CN109706164A - The bridging molecules and its application of minicircle dna expression connection people and monkey CD20 and effector cell's antigen - Google Patents

The bridging molecules and its application of minicircle dna expression connection people and monkey CD20 and effector cell's antigen Download PDF

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Publication number
CN109706164A
CN109706164A CN201711013584.6A CN201711013584A CN109706164A CN 109706164 A CN109706164 A CN 109706164A CN 201711013584 A CN201711013584 A CN 201711013584A CN 109706164 A CN109706164 A CN 109706164A
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cell
seq
variable region
chain variable
carrier
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谢亦武
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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 people and monkey CD20 and effector cell's antigen.More particularly to one kind for expressing people-monkey cross reactivity bispecific antibody (hmBsAb) minicircle dna carrier in vivo.People-monkey bispecific antibody (hmBsAb) can be in conjunction with people or the CD20 molecule of monkey B cell or B system cancer cell, also can be in conjunction with people or the T cell or NK cell of monkey, the treatment for cancer and Other diseases.When applying for Clinical Trials, people-monkey cross reactivity bispecific antibody is conducive to evaluate bispecific antibody class Drug safety and validity by monkey model, to promote the development process of drug.

Description

The bridging molecules of minicircle dna expression connection people and monkey CD20 and effector cell's antigen and It is applied
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of micro-loop (Minicircle, MC) DNA vector, more specifically It is one kind for expressing people-monkey cross reactivity bispecific antibody (Human-Monkey cross-reactive in vivo Bispecific antibody, hmBsAb) minicircle dna carrier.
Background technique
Bispecific antibody (Bispecific antibody, abbreviation BsAb) is one kind of genetic engineering antibody, is a kind of With difunctional antibody hybridization molecule, the variable domains (or Fab section) in two valence antibody are respectively provided with different special Property, it can be with two different ligand bindings.Researcher is when designing BsAb molecule, it is contemplated that BsAb can on target cell Antigen molecule combine, and can be in conjunction with the functional molecular on effector cell.In this way, BsAb molecule is just in target cell (antigen Molecule) and effector cell's (functional molecular) between erect bridge, excite the immune response with guiding performance.Antitumor and anti-immunity The bispecific antibody of competent cell CD16 or CD3 not only have the function of activating T cell or NK cell, but also can pass through Antitumor Fab section specifically binds tumour cell and plays a role, and improves the concentration of local T cell or NK cell, enhancing effect Answer the ability of molecule killing tumour.BsAb has become the research hotspot in antibody engineering field, has in the immunization therapy of tumour Have broad application prospects, is the developing direction in the following antibody drug market.
The preparation of bispecific antibody BsAb at present mainly includes three kinds of methods: chemical coupling method, slush pump underlayer method, Genetic engineering antibody the preparation method.Wherein the method for genetic engineering is current the most frequently used and state-of-the-art technology.Minicircle dna carries Body expression belongs to genetic engineering the preparation method.Micro-loop bispecific antibody MC.BsAb can be expressed using minicircle dna carrier, Expressed bispecific antibody can connect target cell and T cell or NK cell, and triggers T cell or NK cell to kill target thin Born of the same parents.The technology for the minicircle dna carrier expression bispecific antibody invented before being us with the immediate the relevant technologies of the invention MC.BsAb(CN201710245146.6)。
When applying for Clinical Trials, other than the data of small animal model, it is necessary to have big animal, such as the examination of monkey Test data.The bispecific antibody in research and development is human specific mostly at present, the antigen no cross reaction with monkey, it is difficult to utilize Monkey assesses its safety and validity.For Lagothrix in non-human primates, genome is approximate with mankind's height, before being clinical drug The important model animal of safety and efficiency evaluation indispensability.It can be seen that developing a kind of people-monkey cross reactivity bispecific Antibody (Human-Monkey cross-reactive Bispecific antibody, hmBsAb), is conducive to by monkey mould Type evaluates bispecific antibody class Drug safety and validity, to promote the development process of drug.
Summary of the invention
The present invention provides a kind of expression people-monkey cross reactivity target cell-effector cell's bridging molecules (Bridge Between human-monkey cross-reactive target cells and effector cells, hmBTEC) Recombination carrier, the bridging molecules include: the part A with the specific binding of target cell action target spot, and and effector cell The part B of action target spot specific binding.
In one embodiment, the recombination carrier is selected from non-viral gene vector, such as standard plasmid or other Circular expression cassette.Preferably, the recombination carrier is selected from minicircle dna carrier.Wherein, the bridging molecules are selected from albumen Or polypeptide.Preferably, the bridging molecules are selected from bispecific antibody.Wherein, the monkey is selected from machin.
In one embodiment, the recombination carrier is recombinant expression carrier, selected from prokaryotic expression carrier or very Nuclear expression carrier；It is preferred that carrier for expression of eukaryon；It is more preferably used for the recombinant expression carrier of mammalian cell eukaryotic expression.
In one embodiment, the part A and the part B are respectively selected from protein molecular or peptide molecule.It is preferred that , the part A and part B are respectively selected from Fab, Fab ', single-chain antibody (scFv), single domain antibody (VHH), single chain T Cell by Body (scTCR) and other.
