e., after purification.
Fig. 4 is affinity chromatography purifying destination protein collection of illustrative plates（Arrow indicative purpose albumen）.
Fig. 5 be fusion protein anti-CD3-iRGD in various concentrations can respectively with MKN45 cells and T cell surface Acceptor combines.（The combination of flow cytometer detection cell surface and albumen after half an hour, left figure are fusion protein and MKN45 cell surfaces The combination of iRGD acceptors, right figure are the combination of fusion protein and T cell surface C D3 molecules）.
Fig. 6 is effects of the fusion protein anti-CD3-iRGD to T cell surface C D107a phenotypes.（Flow cytometer detection after 24h The combination of cell surface and albumen, CD107a are the marks of T cell killing ability）.
Fig. 7 is effects of the fusion protein anti-CD3-iRGD to T cell surface C D27 phenotypes（CD27 is T cell activation Mark）.
Fig. 8 is the secretion that fusion protein promotes T cell IFN-γ.（T cell IFN-γ is detected by ELISPOT after 24h Secretory volume, right figure are quantitative result）.
Fig. 9 is that fusion protein anti-CD3-iRGD promotes lethal effect of the T cell to MKN45.（Through LDH after 24h and 48h Release experiment detects the lethal effect of T cell）.
Figure 10 is that fusion protein anti-CD3-iRGD promotes T cell penetrating in 3D tumour cells ball in vitro.（6h and Penetration of the T cell in tumour ball is observed after 24h under Laser Scanning Confocal Microscope）.
Figure 11 is that fusion protein anti-CD3-iRGD promotes lethal effect of the T cell to external 3D tumour cells ball.（24h With 48h after the size and form that tumour ball is observed under inverted light microscope）.
Embodiment
Technical scheme is described in detail below in conjunction with the drawings and specific embodiments, but does not limit right of the present invention It is required that protection domain.
Embodiment 1
A kind of CD3 single-chain antibodies-iRGD fusion proteins, the fusion protein is by targetting people CD3 single-chain antibodies and tumour penetrating peptide IRGD is via connection peptide（Such as：GGGGSGGGGSGGGGS）Connection composition.
Further, the fusion protein including but not limited to CD3 single-chain antibodies, single domain antibody, monoclonal antibody and Can activating T cell CD3 functional domains or other can activating T cells molecule, cancer target penetrating peptide including but not limited to iRGD, INGR, and fusogenic peptide, fusion protein or other ornamental equivalents containing foregoing targeting penetrating peptide structure.
Further, the fusion protein has following amino acid sequence：
（1）The protein being made up of the amino acid sequence shown in SEQ ID No.1；Or
（2）With the amino acid sequence homology shown in sequence SEQ ID No.1 in 80%-100% coding identical function protein Amino acid sequence；Or
（3）Amino acid sequence shown in SEQ ID No.1 has on an equal basis through increasing, lacking or replacing one or more amino acid Activity by（1）Derivative albumen.
The expression of fusion protein with antitumor action and purge process are as follows：
1st, expression vector pET28a-anti-CD3-iRGD structure：
The carrier is synthesized by Nanjing Jin Sirui companies, and plasmid carries out nucleic acid electrophoresis after Nco I and the nuclease digestions of Hind III, knot Fruit shows that target gene clip size is 800bp or so, as shown in Figure 1：It can be seen that clip size is with being expected unanimously；And plasmid is entered Row sequence verification.The nucleotide sequence of gene and the amino acid sequence of derivation is sequenced to be analyzed with the result designed. Show that carrier pET28a-anti-CD3-iRGD is successfully constructed by contrast, sequencing result is as shown in Figure 2.
2nd, expression vector pET28a-anti-CD3-iRGD is converted to expression bacterial strain BL21 DE3：
The μ L-5 μ L of 100ng/ μ L expression vectors pET28a-anti-CD3-iRGD 0.1 having been acknowledged are taken to be transformed into calcium chloride system In standby expression bacterial strain BL21 DE3 competent cells, it is applied on the LB flat boards of that resistance of particular card.It is low to select positive colony Glycerine preserves the strain in temperature refrigerator.
