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CN109402168A - Bridging molecules and its application of minicircle dna expression connection HER2 positive cell and effector cell - Google Patents

Bridging molecules and its application of minicircle dna expression connection HER2 positive cell and effector cell Download PDF

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CN109402168A
CN109402168A CN201811277450.XA CN201811277450A CN109402168A CN 109402168 A CN109402168 A CN 109402168A CN 201811277450 A CN201811277450 A CN 201811277450A CN 109402168 A CN109402168 A CN 109402168A
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her2
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carrier
bridging molecules
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谌平
谢亦武
陈志英
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Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
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Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
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Abstract

This application involves bridging molecules and its applications that minicircle dna expresses connection Her2 positive cell and effector cell.More particularly to a kind of for expressing the minicircle dna carrier of anti-Her2 bispecific antibody in vivo.Mediated by Bi-specific Antibodies effector cell (T cell or NK cell) kills Her2 positive cancer cell (target cell).The application discloses the minicircle dna carrier design scheme of Her2 positive cell Yu effector cell's bridging molecules (MC.Her2-BTEC), the treatment suitable for Her2 associated cancer for the first time.

Description

Minicircle dna expression connection HER2 positive cell and effector cell bridging molecules and its Using
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of micro-loop (Minicircle, MC) DNA vector, more specifically To be a kind of for expressing the minicircle dna carrier of the bridging molecules of connection Her2 positive cell and effector cell in vivo.
Background technique
Her2 (human epidermal growth factor receptor-2, human epidermal growth factor receptor 2), again Claim Her2/neu or ErbB2, is the member of I type transmembrane tyrosine kinase receptor family, belongs to one kind of transmembrane protein, excess The generation of heterodimerization between Her2 homodimerization or Her2 and other members of family is often induced when expression or mutation, and then is activated The RAS-MAPK in downstream, the AKT signal path for targeting mTOR, lead to abnormality proliferation and the survival of cell.Have now been found that Her2 It is expressed in kinds cancer cell surface height, including breast cancer, non-small cell lung cancer, salivary-gland carcinoma, gastric cancer, intestinal cancer, cancer of pancreas, wing Guang cancer, carcinoma of endometrium, oophoroma etc. are a kind of cancer immunotherapy target spots of wide spectrum.
Existing Her2 monoclonal antibody medicine listing at present, for example, Trastuzumab Herceptin (Herceptin Trastuzumab) or Pertuzumab handkerchief trastuzumab (Perjeta), is used to treat the cancers such as breast cancer, gastric cancer, and achieves good clinic Therapeutic effect.But, theoretical research in recent years shows that bispecific antibody more preferably, has cancer/tumour treatment curative effect The bispecific antibody of targeting Her2 enters clinical experimental stage, such as Her2-TDB.The Her2 bispecific of existing report is anti- Body (dual anti-) is expressed by traditional gene engineering method in mammalian cells, is obtained later by method for purifying proteins It arrives.In general, the processes such as traditional production preparation, purifying and storage transport are very complicated, and cost is very high, and there are medicines The greater risk of product pollution.In addition, dual anti-half-life period is shorter, generally require to be administered continuously, increases dosage, this virtually increases The financial burden of patient, and there are the risk of drug tolerance.
The present invention expresses bispecific antibody by minicircle dna genophore in vivo, mediate effector cell (T cell or NK cell) kill Her2 positive cancer cell (target cell).It is this to generate Her2 bispecific in vivo in a manner of gene delivery The research of antibody has no relevant report in the prior art.The invention avoids above-mentioned problems of the prior art, and It is safe and efficient, cost is relatively low, patient can bear.It is that minicircle dna carrier expression bispecific is anti-with the immediate technology of the present invention The technology MC.BsAb (CN201710245146.6) of body.
Summary of the invention
The present invention relates to micro-loop (Minicircle, MC) DNA vectors to express connection Her2 positive cell and effect in vivo The bridging molecules of cell, for treating Her2 associated cancer.
The present invention provides a kind of expression Her2 positive cells and effector cell's bridging molecules (Bridge between Her2-positive cells and effector cells, Her2-BTEC) recombination carrier, the bridging molecules Include: the part A with the specific binding of Her2 positive cell action target spot, and specifically bound with effector cell's action target spot Part B.
In an aspect, the recombination carrier is selected from non-viral gene vector, such as standard plasmid or other ring-types Express box.Preferably, the recombination carrier is selected from minicircle dna carrier.
In an aspect, the recombination carrier is selected from recombinant expression carrier.Preferably, the recombination carrier Selected from prokaryotic expression carrier or carrier for expression of eukaryon.It is furthermore preferred that the recombination carrier is selected from carrier for expression of eukaryon.Especially Preferably, the recombination carrier is selected from the recombinant expression carrier for mammalian cell expression.
In an aspect, the bridging molecules are selected from albumen or polypeptide.Preferably, the bridging molecules are selected from double special Property antibody.It is furthermore preferred that the bridging molecules are selected from people-monkey intercrossing bispecific antibody (Human-Monkey cross- Reactive Bispecific Antibody, hm-BsAb).
In an aspect, the part A and the part B are respectively selected from protein molecular or peptide molecule.Preferably, institute State part A and part B be respectively selected from Fab, Fab ', single-chain antibody (scFv), single domain antibody (VHH), single-chain T-cell receptor (scTCR) and it is other.
In an aspect, the Her2 positive cell is selected from Her2 positive cancer cell, Her2 overexpressing cell or other. The effector cell is selected from T cell, NK cell or other.
In an aspect, the action target spot of the part A specific binding is selected from each epitope of Her2.It is described Part B specific binding action target spot be selected from CD3, CD16, CD28,4-1BB, OX40, TCR, CD56, NKG2D, NCR or its It.
In an aspect, the part A is selected from single-chain antibody (scFv), it includes: heavy chain variable region and light chain variable Area, amino acid sequence is as shown in SEQ ID NO:1 and 3.
In an aspect, the part B be selected from single-chain antibody (scFv), (1) it includes: heavy chain variable region and light chain can Become area, amino acid sequence is such as selected from shown in the group of SEQ ID NO:5 and 7 or 9 and 11；The work of the part B specific binding CD3 is selected from target spot；Or (2) it includes heavy chain variable region and light chain variable region, amino acid sequence such as SEQ ID NO:13 Shown in 15；The action target spot of the part B specific binding is selected from CD16.