In one embodiment, the target cell is selected from B cell or B system cancer cell.It is thin that the effector cell is selected from T Born of the same parents, NK cell or other.Preferably, the action target spot of the part A specific binding is selected from: CD20.The part B specificity In conjunction with action target spot be selected from: CD3, CD16, CD28,4-1BB, OX40, TCR, CD56, NKG2D, NCR or other.
In one embodiment, the part A specifically binds CD20 molecule, and the part B specific binding CD3 divides Son.Preferably, the part A includes: heavy chain variable region, and amino acid sequence is as shown in SEQ ID NO:1 and light chain variable Area, amino acid sequence is as shown in SEQ ID NO:3；And the part B includes: heavy chain variable region, amino acid sequence is such as Shown in SEQ ID NO:5 and light chain variable region, amino acid sequence is as shown in SEQ ID NO:7.
In one embodiment, the bridging molecules are selected from people-monkey cross reactivity bispecific of anti-CD20/CD3 Antibody (hmBsAb).The hmBsAb can be in conjunction with people or the CD20 molecule on monkey B cell surface, also can be thin in conjunction with people or monkey T The CD3 molecule of cellular surface.
In one embodiment, the part A specifically binds CD20 molecule, and the part B specifically binds CD16 Molecule.Preferably, the part A includes: heavy chain variable region, and amino acid sequence is as shown in SEQ ID NO:1 and light chain variable Area, amino acid sequence is as shown in SEQ ID NO:3；And the part B includes: heavy chain variable region, amino acid sequence is such as Shown in SEQ ID NO:9 and light chain variable region, amino acid sequence is as shown in SEQ ID NO:11.
In one embodiment, the bridging molecules are selected from people-monkey cross reactivity bispecific of anti-CD20/CD3 Antibody (hmBsAb).The hmBsAb can be in conjunction with people or the CD20 molecule on monkey B cell surface, also can be thin in conjunction with people or monkey NK The CD16 molecule of cellular surface.
In one embodiment, the heavy chain variable region that the part A includes has to be had with sequence shown in SEQ ID NO:1 There is the amino acid sequence of the homology of at least 90%, 95%, 98% or 99%, the light chain variable region for including has and SEQ ID NO: Sequence shown in 3 has the amino acid sequence of at least 90%, 95%, 98% or 99% homology.
In one embodiment, the heavy chain variable region that the part B includes has and sequence shown in SEQ ID NO:5 or 9 The amino acid sequence at least 90%, 95%, 98% or 99% homology is arranged, the light chain variable region for including has and SEQ ID Sequence shown in NO:7 or 11 has the amino acid sequence of at least 90%, 95%, 98% or 99% homology.
The present invention provides a kind of expression people-monkey cross reactivity target cell-effector cell's bridging molecules recombinations Carrier, the recombination carrier include the encoding gene of the bridging molecules.
In one embodiment, the recombination carrier includes: the encoding gene of part A, and/or includes part B Encoding gene.Preferably, the encoding gene of part A includes: the encoding gene of heavy chain variable region, nucleotide sequence such as SEQ Shown in ID NO:2 and/or the encoding gene of light chain variable region, nucleotide sequence is as shown in SEQ ID NO:4.Preferably, portion The encoding gene of point B includes: the encoding gene of heavy chain variable region, nucleotide sequence as shown in SEQ ID NO:6 or 10, and/ Or the encoding gene of light chain variable region, nucleotide sequence is as shown in SEQ ID NO:8 or 12.
In one embodiment, the recombination carrier includes: the encoding gene with above-mentioned part A and/or part B Nucleotide sequence there is the nucleotide sequence of at least 90%, 95%, 98% or 99% homology, and coding obtained bridge joint point Son has function identical with bridging molecules described in embodiment before.It is well known to those skilled in the art, do not changing institute In the case where the amino acid of coding, one or more codons in the coding gene sequence can carry out equal justice replacement, such as One or several codons, such as 1,2,3,4,5,6,8,9,10,15,20,30,40,50 codon.
In one embodiment, the recombination carrier includes the encoding gene of the bridging molecules, nucleotide Sequence is as shown in any one of SEQ ID NO:13-18.
In one embodiment, the recombination carrier includes: the nucleosides with the encoding gene of above-mentioned bridging molecules Acid sequence has the nucleotide sequence of at least 90%, 95%, 98% or 99% homology, and the bridging molecules that coding obtains have 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 1,2,3,4,5,6,8,9,10,15,20,30,40,50 codon.
The present invention provides bridging molecules expressed by a kind of recombination carrier as described in embodiment before.It is excellent Choosing, 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.
The present invention provides the preparation method of recombination carrier described in embodiment before one kind, specific steps packets It includes:
(1) it is anti-that people-monkey cross reactivity anti-CD 20 antibodies, anti-cd 3 antibodies and anti-CD16 are obtained respectively from the prior art Heavy chain variable region (VH) sequence, light chain variable region (VL) sequence of body.
(2) according to above-mentioned VH, VL sequence design at polymorphic bridging molecules, and the expression bridge joint point is constructed The recombination carrier of son.