3rd, anti-CD3-iRGD induced expression and purifying is recombinated：
1）Recombinate anti-CD3-iRGD IPTG induced expressions：
To recombinating anti-CD3-iRGD 1mMIPTG induced expressions, after inducing 4 hours, SDS-PAGE holoprotein electrophoresis, 30KD or so can see clear specific protein band, is being induced without IPTG and IPTG induction contrast displays, anti-be present CD3-iRGD albumen has expressed success.
Holoprotein IPTG induced expressions, experimental procedure are as follows：
a）Take 3 μ L bacterium solutions（Express bacterial strain glycerine and preserve bacterium）Add 3mL LB（Block that resistance）Culture medium, 37 DEG C of 220rpm is overnight Shake bacterium；
b）When surveying OD600 to 0.5；
c）Add the IPTG that concentration is 1mM；
d）37 DEG C of 220rpm shakes bacterium 4 hours；
e）Bacterium solution is taken out, supernatant is removed in 12000g 10min centrifugations；
2）It is urea-denatured with 8M to recombinate anti-CD3-iRGD, dialysis renaturation, and through the FPLC systems of AKTA Purifier 900 System, the purifying of nickel ion affinity chromatograph post：
BL21 DE3 bacterium solutions of the 1000mL after IPTG induced expressions is collected, 10min is centrifuged through 4200 rmin 1, receives Collect thalline.Precipitation is resuspended in PBS（PH 7.4）, carry out ultrasonic degradation in 5mM imidazoles 80mL, ultrasound condition 350W, work 3s, Interval 3s, total time 45min.Ultrasonic degradation thing centrifuges 20 min in 4 DEG C, 12 000 rmin 1.Collection is deposited in Fully dissolved in the albuminous degeneration liquid of the urea containing 8M, and the dialysis renaturation in 6M, 4M, 2M, 0M urea liquid successively, in AKTA The FPLC systems of Purifier 900 purify.5 times of column volume PBS of nickel post（PH7.4）, the imidazoles of 5mmol lL 1 balance after on Sample, through PBS (pH 7.4), the imidazoles of 40 mmolL 1 wash it is miscellaneous after, destination protein PBS (pH 7.4), 500 mmolL 1 imidazoles elutes, such as Fig. 4.Purifying protein after elution is dialysed in PBS (pH 7.4).
4th, with SDS-PAGE method testing goal albumen：
Choose the expression bacterial strain BL21 DE3 pET28a-anti-CD3-iRGD bacterium solutions 1mL of non-induced expression, addition IPTG is lured Lead the bacterium solution 1mL after expressing 4 hours and carry out 12000 rmin 1,1 min of centrifugation, abandon supernatant, 100 μ LddH of precipitation2O weights It is outstanding to be used as " not inducing " and " induction "；The μ L of bacterium solution 100 after ultrasound carry out 12000 rmin 1 and centrifuge 1 min, and supernatant moves to New EP pipes, 100 μ LddH of precipitation2O is resuspended respectively as " supernatant " and " precipitation "；Albumen through dialysis renaturation is as " multiple Property after ", carry out SDS-PAGE electrophoresis, coomassie brilliant blue staining and decolourize after electrophoresis, take pictures, such as Fig. 3.
5th, by MKN45 and the PBMC anti-CD3-iRGD with various concentrations respectively（0、0.1、1、10ug/ml）
30min is incubated on ice, 1mlPBS is washed 1 time, then is separately added into 3ul anti-His streaming antibody, and lucifuge is incubated 30min, 1mlPBS is washed 1 time, 300g centrifugation 5min, is left and taken 100ul volumes and is carried out flow cytometer detection MKN45, PBMC cell surface and anti- CD3-iRGD combination, such as Fig. 5.
6th, MKN45 cells and PBMC cells are pressed into E:T=20：1 is incubated altogether, adds the anti-CD3-iRGD of various concentrations （0、0.1、1、10ug/ml）, the expression of flow cytometer detection T cell surface C D107a, CD27 after 24h.Streaming result is shown, works as albumen When concentration is 0.1ug/ml, T cell surface C D107a（Such as Fig. 6）、CD27（Such as Fig. 7）Expression compared with control group substantially on Adjust.