In an aspect, the heavy chain variable region that the part A includes has with sequence shown in SEQ ID NO:1 at least 90%, sequence shown in the amino acid sequence of 95%, 98% or 99% homology, the light chain variable region for including and SEQ ID NO:3 Amino acid sequence with the homology of at least 90%, 95%, 98% or 99%, and have and part described in embodiment before A has identical function, that is, specifically binds identical action target spot.
In an aspect, the heavy chain variable region that the part B includes has with sequence shown in SEQ ID NO:5 or 9 extremely The amino acid sequence of few 90%, 95%, 98% or 99% homology, the light chain variable region for including have with SEQ ID NO:7 or The amino acid sequence of sequence shown in 11 at least 90%, 95%, 98% or 99% homology, and have and institute in embodiment before Part B is stated with identical function, that is, specifically binds identical action target spot.
It is described the present invention provides a kind of recombination carrier for expressing Her2 positive cell and effector cell's bridging molecules Recombination carrier includes the encoding gene of the bridging molecules.
In an aspect, the recombination carrier includes: the encoding gene of part A, and/or the volume comprising part B Code gene.
In an aspect, part A is selected from single-chain antibody (scFv), and encoding gene includes: heavy chain variable region and light chain The encoding gene of variable region, nucleotide sequence is as shown in SEQ ID NO:2 and 4.
In one aspect, part B is selected from single-chain antibody (scFv), and encoding gene (1) includes: heavy chain variable region and light chain The encoding gene of variable region, nucleotide sequence is as shown in the group for being selected from SEQ ID NO:6 and 8 or 10 and 12；Or (2) packet Contain: the encoding gene of heavy chain variable region and light chain variable region, nucleotide sequence is as shown in SEQ ID NO:14 and 16.
In an aspect, the recombination carrier includes: the core with the encoding gene of above-mentioned part A and/or part B Nucleotide sequence has the nucleotide sequence of at least 90%, 95%, 98% or 99% homology, and the bridging molecules tool that coding obtains There is function identical with bridging molecules described in embodiment before.It is well known to those skilled in the art, it is encoded not changing In the case where amino acid, one or more codons in the coding gene sequence can carry out equal justice replacement, such as one or Several codons, such as 1,2,3,4,5,6,7,8,9,10,15,20,30,40,50 codon.
Bridging molecules expressed by the present invention provides a kind of recombination carrier as described in embodiment before.
In an aspect, the recombination carrier is selected from non-viral gene vector, such as standard plasmid or other ring-types Express box.Preferably, the recombination carrier is selected from minicircle dna carrier.
The present invention provides the preparation method of recombination carrier described in embodiment before one kind, specific steps packets It includes:
(1) light chain variable region (VL) sequence of Her2 antibody, CD3 antibody and CD16 antibody is obtained respectively from the prior art Column, heavy chain variable region (VH) sequence；
(2) it according to VH, VL sequence design in above-mentioned (1) at polymorphic bridging molecules, and constructs described in expression The recombination carrier of bridging molecules；
Optional,
(3) expression of the recombination carrier is identified in vivo and in vitro, and is detected expression product and mediated effector cell To the fragmentation effect of Her2 positive cell.
In an aspect, the Her2 positive cell is selected from Her2 positive cancer cell, Her2 overexpressing cell or other. The effector cell is selected from T cell, NK cell or other.
In an aspect, the recombination carrier is selected from non-viral gene vector, such as standard plasmid or other ring-types Express box.Preferably, the recombination carrier is selected from minicircle dna carrier.
In an aspect, the bridging molecules are selected from albumen or polypeptide.Preferably, the bridging molecules are selected from double special Property antibody.Particularly preferred, the bridging molecules are selected from people-monkey intercrossing bispecific antibody (Human-Monkey cross- Reactive Bispecific Antibody, hm-BsAb).
The present invention provides a kind of host cell, the recombination carrier described in embodiment comprising before, or by before The obtained recombination carrier of preparation method described in embodiment.
In one embodiment, the host cell includes that bacterial cell, yeast cell, insect cell or lactation are dynamic Object cell.
The present invention provides the preparation method of bridging molecules described in embodiment before one kind, specific steps include:
(1) the recombination carrier is constructed；
(2) by the recombination vector introduction host cell；
(3) under conditions suitable for the expression, host cell is cultivated, inducing expression isolates and purifies, and obtains the bridge joint point Son.
The present invention provides a kind of pharmaceutical composition, the recombination carrier described in embodiment comprising before, or by it The obtained recombination carrier of preparation method described in preceding embodiment, or the recombination as described in embodiment before carry Bridging molecules expressed by body and pharmaceutically acceptable carrier.
In an aspect, pharmaceutical preparation can be made in described pharmaceutical composition according to conventional methods.In production process, preferably Recombination carrier or bispecific antibody are mixed with pharmaceutically acceptable carrier or diluted with carrier.When carrier is as dilute It can be solid, semisolid or liquid when releasing agent.Preparation is selected from tablet, pill, pulvis, capsule, suspension, emulsion, solution The forms such as agent, aerosol, injection solution.Suitable carrier, excipient or diluent include water, lactose, glucose, sucrose, Sorbierite, mannitol, calcium silicates, cellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, nipasol, talcum Powder, magnesium stearate and mineral oil etc..Preparation can also include filler, anticoagulant, lubricant, moisturizer, flavoring agent, emulsification Agent, preservative etc..
The present invention provides recombination carrier described in embodiment before, the bridging molecules, the host cells Or described pharmaceutical composition is preparing the purposes in the drug for treating Her2 associated cancer.Preferably, the Her2 is related Cancer is selected from breast cancer, non-small cell lung cancer, salivary-gland carcinoma, gastric cancer, intestinal cancer, cancer of pancreas, bladder cancer, carcinoma of endometrium, ovary Cancer.Particularly preferred, the Her2 associated cancer is selected from breast cancer, gastric cancer.