Optional, (3) identify the expression of the bridging molecules in vivo and in vitro, and detect expression product mediate T cell Or NK cell is to the fragmentation effect of CD20 positive cell.
In one embodiment, the recombination carrier is selected from non-viral gene vector, such as standard plasmid or other Circular expression cassette.Preferably, the recombination carrier is selected from minicircle dna carrier.Preferably, the bridging molecules are selected from double Specific antibody.It is furthermore preferred that the recombination carrier is selected from the minicircle dna carrier of expression bispecific antibody (MC.BsAb).Wherein, the monkey is selected from machin.
The present invention provides a kind of host cells, comprising recombination carrier described in embodiment before, or by it The obtained recombination carrier of preparation method described in preceding 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 bispecific antibody described in embodiment before one kind, specific steps packets It includes:
(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 bispecific Antibody.
The present invention provides a kind of pharmaceutical compositions, comprising recombination carrier described in embodiment before, or by The obtained recombination carrier of preparation method described in embodiment before, or the recombination as described in embodiment before Bridging molecules expressed by genophore and pharmaceutically acceptable carrier.
In one embodiment, pharmaceutical preparation can be made in described pharmaceutical composition according to conventional methods.In production process, It is preferred that recombination carrier or bispecific antibody are mixed with pharmaceutically acceptable carrier or diluted with carrier.When carrier is made It can be solid, semisolid or liquid when for diluent.Preparation be selected from tablet, pill, pulvis, capsule, suspension, emulsion, The forms such as solution, aerosol, capsule, injection solution.Suitable carrier, excipient or diluent include water, lactose, grape Sugar, sucrose, sorbierite, mannitol, calcium silicates, cellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, hydroxybenzoic acid Propyl ester, talcum powder, magnesium stearate and mineral oil etc..Preparation can also include filler, anticoagulant, lubricant, moisturizer, tune Taste agent, emulsifier, preservative etc..
The present invention provides recombination carriers described in embodiment before one kind, bridging molecules, described Host cell or the pharmaceutical composition preparation for treating cancer, infection or Other diseases drug in purposes.
The positive effect of the present invention includes:
When applying for Clinical Trials, need to have big animal, such as the test data of monkey.People of the invention-monkey is intersected Reactive bispecific antibody can either can intersect the antigen molecule of identification monkey in conjunction with the antigen molecule of people, be conducive to borrow Help non-human primate model evaluation bispecific antibody class Drug safety and validity, with promote the exploitation of drug into Journey.
Detailed description of the invention
Fig. 1: anti-CD20 (scFv)/CD3 (scFv) bispecific antibody minicircle dna carrier structure figure is expressed.
Fig. 2: the minicircle dna carrier knot of anti-CD20/CD3 people-monkey cross reactivity bridging molecules hmBTEC (DART) is expressed Composition.
Fig. 3: the minicircle dna carrier of anti-CD20/CD3 people-monkey cross reactivity bridging molecules hmBTEC (DART-Fc) is expressed Structure chart.
Fig. 4: anti-CD20 (scFv)/CD16 (scFv) bispecific antibody minicircle dna carrier structure figure is expressed.
Fig. 5: the minicircle dna carrier knot of anti-CD20/CD16 people-monkey cross reactivity bridging molecules hmBTEC (DART) is expressed Composition.
Fig. 6: the minicircle dna for expressing anti-CD20/CD16 people-monkey cross reactivity bridging molecules hmBTEC (DART-Fc) carries Body structure chart.
Fig. 7: using the expression of PAGE detection CD20/CD3 people-monkey cross reactivity bridging molecules hmBTEC.
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, anti-CD20 people-monkey cross reactivity bridging molecules (CD20.BTEC, hereinafter referred to as " hmBTEC ")
Design hmBTEC simultaneously constructs corresponding minicircle dna expression vector, and hmBTEC expression cassette includes various structures, and point column are such as Under:
(1) scFv-scFv structure:
Wherein, SP is signal peptide (Signal Peptide)；Linker is catenation sequence.anti-CD20.VHAmino acid Sequence is as shown in SEQ ID NO:1, anti-CD20.VLAmino acid sequence as shown in SEQ ID NO:3；anti-CD3.VH's Amino acid sequence is as shown in SEQ ID NO:5, anti-CD3.VLAmino acid sequence as shown in SEQ ID NO:7；anti- CD16.VHAmino acid sequence as shown in SEQ ID NO:9, anti-CD16.VLAmino acid sequence such as SEQ ID NO:11 institute Show.It wherein further include the encoding gene of above-mentioned each element；The encoding gene of scFv-scFv bispecific antibody such as SEQ ID Shown in NO:13 or 16.
(2) bispecific antibody DART (Dual-Affinity Re-Targeting antibody) structure:
Or
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.
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..It wherein further include the encoding gene of above-mentioned each element；DART bispecific antibody Encoding gene is as shown in SEQ ID NO:14 or 17.
(3) bispecific antibody DART-Fc structure:
Or
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.
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..It wherein further include the encoding gene of above-mentioned each element；The encoding gene of DART-Fc bispecific antibody is such as Shown in SEQ ID NO:15 or 18.