7th, MKN45 cells and PBMC cells are pressed into E:T=20：1 is incubated altogether, adds the anti-CD3-iRGD of various concentrations （0、0.1、1、10ug/ml）, ELISPOT results, when protein concentration is 5ug/ml and 10ug/ml, secretion of gamma-IFN are surveyed after 24h T cell number showed increased, such as Fig. 8.
8th, LDH release experiments：MKN45 cells and PBMC cells are pressed into E:T=20：1 or 40:After 1 incubation altogether, 24h and 48h 100ul supernatants are taken, 100ulLDH reaction solutions is added, the absorbance at A492nm, such as Fig. 9 is surveyed after lucifuge effect 30min.
9th, MKN45 cells are added in 96 orifice plates of ultralow absorption by 1000/ml concentration, cell balling-up after 2 days will The PBMC of CFSE marks presses E:T=4：1 adds in 96 orifice plates, and experimental group adds 10ug/ml anti-CD3-iRGD albumen simultaneously, Penetration of the T cell in tumour ball is observed after 6h and 24h under Laser Scanning Confocal Microscope（Such as Figure 10）.Or will be unlabelled PBMC presses E:T=5：1、10：1、20:1 adds in 96 orifice plates, and 24h and 48h are after observation tumour ball under inverted light microscope Size and form（Such as Figure 11）.As a result show, anti-CD3-iRGD can promote T cell penetrating and killing in 3D tumours ball level Wound acts on.
Sequence table
<110>Nanjing drum tower hospital
<120>CD3 single-chain antibody-iRGD fusion proteins, preparation and its application as antineoplastic
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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 (6)

  1. A kind of 1. CD3 single-chain antibodies-iRGD fusion proteins, it is characterised in that：The fusion protein is by targetting people's CD3 single-chain antibodies With tumour penetrating peptide iRGD via connection peptide connection composition.
  2. 2. CD3 single-chain antibodies-iRGD fusion proteins according to claim 1, it is characterised in that：The fusion protein includes But being not limited to CD3 single-chain antibodies, single domain antibody, monoclonal antibody and the CD3 functional domains of energy activating T cell or other can activate The molecule of T cell, cancer target penetrating peptide is including but not limited to iRGD, iNGR, and melting containing foregoing targeting penetrating peptide structure Close peptide, fusion protein or other ornamental equivalents.
  3. 3. CD3 single-chain antibodies-iRGD fusion proteins according to claim 1, it is characterised in that：The fusion protein has Following amino acid sequence：
    The protein being made up of the amino acid sequence shown in SEQ ID No.1；Or
    The amino of identical function protein is encoded in 80%-100% with the amino acid sequence homology shown in sequence SEQ ID No.1 Acid sequence；Or
    Amino acid sequence shown in SEQ ID No.1 has equal live through increasing, lacking or replacing one or more amino acid Property by（1）Derivative albumen.
  4. 4. the preparation method of CD3 single-chain antibodies-iRGD fusion proteins described in a kind of claim 1, it is characterised in that including following Step：
    Expression vector pET28a-anti-CD3-iRGD structure；
    Expression vector pET28a-anti-CD3-iRGD is converted to expression bacterial strain BL21 DE3；
    Recombinate anti-CD3-iRGD induced expression and purifying；
    To recombinating anti-CD3-iRGD 1mM isopropyl-β-D-thiogalactosides（IPTG）Induction 4 hours, collect bacterium solution Ultrasonication is carried out, collected after centrifugation precipitation, is precipitated with urea-denatured dose of dissolving of 8M, and it is molten in 6M, 4M, 2M, 0M urea successively Dialysis renaturation in liquid, restructuring anti-CD3-iRGD are pure through the FPLC systems of AKTA Purifier 900, nickel ion affinity chromatograph post Change：With SDS-PAGE method testing goal albumen.
  5. 5. application of the CD3 single-chain antibodies-iRGD fusion proteins according to claim 1 as antineoplastic.
  6. 6. application of the CD3 single-chain antibodies-iRGD fusion proteins according to claim 5 as antineoplastic, its feature It is, the tumour is including but not limited to tumours such as breast cancer, stomach cancer, liver cancer, intestinal cancer, cancer of pancreas, lung cancer, cervical carcinomas.
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EP3934680A4 (en) * 2019-03-08 2022-12-07 Cend Therapeutics, Inc. Low-dose cytokine co-administered with irgd for treating cancer

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