The positive effect of the present invention includes: Her2 positive cell and the effector cell of the first public Her2 specificity of the present invention Minicircle dna carrier (MC.Her2-BTEC) design scheme of bridging molecules, the treatment suitable for Her2 associated cancer.Her2- BTEC can increase substantially effector cell to the lethal effect of Her2 positive cell.Cytotoxic effect is dense with Her2-BTEC The increase of degree and constantly increase.Compared with untreated lotus knurl control mice, Her2-BTEC treatment can significantly reduce tumor load And extend the life cycle of tumor-bearing mice.Her2-BTEC of the invention is other than combining the antigen molecule of people, moreover it is possible to intersect identification The antigen of monkey is conducive to pre-clinical safety of BTEC (BsAb, the bispecific antibody) drug based on non-human primate model Property and efficiency evaluation.As it can be seen that Her2-BTEC of the invention, minicircle dna carrier are for preventing and treating Her2 associated cancer tool There is good prospect, and provides new Clinical Thinking.
Detailed description of the invention
Fig. 1: pMC.Her2-BTEC minicircle dna matrix grain Vector map.
The preparation of Fig. 2: MC.Her2-BTEC micro-loop is expressed with BTEC；(left side) agarose gel electrophoresis detects MC；(right side) SDS- PAGE detects BTEC protein expression；Lane A: cell conditioned medium；Lane B: liquid is flowed through；Lane C:20mM imidazole elution；Lane D:50mM imidazole elution；Lane E:200mM imidazole elution；The elution of Lane F:500mM imidazoles.
Fig. 3: Her2-BTEC mediates effector cell (T cell) to the cytotoxicity of target cell (human ovarian cancer SKOV3 cell) Effect；The oophoroma SKOV3 cell of T cell and the Her2 positive is incubated for 12 hours altogether, effect target ratio 10:1, LDH method for releasing detection The cell killing rate that Her2-BTEC is mediated.
Fig. 4: the result of human ovarian cancer transplantable tumor mouse Experiment on therapy.
Specific embodiment
Following experimental methods are conventional method unless otherwise instructed, used experimental material unless otherwise instructed, It can easily be obtained from commercial company.Without departing from the spirit of the invention, those skilled in the art combine well-known technique, Many modifications can be made to the present invention, such modification also falls into the scope of the present invention.
The building of embodiment 1, Her2 positive cell and effector cell's bridging molecules (Her2-BTEC)
Design Her2-BTEC simultaneously constructs corresponding minicircle dna expression vector, and Her2-BTEC expression cassette includes various structures, Divide column as follows:
(1) scFv-scFv structure:
Wherein, SP is signal peptide (Signal Peptide)；Linker is catenation sequence.VLRepresent light chain variable region, VHGeneration Table heavy chain variable region.The position anti-Her2scFv, antiCD3 (or antiCD16) scFv is interchangeable.
Wherein, antiHer2.VHAmino acid sequence as shown in SEQ ID NO:1, the nucleotide sequence of encoding gene is such as Shown in SEQ ID NO:2；antiHer2.VLAmino acid sequence as shown in SEQ ID NO:3, the nucleotide sequence of encoding gene As shown in SEQ ID NO:4；antiCD3.VHAmino acid sequence as shown in SEQ ID NO:5 or 9, the nucleotide of encoding gene Sequence is as shown in SEQ ID NO:6 or 10；antiCD3.VLAmino acid sequence as shown in SEQ ID NO:7 or 11, encode base The nucleotide sequence of cause is as shown in SEQ ID NO:8 or 12；antiCD16.VHAmino acid sequence such as SEQ ID NO:13 institute Show, the nucleotide sequence of encoding gene is as shown in SEQ ID NO:14；antiCD16.VLAmino acid sequence such as SEQ ID NO: Shown in 15, the nucleotide sequence of encoding gene is as shown in SEQ ID NO:16.
(2) bispecific antibody DART (Dual-Affinity Re-Targeting antibody) structure:
DART expression cassette is translated, two chains (Chain 1, Chain 2) are formed after shearing；This two chains pass through pairing E/K-coli forms stable heterodimer.The position Chain1, Chain2 is interchangeable.
Wherein, SP is signal peptide (Signal Peptide)；Linker is catenation sequence, and Furin is that furin protease is cut Enzyme site, 2A are 2A self cleavage site.The amino acid sequence of Furin shearing site is R-X- [R/K]-R (such as RRKR), and X refers to Act as a kind of amino acid；The helical structure that E/K coli is opposite for electrical property, matches, and K coli and E coli interchanging positions； 2A includes E2A, F2A, P2A and T2A etc..
Wherein, antiHer2.VH、antiHer2.VL、antiCD3.VH、antiCD3.VL、antiCD16.VH、 antiCD16.VLThe amino acid sequence and its encoding gene of each element are consistent in above-mentioned (1).
(3) bispecific antibody DART-Fc structure:
DART-Fc expression cassette is translated, two chains (Chain 1, Chain 2) are formed after shearing；Chain 1 and Chain 2 E/K-coli and Fc " knob-into-hole " structure by matching forms stable heterodimer."knob-into- Hole " design both can avoid the generation of homodimer, moreover it is possible to increase half-life period.The position Chain1, Chain2 is interchangeable.
Wherein, SP is signal peptide (Signal Peptide)；Linker is catenation sequence, and Furin is that furin protease is cut Enzyme site, 2A are 2A self cleavage site.The amino acid sequence of Furin shearing site is R-X- [R/K]-R (such as RRKR), and X refers to Act as a kind of amino acid；The helical structure that E/K coli is opposite for electrical property, matches, and K coli and E coli interchanging positions； Fc-knob and Fc-hole is respectively Fc sections of CH2-CH3 mutant of IgG, their also interchanging positions；2A include E2A, F2A, P2A and T2A etc..
Wherein, antiHer2.VH、antiHer2.VL、antiCD3.VH、antiCD3.VL、antiCD16.VH、 antiCD16.VLThe amino acid sequence and its encoding gene of each element are consistent in above-mentioned (1).
Embodiment 2, the building of Her2-BTEC minicircle dna carrier and the preparation of micro-loop (MC)
One, experimental method
1, the encoding gene of Her2-BTEC is synthesized, for convenience of BTEC protein purification and detection, in C-terminal insertion 6xHis mark Label.
2, suitable site double digestion is selected to linearize minicircle dna empty carrier pMC.BESPX.
3, using seamless clone or conventional cloning methods (e.g., digestion-connection) by the encoding gene of Her2-BTEC It is inserted into the empty carrier pMC.BESPX of linearisation, is built into minicircle dna carrier pMC.Her2-BTEC.