Embodiment 2, the building of minicircle dna carrier and the preparation of micro-loop (MC)
(a) encoding gene of hmBTEC is synthesized.
(b) suitable site double digestion minicircle dna empty carrier pMC.BESPX is selected.
(c) encoding gene of hmBTEC is inserted using seamless clone or conventional cloning methods (e.g., digestion-connection) In empty carrier pMC.BESPX after entering double digestion, it is built into minicircle dna carrier (pMC.hmBTEC).
(d) carrier pMC.hmBTEC converts Escherichia coli E coli.ZYCY10P3S2T, obtains by standard micro-loop preparation method To micro-loop (MC.hmBTEC).
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.
The expression and purifying of embodiment 3, hmBTEC
One, expression of the hmBTEC 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 hmBTEC
It collects cell culture supernatant and carries out low temperature ultracentrifugation, take supernatant, then purify using His-Tag affine resin (cOmplete His-Tag Purification Resin, Roche), albumen after purification use PAGE or Western Blot qualitative detection, and protein concentration is detected using Bradford standard measure.Purified product is placed in -20 DEG C or -80 DEG C long-term protect It deposits.
The Binding experiment of embodiment 4, hmBTEC
4.1, the combination of hmBTEC and human effector cell, target cell
By taking anti-CD20/CD3-hmBTEC and anti-CD20/CD16-hmBTEC as an example, using Flow cytometry hmBTEC With the combination activity of target cell (Targets, T), effector cell (Effector, E), include the following steps:
(a) cell culture: target cell (CD20 positive cell, such as Raji cell)；(T cell or NK are thin by effector cell Born of the same parents), including people CD3+T cell (such as JurKat cell or CIK cell), NK cells of human beings (CD16+)。
(b) collect cell: attached cell is digested using pancreatin, and centrifugation is abandoned supernatant and obtained carefully after adding serum-containing media to neutralize Born of the same parents；Suspension cell is directly collected by centrifugation.
(c) two kinds of cells are washed 2 times with pre-cooling PBS, and 200g is centrifuged 4min, are collected cell, are counted respectively.
(d) each experimental group distributes 1 × 10 respectively5A target cell and effector cell, are grouped as follows:
Blank group (hmBTEC is not added) and hmBTEC group
(e) 100 μ L/ group of hmBTEC solution is added and is incubated for 30min on ice.
(f) 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.
(g) 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 Observation combines situation.
4.2, the combination of hmBTEC and machin effector cell, target cell
By taking anti-CD20/CD3-hmBTEC and anti-CD20/CD16-hmBTEC as an example, detected respectively using flow cytometry The combination of hmBTEC and machin target cell (Targets, T), effector cell (Effector, E) are active, described in step and 4.1 Almost the same, difference is that effector cell in this example, target cell replace with the cell in machin source.Specifically, target cell is Machin CD20+B cell, effector cell are machin CD3+T cell or NK cell (CD16+)。
Embodiment 5, hmBTEC mediate effector cell to kill target cell
5.1, by taking anti-CD20/CD3-hmBTEC and anti-CD20/CD16-hmBTEC as an example, hmBTEC is measured using cck8 method Mediate human effector cell to the lethal effect of people target cell (specific effector cell/target cell is referring to 4.1).
(a) 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 simultaneously (as shown in table 1；Every group of 3 multiple holes)；
1 hmBTEC of table mediates the different grouping of effector cell killing target cell
Group T(2×104The hole cells/) E(1.6×105The hole cells/) hmBTEC(10ng/mL)
1 (T) of group + - -
2 (E) of group - + -
3 (T&E) of group + + -
4 (hmBTEC) of group + + +
5 (blank) of group - - -
(b) fresh opti-MEM culture medium, 100 holes μ L/ are replaced within second day.
(c) effector cell (E) counts and is resuspended in opti-MEM culture medium, according to T: E=1: 8 ratio, according to (a) The effector cell (1.6 × 10 of respective numbers is added in middle grouping5/ hole, 100 holes μ L/).
(d) according to grouping, the hmBTEC (final concentration of 10ng/mL) of purifying is added in every hole in corresponding hole, is mixed.
(e) 37 DEG C of cell incubators are incubated for 6 hours.
(f) cck8 (Dojindo, Japan) reagent (20 hole μ L/) is added, after 37 DEG C of cell incubators are incubated for 2 hours, Absorbance is measured at 450nm.
