CN109402168A

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

Info

Publication number: CN109402168A
Application number: CN201811277450.XA
Authority: CN
Inventors: 谌平; 谢亦武; 陈志英
Original assignee: Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
Current assignee: Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
Priority date: 2018-10-30
Filing date: 2018-10-30
Publication date: 2019-03-01
Also published as: WO2020088365A1

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 (V_HH), 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.

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 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.V_LRepresent light chain variable region, V_HGeneration Table heavy chain variable region.The position anti-Her2scFv, antiCD3 (or antiCD16) scFv is interchangeable.

Wherein, antiHer2.V_HAmino 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.V_LAmino acid sequence as shown in SEQ ID NO:3, the nucleotide sequence of encoding gene As shown in SEQ ID NO:4；antiCD3.V_HAmino 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.V_LAmino 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.V_HAmino 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.V_LAmino 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.V_H、antiHer2.V_L、antiCD3.V_H、antiCD3.V_L、antiCD16.V_H、 antiCD16.V_LThe 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..

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

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 (V_HH), 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.