4, minicircle dna carrier pMC.Her2-BTEC converts Escherichia coli E coli.ZYCY10P3S2T, by standard micro-loop system Preparation Method obtains micro-loop MC.Her2-BTEC.
Wherein, minicircle dna empty carrier pMC.BESPX, engineering bacteria E coli.ZYCY10P3S2T and micro-loop preparation method See reference document Nat Biotechnol.2010,28:1287-1289.
Two, experimental result
Fig. 2 (left side) shows the agarose gel electrophoresis testing result of MC.Her2-BTEC micro-loop preparation.By in figure it is found that By the construction method of Her2-BTEC minicircle dna carrier, micro-loop MC.Her2-BTEC can be prepared.
Embodiment 3, the expression of Her2-BTEC, purifying
One, expression of the Her2-BTEC in 293T cell
Above-mentioned minicircle dna is transfected by 293T cell using superfect plasmid transfection kit (Invitrogen company), 293T cells and supernatant is collected respectively after cultivating three days in serum free medium.
Two, the purifying of Her2-BTEC
1, it collects cell culture supernatant and carries out low temperature ultracentrifugation, take supernatant.
2, Her2-BTEC using His-Tag affine resin (cOmplete His-Tag Purification Resin, Roche it) purifies.
3, albumen after purification uses PAGE or Western Blot qualitative detection, and is detected using Bradford standard measure Protein concentration.
4, purified product is placed in -20 DEG C or -80 DEG C of long-term preservations.
Three, experimental result
Fig. 2 (right side) shows the SDS-PAGE testing result of Her2-BTEC expression.By in figure it is found that by cell transfecting, Protein expression, purifying method, Her2-BTEC can be prepared.
The Binding experiment of embodiment 4, Her2-BTEC
Utilize Flow cytometry Her2-BTEC and target cell (Target, T), effector cell (Effector, E) In conjunction with activity, include the following steps:
1, cell culture: target cell (Her2 positive cell, such as SKOV3 cell)；(T cell or NK are thin by effector cell Born of the same parents).
2, collect cell: attached cell is digested using pancreatin, and centrifugation abandons supernatant and obtains cell after adding serum-containing media to neutralize； Suspension cell is directly collected by centrifugation.
3, two kinds of cells are washed 2 times with pre-cooling PBS, and 200g is centrifuged 4min, are collected cell, are counted respectively.
4, each experimental group distributes 1 × 10 respectively5A target cell and effector cell, are grouped as follows:
Blank group (Her2-BTEC is not added) and Her2-BTEC group
5,100 μ L/ group of Her2-BTEC solution is added, is incubated for 30min on ice.
6,1mL pre-cooling PBS washing is added, 200g is centrifuged 4min, collects cell, adds pre-cooling and mixed anti-flag in advance The 100 μ L of PBS of antibody, is incubated for 30min on ice.
7,1mL pre-cooling PBS washing is added, 200g is centrifuged 4min, collects cell, 100 μ l PBS are added and are resuspended, up flow type is seen It examines and combines situation.
Embodiment 5, Her2-BTEC mediate effector cell to kill target cell
One, experimental group
Mediate effector cell (Effector, E) thin to target using LDH (lactic dehydrogenase) method for releasing measurement Her2-BTEC The lethal effect of born of the same parents (Target, T).
Experimental material: effector cell's (T cell or NK cell), target cell (Her2 positive cell, such as human ovarian cancer SKOV3 cell), Her2-BTEC (Her2 positive cell and effector cell's bridging molecules).
Table 1Her2-BTEC mediates the different grouping of effector cell killing target cell
Two, experimental method
1, shift to an earlier date 1 day target cell is inoculated on 96 orifice plates, cell inoculation amount is 2 × 104/ hole；Grouping is set (such as simultaneously Shown in table 1；Every group of 3 multiple holes).
2, fresh opti-MEM culture medium, 100 holes μ L/ are replaced within second day.
3, effector cell (E) counts and is resuspended in opti-MEM culture medium, according to T: E=1: 10 ratio, according to (a) The effector cell (2.0 × 10 of respective numbers is added in middle grouping5/ hole, 100 holes μ L/).
4, according to grouping, the Her2-BTEC of respective concentration is added in every hole in corresponding hole, is mixed；In target cell maximum It also needs that cell pyrolysis liquid smudge cells are added in group corresponding aperture, sufficiently to discharge LDH.
5,37 DEG C of cell incubators are incubated for 12 hours.
6, it according to LDH kit (Promega, the U.S.) specification, detects LDH burst size and calculates cell killing rate, it is public Formula is as follows:
Cell killing rate=(experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell is spontaneous) x 100%
Two, experimental result
Fig. 3 shows that Her2-BTEC mediates effector cell (T cell) to the thin of target cell (human ovarian cancer SKOV3 cell) Cellular toxicity effect.By in figure it is found that cytotoxic effect constantly increases with the increase of Her2-BTEC concentration.
Embodiment 6, Her2 positive graft tumor mouse Experiment on therapy
One, experimental method
1, NOD/SCID mouse inoculation band firefly luciferase (firefly luciferase, luc) of immune deficiency is marked The Her2 positive tumor cell (such as SKOV3-luc) of note.
2, after inoculated tumour cell 7 days, with small animal living body imaging system (In Vivo Imaging System, IVIS) Record luciferase fluorescence intensity, monitoring mouse tumor formation situation；While grouping is set, including control group (Control), T thin Born of the same parents group and three groups of experimental group, every group of 5 mouse.
3, control group does not apply any treatment, T cell group injects that human T-cell, experimental group is in regulation in stipulated time point Between point injection MC.Her2-BTEC minicircle dna and inject human T-cell.
4, after implementing different treatment processing by grouping, periodic monitoring Survival simultaneously grows feelings with IVIS system tracking of knub Condition (record tumor-bearing mice luciferase fluorescence intensity).
Two, experimental result
Fig. 4 shows the result of human ovarian cancer transplantable tumor mouse Experiment on therapy.By in figure it is found that with untreated lotus knurl pair It is compared according to mouse, Her2-BTEC treatment can significantly reduce tumor load and extend the life cycle of tumor-bearing mice.