(g) it according to cck8 kit (Dojindo, Japan) specification, counts number of viable cells and calculates killing-efficiency:
Killing rate=[((T+E)-T&E)/T] × 100%
Wherein, T represents target cell numbers living；E represents effector cell's quantity living；Then T+E be equal to living target cell and The sum of effector cell；T&E represents living cells quantity of the target cell after effector cell kills.5.2, with anti-CD20/CD3- For hmBTEC and anti-CD20/CD16-hmBTEC, mediate machin effector cell to machin using cck8 method measurement hmBTEC The lethal effect of target cell (specific effector cell/target cell is with 5.2).Cck8 method experimental procedure same as above 5.1.Sequence table
SEQUENCE LISTING
<110>Shenzhen Xin Nuo micro-loop Biotechnology Co., Ltd
<120>bridging molecules and its application of minicircle dna expression connection people and monkey CD20 and effector cell's antigen
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 119
<212> PRT
<213>anti-CD20 VH amino acid sequence
<400> 1
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 2
<211> 357
<212> DNA
<213>encoding gene of anti-CD20 VH
<400> 2
caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60
tcctgcaagg cttcaggata cgccttcagc tattcttgga tcaattgggt gcggcaggcg 120
cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180
aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300
tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctcc 357
<210> 3
<211> 112
<212> PRT
<213>anti-CD20 VL amino acid sequence
<400> 3
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
85 90 95
Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 4
<211> 336
<212> DNA
<213>encoding gene of anti-CD20 VL
<400> 4
gatatcgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc 60
attagctgca ggtctagcaa gagcctcttg cacagcaatg gcatcactta tttgtattgg 120
tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc caaccttgtc 180
tctggcgtcc ctgaccggtt ctcaggatcc gggtcaggca ctgatttcac actgaaaatc 240
agcagggtgg aggctgagga tgttggagtt tattactgcg ctcagaatct agaacttcct 300
tacaccttcg gcggagggac caaggtggag atcaaa 336
<210> 5
<211> 125
<212> PRT
<213>AntiCD3 McAb VH 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 Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp 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>encoding gene of AntiCD3 McAb VH
<400> 6
gaggtgcaac tcgtcgaatc aggagggggc ctggtccaac ccggaggaag cctgaggctc 60
agttgtgctg cctcaggctt taccttcaat acttacgcga tgaactgggt aagacaagct 120
cctgggaagg ggcttgaatg ggtggctcgc attcggtcaa agtataataa ttatgcgacc 180
tactatgccg attctgtaaa ggatagattc acaataagcc gagacgacag caaaaactca 240
ctttaccttc agatgaactc actcaaaaca gaggacacag cagtgtatta ctgtgtgaga 300
cacgggaact tcgggaactc ttatgtcagt tggtttgcgt actggggtca aggcacactc 360
gtgaccgtat catca 375
<210> 7
<211> 109
<212> PRT
<213>AntiCD3 McAb VL amino acid sequence
<400> 7
Gln Ala Trp Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr
1 5 10 15
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn
20 25 30
Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu
35 40 45
Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe Ser
50 55 60
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln
65 70 75 80
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu
85 90 95
Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 8
<211> 327
<212> DNA
<213>encoding gene of AntiCD3 McAb VL
<400> 8
caagcctgga ctcaggagcc atcccttaca gtgtctcctg gaggaactgt aacactcacc 60
tgcagatcat caaccggggc tgtgacaacg agcaactacg ctaattgggt ccaacagaag 120
cctggtcaag ctccgcgagg attgatcggg ggaacaaaca aacgcgcacc ttggacacct 180
gcacgcttta gcggcagtct cctgggaggt aaggcggccc tgactattac cggagcgcag 240
gctgaagatg aagctgatta ttattgtgct ctttggtata gcaatctttg ggtattcggt 300
gggggaacca agctcactgt gctcggc 327
<210> 9
<211> 120
<212> PRT
<213>anti-CD16 VH amino acid sequence
<400> 9
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> 10