Sequence table
SEQ ID NO:1 anti-Her2.VH amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFT ISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID NO:2 anti-Her2.VH nucleotide sequence
gaagtgcagctggtggagtcaggaggaggactggtgcagccaggaggatctctgagactgtcttgcgc cgccagcggcttcaacatcaaggacacctacatccattgggtccggcaggctccaggaaaaggactcgagtgggtg gccagaatctaccccaccaacggctacacccgctacgccgatagcgtgaaaggccggttcaccatcagcgccgata ccagcaagaacaccgcctacctgcagatgaacagcctgagagccgaggacaccgccgtgtactattgtagccggtg gggaggagacggcttctacgccatggactattggggccagggaacactggtgacagtgtcttcc
SEQ ID NO:3 anti-Her2.VL amino acid sequence
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGT DFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKLEIK
SEQ ID NO:4 anti-Her2.VL nucleotide sequence
gacatccagatgacccagagccctagctctctgagcgccagcgtgggcgatagagtgaccatcacctg cagagcctctcaggacgtgaacaccgcagtggcttggtaccagcagaagccagggaaggcccctaagctgctgatc tacagcgcctctttcctgtacagcggcgtgcctagcaggttcagcggaagcagaagcggcaccgatttcaccctga ccatcagctctctgcagccagaggacttcgccacctactactgccagcagcactacacaacccctcctacctttgg ccagggcacaaagctggagatcaaa
SEQ ID NO:5 anti-CD3.VH-1 amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGR FTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS
SEQ ID NO:6 anti-CD3.VH-1 nucleotide sequence
gaagtgcagctggtggaaagcggaggaggactggtgcagccaggaggatctctgagactgtcttgcgcc gccagcggctttacattcagcacctacgccatgaattgggtccggcaggctccaggaaaaggactcgagtgggtcg gaaggatccggagcaagtacaacaactacgccacctactacgccgacagcgtgaagggcaggttcaccatcagccg ggacgacagcaagaacagcctgtacctgcagatgaacagcctgaagaccgaggacacagccgtgtactattgcgtg cgccacggcaacttcggcaacagctacgtcagctggttcgcctattgggggcagggaacactggtgacagtgtcta gc
SEQ ID NO:7 anti-CD3.VL-1 amino acid sequence
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLL GGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG
SEQ ID NO:8 anti-CD3.VL-1 nucleotide sequence
caggcagtggtgacccaggaaccttctctgaccgtgtctccaggcggaacagtgacactgacctgcag aagcagcacaggcgccgtgacaaccagcaactacgccaattgggtgcagcagaagccaggacaggcccctagaggc ctgattggaggcacaaacaagagagccccttggaccccagccagattctccggatctctgctgggaggcaaagccg ccctgacaatcacaggagctcaggccgaagacgaggccgactactattgcgccctctggtacagcaacctctgggt gttcggcggaggaacaaagctgacagtgctggga
SEQ ID NO:9 anti-CD3.VH-2 amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKDLEWVALINPYKGVSTYNQKFKDRFT ISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS
SEQ ID NO:10 anti-CD3.VH-2 nucleotide sequence
gaggtgcagctggtggagtccggaggaggactggtgcagccaggaggcagcctgagactgtcctgtgc cgcctccggctattcttttaccggctacacaatgaattgggtgaggcaggcaccaggcaaggatctggagtgggtg gccctgatcaacccttataagggcgtgtccacctacaatcagaagttcaaggatcggtttaccatctctgtggaca agagcaagaacacagcctatctgcagatgaatagcctgcgcgctgaagacaccgccgtgtactactgtgcccggtc cggctactatggcgattctgactggtacttcgacgtgtggggccagggcaccctggtcacagtgtctagc
SEQ ID NO:11 anti-CD3.VL-2 amino acid sequence
DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGS GTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLELK
SEQ ID NO:12 anti-CD3.VL-2 nucleotide sequence
gacatccagatgacccagtccccatcctctctgtctgccagcgtgggcgatcgggtgaccatcacatg tagagcctctcaggacatcaggaactatctgaattggtaccagcagaagccaggcaaggcccccaagctgctgatc tactatacctccaggctggagtctggagtgcctagccggttttccggctccggaagcggaaccgattacaccctga caatcagctccctgcagccagaggacttcgccacatactattgccagcagggcaataccctgccctggacatttgg ccagggcaccaagctggagctgaag
SEQ ID NO:13 anti-CD16.VH amino acid sequence
QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS
SEQ ID NO:14 anti-CD16.VH nucleotide sequence
caagtccagcttgtgcagtcaggtgctgaggttaaaaaaccaggagaaagtctgaaggtatcatgtaa ggcctcaggttatacgtttacttcatattacatgcactgggtgcgacaggctcctggtcaggggttggagtggatg ggaatcatcaatccatccggtggtagcaccagctacgcacaaaagtttcagggtcgcgtgacaatgacaagagaca cgtccacgtccacggtctacatggaattgtcatccctgagatcagaggacaccgccgtatattattgtgcacgcgg cagtgcttactactacgatttcgcagattattgggggcaggggactttggttacagtctcctcc
SEQ ID NO:15 anti-CD16.VL amino acid sequence
SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNT ATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL
SEQ ID NO:16 anti-CD16.VL nucleotide sequence
tcctacgttctcactcagcccagtagtgtctcagttgctccagggcaaacggccacgattagttgcgg aggtcacaacataggcagtaagaatgtacattggtaccaacagcgaccaggccagagccccgttttggtcatctat caggataataagcggccaagtggaataccggagcggttcagcggtagtaacagtgggaacaccgccactctgacta tatccggtacgcaagctatggacgaagcagactactattgccaggtgtgggataactacagcgtactgttcggagg cgggacgaaacttacagtcttg
SEQ ID NO:17 Fc-knob amino acid sequence
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:18 Fc-knob nucleotide sequence
gctccagaagcagcaggaggacctagcgtgttcctgttccctcccaagcctaaggacaccctgatgat cagccggaccccagaagtgacttgcgtggtggtggacgtgtcccacgaagaccccgaggtcaagttcaattggtac gtggacggagtggaggtgcacaacgctaagaccaagcccagggaggagcagtacaacagcacctacagggtggtgt ccgtgctgacagtgctgcaccaggattggctgaacggcaaggagtacaagtgcaaggtgtccaacaaggccctgcc agcccctatcgagaagaccatcagcaaggccaagggccagcctagagaacctcaggtgtacaccctgccccctagc agagaggagatgaccaagaaccaggtctccctctggtgcctggtgaagggcttctaccctagcgacatcgccgtgg agtgggaatctaacggtcagccagagaacaactacaagaccacccccccagtgctggacagcgacggcagcttctt cctgtacagcaagctgaccgtggacaaaagccgctggcagcagggcaacgtgttctcttgcagcgtgatgcacgag gccctgcacaaccactacacccagaagagcctgagcctgagcccaggaaag
SEQ ID NO:19 Fc-hole amino acid sequence