<211> 360
<212> DNA
<213>encoding gene of anti-CD16 VH
<400> 10
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> 11
<211> 106
<212> PRT
<213>anti-CD16 VL amino acid sequence
<400> 11
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> 12
<211> 318
<212> DNA
<213>encoding gene of anti-CD16 VL
<400> 12
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> 13
<211> 1611
<212> DNA
<213>encoding gene of anti-CD20/CD3 (scFv-scFv) bispecific antibody
<400> 13
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactccgac 60
tacaaagatg atgacgataa ggatatcgtg atgacccaga ctccactctc cctgcccgtc 120
acccctggag agcccgccag cattagctgc aggtctagca agagcctctt gcacagcaat 180
ggcatcactt atttgtattg gtacctgcaa aagccagggc agtctccaca gctcctgatt 240
tatcaaatgt ccaaccttgt ctctggcgtc cctgaccggt tctcaggatc cgggtcaggc 300
actgatttca cactgaaaat cagcagggtg gaggctgagg atgttggagt ttattactgc 360
gctcagaatc tagaacttcc ttacaccttc ggcggaggga ccaaggtgga gatcaaaggt 420
ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctcaggtgca attggtgcag 480
tctggcgctg aagttaagaa gcctgggagt tcagtgaagg tctcctgcaa ggcttcagga 540
tacgccttca gctattcttg gatcaattgg gtgcggcagg cgcctggaca agggctcgag 600
tggatgggac ggatctttcc cggcgatggg gatactgact acaatgggaa attcaagggc 660
agagtcacaa ttaccgccga caaatccact agcacagcct atatggagct gagcagcctg 720
agatctgagg acacggccgt gtattactgt gcaagaaatg tctttgatgg ttactggctt 780
gtttactggg gccagggaac cctggtcacc gtctcctccg gaggtggtgg ctccgaggtg 840
caactcgtcg aatcaggagg gggcctggtc caacccggag gaagcctgag gctcagttgt 900
gctgcctcag gctttacctt caatacttac gcgatgaact gggtaagaca agctcctggg 960
aaggggcttg aatgggtggc tcgcattcgg tcaaagtata ataattatgc gacctactat 1020
gccgattctg taaaggatag attcacaata agccgagacg acagcaaaaa ctcactttac 1080
cttcagatga actcactcaa aacagaggac acagcagtgt attactgtgt gagacacggg 1140
aacttcggga actcttatgt cagttggttt gcgtactggg gtcaaggcac actcgtgacc 1200
gtatcatcag gcgaaggtac tagtactggt tctggtggaa gtggaggttc aggtggagca 1260
gaccaagcct ggactcagga gccatccctt acagtgtctc ctggaggaac tgtaacactc 1320
acctgcagat catcaaccgg ggctgtgaca acgagcaact acgctaattg ggtccaacag 1380
aagcctggtc aagctccgcg aggattgatc gggggaacaa acaaacgcgc accttggaca 1440
cctgcacgct ttagcggcag tctcctggga ggtaaggcgg ccctgactat taccggagcg 1500
caggctgaag atgaagctga ttattattgt gctctttggt atagcaatct ttgggtattc 1560
ggtgggggaa ccaagctcac tgtgctcggc catcatcacc atcatcatta g 1611
<210> 14
<211> 1860
<212> DNA
<213>encoding gene of anti-CD20/CD3-hmBTEC (DART)
<400> 14
atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60
caggcagtgg tgacccagga accttctctg accgtgtctc caggcggaac agtgacactg 120
acctgcagaa gcagcacagg cgccgtgaca accagcaact acgccaattg ggtgcagcag 180
aagccaggac aggcccctag aggcctgatt ggaggcacaa acaagagagc cccttggacc 240
ccagccagat tctccggatc tctgctggga ggcaaagccg ccctgacaat cacaggagct 300
caggccgaag acgaggccga ctactattgc gccctctggt acagcaacct ctgggtgttc 360
ggcggaggaa caaagctgac agtgctggga ggaggaggaa gcggaggagg aggacaggtg 420
caattggtgc agtctggcgc tgaagttaag aagcctggga gttcagtgaa ggtctcctgc 480
aaggcttcag gatacgcctt cagctattct tggatcaatt gggtgcggca ggcgcctgga 540
caagggctcg agtggatggg acggatcttt cccggcgatg gggatactga ctacaatggg 600
aaattcaagg gcagagtcac aattaccgcc gacaaatcca ctagcacagc ctatatggag 660
ctgagcagcc tgagatctga ggacacggcc gtgtattact gtgcaagaaa tgtctttgat 720
ggttactggc ttgtttactg gggccaggga accctggtca ccgtctcctc cgctagcacc 780
aagggcaagg tggcagcttg caaggagaag gtggccgctc tgaaggagaa agtggccgct 840
ctgaaggaga aagtggccgc cctgaaggag agaagaaaga gaggcagcgg cgagggaaga 900
ggatctctgc tgacttgcgg cgacgtggaa gagaatcccg gccctatgga gaccgacaca 960
ctcctcctct gggtgctgct cctctgggtg ccaggatcta caggcgatat cgtgatgacc 1020
cagactccac tctccctgcc cgtcacccct ggagagcccg ccagcattag ctgcaggtct 1080
agcaagagcc tcttgcacag caatggcatc acttatttgt attggtacct gcaaaagcca 1140
gggcagtctc cacagctcct gatttatcaa atgtccaacc ttgtctctgg cgtccctgac 1200
cggttctcag gatccgggtc aggcactgat ttcacactga aaatcagcag ggtggaggct 1260