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK
SEQ ID NO:20 Fc-hole nucleotide sequence
gctccagaagcagcaggaggacctagcgtgttcctgttccctcccaagcctaaggacaccctgatgat cagccggaccccagaagtgacttgcgtggtggtggacgtgtcccacgaagaccccgaggtcaagttcaattggtac gtggacggagtggaggtgcacaacgctaagaccaagcccagggaggagcagtacaacagcacctacagggtggtgt ccgtgctgacagtgctgcaccaggattggctgaacggcaaggagtacaagtgcaaggtgtccaacaaggccctgcc agcccctatcgagaagaccatcagcaaggccaagggccagcctagagaacctcaggtgtacaccctgccccctagc agagaggagatgaccaagaaccaggtgtccctgtcttgcgccgtgaagggcttctaccctagcgacatcgccgtgg agtgggaatctaacggtcagccagagaacaactacaagaccaccccccccgtgctggatagcgacggcagcttctt cctggtgtccaagctgaccgtggacaaaagccgctggcagcagggcaacgtgttctcttgcagcgtgatgcacgag gccctgcataacagatacacccagaagagcctgagcctgagcccaggaaag
SEQ ID NO:21 E4coli amino acid sequence
EVAACEKEVAALEKEVAALEKEVAALEK
SEQ ID NO:22 E4coli nucleotide sequence
gaagtggcagcttgcgagaaggaagtggccgctctggagaaggaagtggccgctctggaaaaggaagt ggcagccctggagaag
SEQ ID NO:23 K4coli amino acid sequence
KVAACKEKVAALKEKVAALKEKVAALKE
SEQ ID NO:24 K4coli nucleotide sequence
aaggtggcagcttgcaaggagaaggtggccgctctgaaggagaaagtggccgctctgaaggagaaagt ggccgccctgaaggag
SEQUENCE LISTING
<110>Shenzhen Xin Nuo micro-loop Biotechnology Co., Ltd
<120>bridging molecules and its application of minicircle dna expression connection HER2 positive cell and effector cell
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 120
<212> PRT
<213>anti-Her2.VH amino acid sequence
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 2
<211> 360
<212> DNA
<213>anti-Her2.VH nucleotide sequence
<400> 2
gaagtgcagc tggtggagtc aggaggagga ctggtgcagc caggaggatc tctgagactg 60
tcttgcgccg ccagcggctt caacatcaag gacacctaca tccattgggt ccggcaggct 120
ccaggaaaag gactcgagtg ggtggccaga atctacccca ccaacggcta cacccgctac 180
gccgatagcg tgaaaggccg gttcaccatc agcgccgata ccagcaagaa caccgcctac 240
ctgcagatga acagcctgag agccgaggac accgccgtgt actattgtag ccggtgggga 300
ggagacggct tctacgccat ggactattgg ggccagggaa cactggtgac agtgtcttcc 360
<210> 3
<211> 107
<212> PRT
<213>anti-Her2.VL amino acid sequence
<400> 3
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 4
<211> 321
<212> DNA
<213>anti-Her2.VL nucleotide sequence
<400> 4
gacatccaga tgacccagag ccctagctct ctgagcgcca gcgtgggcga tagagtgacc 60
atcacctgca gagcctctca ggacgtgaac accgcagtgg cttggtacca gcagaagcca 120
gggaaggccc ctaagctgct gatctacagc gcctctttcc tgtacagcgg cgtgcctagc 180
aggttcagcg gaagcagaag cggcaccgat ttcaccctga ccatcagctc tctgcagcca 240
gaggacttcg ccacctacta ctgccagcag cactacacaa cccctcctac ctttggccag 300
ggcacaaagc tggagatcaa a 321
<210> 5
<211> 125
<212> PRT
<213>anti-CD3.VH-1 amino acid sequence
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 375
<212> DNA
<213>anti-CD3.VH-1 nucleotide sequence
<400> 6
gaagtgcagc tggtggaaag cggaggagga ctggtgcagc caggaggatc tctgagactg 60
tcttgcgccg ccagcggctt tacattcagc acctacgcca tgaattgggt ccggcaggct 120
ccaggaaaag gactcgagtg ggtcggaagg atccggagca agtacaacaa ctacgccacc 180
tactacgccg acagcgtgaa gggcaggttc accatcagcc gggacgacag caagaacagc 240
ctgtacctgc agatgaacag cctgaagacc gaggacacag ccgtgtacta ttgcgtgcgc 300
cacggcaact tcggcaacag ctacgtcagc tggttcgcct attgggggca gggaacactg 360
gtgacagtgt ctagc 375
<210> 7
<211> 110
<212> PRT
<213>anti-CD3.VL-1 amino acid sequence
<400> 7
Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110
<210> 8
<211> 330
<212> DNA
<213>anti-CD3.VL-1 nucleotide sequence
<400> 8
caggcagtgg tgacccagga accttctctg accgtgtctc caggcggaac agtgacactg 60
acctgcagaa gcagcacagg cgccgtgaca accagcaact acgccaattg ggtgcagcag 120
aagccaggac aggcccctag aggcctgatt ggaggcacaa acaagagagc cccttggacc 180
ccagccagat tctccggatc tctgctggga ggcaaagccg ccctgacaat cacaggagct 240
caggccgaag acgaggccga ctactattgc gccctctggt acagcaacct ctgggtgttc 300
ggcggaggaa caaagctgac agtgctggga 330
<210> 9
<211> 122
<212> PRT
<213>anti-CD3.VH-2 amino acid sequence
<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Asp Leu Glu Trp Val
35 40 45
Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 10
<211> 366
<212> DNA
<213>anti-CD3.VH-2 nucleotide sequence
<400> 10
gaggtgcagc tggtggagtc cggaggagga ctggtgcagc caggaggcag cctgagactg 60
tcctgtgccg cctccggcta ttcttttacc ggctacacaa tgaattgggt gaggcaggca 120
ccaggcaagg atctggagtg ggtggccctg atcaaccctt ataagggcgt gtccacctac 180
aatcagaagt tcaaggatcg gtttaccatc tctgtggaca agagcaagaa cacagcctat 240
ctgcagatga atagcctgcg cgctgaagac accgccgtgt actactgtgc ccggtccggc 300
tactatggcg attctgactg gtacttcgac gtgtggggcc agggcaccct ggtcacagtg 360
tctagc 366
<210> 11
<211> 107
<212> PRT
<213>anti-CD3.VL-2 amino acid sequence
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 12
<211> 321
<212> DNA
<213>anti-CD3.VL-2 nucleotide sequence
<400> 12
gacatccaga tgacccagtc cccatcctct ctgtctgcca gcgtgggcga tcgggtgacc 60
atcacatgta gagcctctca ggacatcagg aactatctga attggtacca gcagaagcca 120
ggcaaggccc ccaagctgct gatctactat acctccaggc tggagtctgg agtgcctagc 180
cggttttccg gctccggaag cggaaccgat tacaccctga caatcagctc cctgcagcca 240
gaggacttcg ccacatacta ttgccagcag ggcaataccc tgccctggac atttggccag 300
ggcaccaagc tggagctgaa g 321
<210> 13
<211> 120
<212> PRT
<213>anti-CD16.VH amino acid sequence
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 14
<211> 360
<212> DNA