gaggatgttg gagtttatta ctgcgctcag aatctagaac ttccttacac cttcggcgga 1320
gggaccaagg tggagatcaa aggcggcgga agcggaggag gaggagaagt gcagctggtg 1380
gaaagcggag gaggactggt gcagccagga ggatctctga gactgtcttg cgccgccagc 1440
ggctttacat tcagcaccta cgccatgaat tgggtccggc aggctccagg aaaaggactc 1500
gagtgggtcg gaaggatccg gagcaagtac aacaactacg ccacctacta cgccgacagc 1560
gtgaagggca ggttcaccat cagccgggac gacagcaaga acagcctgta cctgcagatg 1620
aacagcctga agaccgagga cacagccgtg tactattgcg tgcgccacgg caacttcggc 1680
aacagctacg tcagctggtt cgcctattgg gggcagggaa cactggtgac agtgtctagc 1740
gccagcacca aaggcgaagt ggcagcttgc gagaaggaag tggccgctct ggagaaggaa 1800
gtggccgctc tggaaaagga agtggcagcc ctggagaagc atcatcacca tcatcattga 1860
<210> 15
<211> 3240
<212> DNA
<213>encoding gene of anti-CD20/CD3-hmBTEC (DART-Fc)
<400> 15
atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60
caggcagtgg tgacccagga accttctctg accgtgtctc caggcggaac agtgacactg 120
acctgcagaa gcagcacagg cgccgtgaca accagcaact acgccaattg ggtgcagcag 180
aagccaggac aggcccctag aggcctgatt ggaggcacaa acaagagagc cccttggacc 240
ccagccagat tctccggatc tctgctggga ggcaaagccg ccctgacaat cacaggagct 300
caggccgaag acgaggccga ctactattgc gccctctggt acagcaacct ctgggtgttc 360
ggcggaggaa caaagctgac agtgctggga ggaggaggaa gcggaggagg aggacaggtg 420
caattggtgc agtctggcgc tgaagttaag aagcctggga gttcagtgaa ggtctcctgc 480
aaggcttcag gatacgcctt cagctattct tggatcaatt gggtgcggca ggcgcctgga 540
caagggctcg agtggatggg acggatcttt cccggcgatg gggatactga ctacaatggg 600
aaattcaagg gcagagtcac aattaccgcc gacaaatcca ctagcacagc ctatatggag 660
ctgagcagcc tgagatctga ggacacggcc gtgtattact gtgcaagaaa tgtctttgat 720
ggttactggc ttgtttactg gggccaggga accctggtca ccgtctcctc cgctagcacc 780
aagggcaagg tggcagcttg caaggagaag gtggccgctc tgaaggagaa agtggccgct 840
ctgaaggaga aagtggccgc cctgaaggag ggaggcggcg ataagacaca cacttgccct 900
ccttgtccag ctccagaagc agcaggagga cctagcgtgt tcctgttccc tcccaagcct 960
aaggacaccc tgatgatcag ccggacccca gaagtgactt gcgtggtggt ggacgtgtcc 1020
cacgaagacc ccgaggtcaa gttcaattgg tacgtggacg gagtggaggt gcacaacgct 1080
aagaccaagc ccagggagga gcagtacaac agcacctaca gggtggtgtc cgtgctgaca 1140
gtgctgcacc aggattggct gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc 1200
ctgccagccc ctatcgagaa gaccatcagc aaggccaagg gccagcctag agaacctcag 1260
gtgtacaccc tgccccctag cagagaggag atgaccaaga accaggtctc cctctggtgc 1320
ctggtgaagg gcttctaccc tagcgacatc gccgtggagt gggaatctaa cggtcagcca 1380
gagaacaact acaagaccac ccccccagtg ctggacagcg acggcagctt cttcctgtac 1440
agcaagctga ccgtggacaa aagccgctgg cagcagggca acgtgttctc ttgcagcgtg 1500
atgcacgagg ccctgcacaa ccactacacc cagaagagcc tgagcctgag cccaggaaag 1560
agaagaaaga gaggcagcgg cgagggaaga ggatctctgc tgacttgcgg cgacgtggaa 1620
gagaatcccg gccctatgga gaccgacaca ctcctcctct gggtgctgct cctctgggtg 1680
ccaggatcta caggcgatat cgtgatgacc cagactccac tctccctgcc cgtcacccct 1740
ggagagcccg ccagcattag ctgcaggtct agcaagagcc tcttgcacag caatggcatc 1800
acttatttgt attggtacct gcaaaagcca gggcagtctc cacagctcct gatttatcaa 1860
atgtccaacc ttgtctctgg cgtccctgac cggttctcag gatccgggtc aggcactgat 1920
ttcacactga aaatcagcag ggtggaggct gaggatgttg gagtttatta ctgcgctcag 1980
aatctagaac ttccttacac cttcggcgga gggaccaagg tggagatcaa aggcggcgga 2040
agcggaggag gaggagaagt gcagctggtg gaaagcggag gaggactggt gcagccagga 2100
ggatctctga gactgtcttg cgccgccagc ggctttacat tcagcaccta cgccatgaat 2160
tgggtccggc aggctccagg aaaaggactc gagtgggtcg gaaggatccg gagcaagtac 2220
aacaactacg ccacctacta cgccgacagc gtgaagggca ggttcaccat cagccgggac 2280
gacagcaaga acagcctgta cctgcagatg aacagcctga agaccgagga cacagccgtg 2340
tactattgcg tgcgccacgg caacttcggc aacagctacg tcagctggtt cgcctattgg 2400
gggcagggaa cactggtgac agtgtctagc gccagcacca aaggcgaagt ggcagcttgc 2460
gagaaggaag tggccgctct ggagaaggaa gtggccgctc tggaaaagga agtggcagcc 2520
ctggagaagg gaggcggcga taagacacac acttgccctc cttgtccagc tccagaagca 2580