<213>anti-CD16.VH nucleotide sequence
<400> 14
caagtccagc ttgtgcagtc aggtgctgag gttaaaaaac caggagaaag tctgaaggta 60
tcatgtaagg cctcaggtta tacgtttact tcatattaca tgcactgggt gcgacaggct 120
cctggtcagg ggttggagtg gatgggaatc atcaatccat ccggtggtag caccagctac 180
gcacaaaagt ttcagggtcg cgtgacaatg acaagagaca cgtccacgtc cacggtctac 240
atggaattgt catccctgag atcagaggac accgccgtat attattgtgc acgcggcagt 300
gcttactact acgatttcgc agattattgg gggcagggga ctttggttac agtctcctcc 360
<210> 15
<211> 106
<212> PRT
<213>anti-CD16.VL amino acid sequence
<400> 15
Ser Tyr Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val
20 25 30
His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45
Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 16
<211> 318
<212> DNA
<213>anti-CD16.VL nucleotide sequence
<400> 16
tcctacgttc tcactcagcc cagtagtgtc tcagttgctc cagggcaaac ggccacgatt 60
agttgcggag gtcacaacat aggcagtaag aatgtacatt ggtaccaaca gcgaccaggc 120
cagagccccg ttttggtcat ctatcaggat aataagcggc caagtggaat accggagcgg 180
ttcagcggta gtaacagtgg gaacaccgcc actctgacta tatccggtac gcaagctatg 240
gacgaagcag actactattg ccaggtgtgg gataactaca gcgtactgtt cggaggcggg 300
acgaaactta cagtcttg 318
<210> 17
<211> 217
<212> PRT
<213>Fc-knob amino acid sequence
<400> 17
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 18
<211> 651
<212> DNA
<213>Fc-knob nucleotide sequence
<400> 18
gctccagaag cagcaggagg acctagcgtg ttcctgttcc ctcccaagcc taaggacacc 60
ctgatgatca gccggacccc agaagtgact tgcgtggtgg tggacgtgtc ccacgaagac 120
cccgaggtca agttcaattg gtacgtggac ggagtggagg tgcacaacgc taagaccaag 180
cccagggagg agcagtacaa cagcacctac agggtggtgt ccgtgctgac agtgctgcac 240
caggattggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaggc cctgccagcc 300
cctatcgaga agaccatcag caaggccaag ggccagccta gagaacctca ggtgtacacc 360
ctgcccccta gcagagagga gatgaccaag aaccaggtct ccctctggtg cctggtgaag 420
ggcttctacc ctagcgacat cgccgtggag tgggaatcta acggtcagcc agagaacaac 480
tacaagacca cccccccagt gctggacagc gacggcagct tcttcctgta cagcaagctg 540
accgtggaca aaagccgctg gcagcagggc aacgtgttct cttgcagcgt gatgcacgag 600
gccctgcaca accactacac ccagaagagc ctgagcctga gcccaggaaa g 651
<210> 19
<211> 217
<212> PRT
<213>Fc-hole amino acid sequence
<400> 19
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 20
<211> 651
<212> DNA
<213>Fc-hole nucleotide sequence
<400> 20
gctccagaag cagcaggagg acctagcgtg ttcctgttcc ctcccaagcc taaggacacc 60
ctgatgatca gccggacccc agaagtgact tgcgtggtgg tggacgtgtc ccacgaagac 120
cccgaggtca agttcaattg gtacgtggac ggagtggagg tgcacaacgc taagaccaag 180
cccagggagg agcagtacaa cagcacctac agggtggtgt ccgtgctgac agtgctgcac 240
caggattggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaggc cctgccagcc 300
cctatcgaga agaccatcag caaggccaag ggccagccta gagaacctca ggtgtacacc 360
ctgcccccta gcagagagga gatgaccaag aaccaggtgt ccctgtcttg cgccgtgaag 420
ggcttctacc ctagcgacat cgccgtggag tgggaatcta acggtcagcc agagaacaac 480
tacaagacca ccccccccgt gctggatagc gacggcagct tcttcctggt gtccaagctg 540
accgtggaca aaagccgctg gcagcagggc aacgtgttct cttgcagcgt gatgcacgag 600
gccctgcata acagatacac ccagaagagc ctgagcctga gcccaggaaa g 651
<210> 21
<211> 28
<212> PRT
<213>E4 coli amino acid sequence
<400> 21
Glu Val Ala Ala Cys Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val
1 5 10 15
Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys
20 25
<210> 22
<211> 84
<212> DNA
<213>E4 coli nucleotide sequence
<400> 22
gaagtggcag cttgcgagaa ggaagtggcc gctctggaga aggaagtggc cgctctggaa 60
aaggaagtgg cagccctgga gaag 84
<210> 23
<211> 28
<212> PRT
<213>K4 coli amino acid sequence
<400> 23
Lys Val Ala Ala Cys Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val
1 5 10 15
Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu
20 25
<210> 24
<211> 84
<212> DNA
<213>K4 coli nucleotide sequence
<400> 24
aaggtggcag cttgcaagga gaaggtggcc gctctgaagg agaaagtggc cgctctgaag 60
gagaaagtgg ccgccctgaa ggag 84

Claims (10)

1. a kind of expression Her2 positive cell and effector cell's bridging molecules (Bridge between Her2-positive Cells and effector cells, Her2-BTEC) recombination carrier, which is characterized in that the bridging molecules packet Contain: the part A with the specific binding of Her2 positive cell action target spot, and the portion with the specific binding of effector cell's action target spot Divide B；
Preferably, the recombination carrier is selected from non-viral gene vector, such as standard plasmid or other circular expression cassettes；More Preferably, the recombination carrier is selected from minicircle dna carrier；
Further preferably, the bridging molecules are selected from albumen or polypeptide；It is furthermore preferred that the bridging molecules are anti-selected from bispecific Body；Particularly preferred, the bridging molecules are selected from people-monkey intercrossing bispecific antibody (Human-Monkey cross- Reactive Bispecific Antibody, hm-BsAb)；
It is furthermore preferred that the part A and the part B are respectively selected from protein molecular or peptide molecule；It is particularly preferred, the portion Point A and part B is respectively selected from Fab, Fab ', single-chain antibody (scFv), single domain antibody (VHH), single-chain T-cell receptor (scTCR) With it is other.
2. recombination carrier as described in claim 1, the Her2 positive cell is selected from Her2 positive cancer cell, Her2 crosses table Up to cell or other；The effector cell is selected from T cell, NK cell or other；
Preferably, the action target spot of the part A specific binding is selected from each epitope of Her2；Preferably, the portion The action target spot of B specific binding is divided to be selected from CD3, CD16, CD28,4-1BB, OX40, TCR, CD56, NKG2D, NCR or other.
3. recombination carrier as claimed in claim 1 or 2, the part A is selected from single-chain antibody (scFv), and it includes heavy chains can Become area and light chain variable region, amino acid sequence is as shown in SEQ ID NO:1 and 3；
And/or the part B is selected from single-chain antibody (scFv), (1) it includes heavy chain variable region and light chain variable region, amino Acid sequence is such as selected from shown in the group of SEQ ID NO:5 and 7 or 9 and 11；The action target spot of the part B specific binding is selected from CD3；Or (2) it includes: heavy chain variable region and light chain variable region, amino acid sequence is as shown in SEQ ID NO:13 and 15； The action target spot of the part B specific binding is selected from CD16.
4. the recombination carrier as described in any one of claim 1-3, the recombination carrier includes the bridging molecules Encoding gene；
Preferably, the recombination carrier includes: the encoding gene of part A, and/or the encoding gene comprising part B；
It is furthermore preferred that part A is selected from single-chain antibody (scFv), encoding gene includes: heavy chain variable region and light chain variable region Encoding gene, nucleotide sequence is as shown in SEQ ID NO:2 and 4；
It is furthermore preferred that part B is selected from single-chain antibody (scFv), encoding gene (1) includes: heavy chain variable region and light chain variable region Encoding gene, nucleotide sequence is as shown in the group for being selected from SEQ ID NO:6 and 8 or 10 and 12；Or (2) include: heavy chain The encoding gene of variable region and light chain variable region, nucleotide sequence is as shown in SEQ ID NO:14 and 16.
5. bridging molecules expressed by the recombination carrier as described in any one of claim 1-4.
6. the preparation method of recombination carrier, specific steps include: as described in any one of claim 1-4
(1) Her2 antibody, light chain variable region (VL) sequence of CD3 antibody and CD16 antibody, again are obtained respectively from the prior art Chain variable region (VH) sequence；
(2) according to VH, VL sequence design in above-mentioned (1) at polymorphic bridging molecules, and the expression bridge joint is constructed The recombination carrier of molecule；
Optional,
(3) expression of the recombination carrier is identified in vivo and in vitro, and is detected expression product and mediated effector cell couple The fragmentation effect of Her2 positive cell.
7. preparation method as claimed in claim 6, the Her2 positive cell is selected from Her2 positive cancer cell, Her2 is overexpressed carefully Born of the same parents are other.The effector cell is selected from T cell, NK cell or other；
Preferably, the recombination carrier is selected from non-viral gene vector, it is furthermore preferred that the recombination carrier is selected from micro- Circular DNA carrier；
Further preferably, the bridging molecules are selected from albumen or polypeptide；It is furthermore preferred that the bridging molecules are anti-selected from bispecific Body；Particularly preferred, the bridging molecules are selected from people-monkey intercrossing bispecific antibody (Human-Monkey cross- Reactive Bispecific Antibody, hm-BsAb).
8. a kind of host cell, it includes recombination carriers described in any one of claim 1-4, or by claim 6 or 7 The obtained recombination carrier of preparation method；
Preferably, the host cell includes bacterial cell, yeast cell, insect cell or mammalian cell.
9. a kind of pharmaceutical composition, it includes the recombination carriers as described in any one of claim 1-4, or such as claim 5 bridging molecules, or by the obtained recombination carrier of the preparation method of claim 6 or 7, and can pharmaceutically connect The carrier received.
10. the recombination carrier as described in any one of claim 1-4, as obtained by the preparation method of claim 6 or 7 Recombination carrier, bridging molecules as claimed in claim 5, host cell as claimed in claim 8, or such as claim 9 Described pharmaceutical composition is preparing the purposes in the drug for treating Her2 associated cancer；
Preferably, the Her2 associated cancer be selected from breast cancer, non-small cell lung cancer, salivary-gland carcinoma, gastric cancer, intestinal cancer, cancer of pancreas, Bladder cancer, carcinoma of endometrium, oophoroma；Particularly preferred, the Her2 associated cancer is selected from breast cancer, gastric cancer.
CN201811277450.XA 2018-10-30 2018-10-30 Bridging molecules and its application of minicircle dna expression connection HER2 positive cell and effector cell Pending CN109402168A (en)

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