gcaggaggac ctagcgtgtt cctgttccct cccaagccta aggacaccct gatgatcagc 2640
cggaccccag aagtgacttg cgtggtggtg gacgtgtccc acgaagaccc cgaggtcaag 2700
ttcaattggt acgtggacgg agtggaggtg cacaacgcta agaccaagcc cagggaggag 2760
cagtacaaca gcacctacag ggtggtgtcc gtgctgacag tgctgcacca ggattggctg 2820
aacggcaagg agtacaagtg caaggtgtcc aacaaggccc tgccagcccc tatcgagaag 2880
accatcagca aggccaaggg ccagcctaga gaacctcagg tgtacaccct gccccctagc 2940
agagaggaga tgaccaagaa ccaggtgtcc ctgtcttgcg ccgtgaaggg cttctaccct 3000
agcgacatcg ccgtggagtg ggaatctaac ggtcagccag agaacaacta caagaccacc 3060
ccccccgtgc tggatagcga cggcagcttc ttcctggtgt ccaagctgac cgtggacaaa 3120
agccgctggc agcagggcaa cgtgttctct tgcagcgtga tgcacgaggc cctgcataac 3180
agatacaccc agaagagcct gagcctgagc ccaggaaagc atcatcacca tcatcattga 3240
<210> 16
<211> 1587
<212> DNA
<213>encoding gene of anti-CD20/CD16 (scFv-scFv) bispecific antibody
<400> 16
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactccgac 60
tacaaagatg atgacgataa ggatatcgtg atgacccaga ctccactctc cctgcccgtc 120
acccctggag agcccgccag cattagctgc aggtctagca agagcctctt gcacagcaat 180
ggcatcactt atttgtattg gtacctgcaa aagccagggc agtctccaca gctcctgatt 240
tatcaaatgt ccaaccttgt ctctggcgtc cctgaccggt tctcaggatc cgggtcaggc 300
actgatttca cactgaaaat cagcagggtg gaggctgagg atgttggagt ttattactgc 360
gctcagaatc tagaacttcc ttacaccttc ggcggaggga ccaaggtgga gatcaaaggt 420
ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctcaggtgca attggtgcag 480
tctggcgctg aagttaagaa gcctgggagt tcagtgaagg tctcctgcaa ggcttcagga 540
tacgccttca gctattcttg gatcaattgg gtgcggcagg cgcctggaca agggctcgag 600
tggatgggac ggatctttcc cggcgatggg gatactgact acaatgggaa attcaagggc 660
agagtcacaa ttaccgccga caaatccact agcacagcct atatggagct gagcagcctg 720
agatctgagg acacggccgt gtattactgt gcaagaaatg tctttgatgg ttactggctt 780
gtttactggg gccagggaac cctggtcacc gtctcctccg gaggtggtgg ctcccaagtc 840
cagcttgtgc agtcaggtgc tgaggttaaa aaaccaggag aaagtctgaa ggtatcatgt 900
aaggcctcag gttatacgtt tacttcatat tacatgcact gggtgcgaca ggctcctggt 960
caggggttgg agtggatggg aatcatcaat ccatccggtg gtagcaccag ctacgcacaa 1020
aagtttcagg gtcgcgtgac aatgacaaga gacacgtcca cgtccacggt ctacatggaa 1080
ttgtcatccc tgagatcaga ggacaccgcc gtatattatt gtgcacgcgg cagtgcttac 1140
tactacgatt tcgcagatta ttgggggcag gggactttgg ttacagtctc ctccggcgaa 1200
ggtactagta ctggttctgg tggaagtgga ggttcaggtg gagcagactc ctacgttctc 1260
actcagccca gtagtgtctc agttgctcca gggcaaacgg ccacgattag ttgcggaggt 1320
cacaacatag gcagtaagaa tgtacattgg taccaacagc gaccaggcca gagccccgtt 1380
ttggtcatct atcaggataa taagcggcca agtggaatac cggagcggtt cagcggtagt 1440
aacagtggga acaccgccac tctgactata tccggtacgc aagctatgga cgaagcagac 1500
tactattgcc aggtgtggga taactacagc gtactgttcg gaggcgggac gaaacttaca 1560
gtcttgcatc atcaccatca tcattag 1587
<210> 17
<211> 1833
<212> DNA
<213>encoding gene of anti-CD20/CD16-hmBTEC (DART)
<400> 17
atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60
tcctacgttc tcactcagcc cagtagtgtc tcagttgctc cagggcaaac ggccacgatt 120
agttgcggag gtcacaacat aggcagtaag aatgtacatt ggtaccaaca gcgaccaggc 180
cagagccccg ttttggtcat ctatcaggat aataagcggc caagtggaat accggagcgg 240
ttcagcggta gtaacagtgg gaacaccgcc actctgacta tatccggtac gcaagctatg 300
gacgaagcag actactattg ccaggtgtgg gataactaca gcgtactgtt cggaggcggg 360
acgaaactta cagtcttggg aggaggaagc ggaggaggag gacaggtgca attggtgcag 420
tctggcgctg aagttaagaa gcctgggagt tcagtgaagg tctcctgcaa ggcttcagga 480
tacgccttca gctattcttg gatcaattgg gtgcggcagg cgcctggaca agggctcgag 540
tggatgggac ggatctttcc cggcgatggg gatactgact acaatgggaa attcaagggc 600
agagtcacaa ttaccgccga caaatccact agcacagcct atatggagct gagcagcctg 660
agatctgagg acacggccgt gtattactgt gcaagaaatg tctttgatgg ttactggctt 720
gtttactggg gccagggaac cctggtcacc gtctcctccg ctagcaccaa gggcaaggtg 780
gcagcttgca aggagaaggt ggccgctctg aaggagaaag tggccgctct gaaggagaaa 840
gtggccgccc tgaaggagag aagaaagaga ggcagcggcg agggaagagg atctctgctg 900
acttgcggcg acgtggaaga gaatcccggc cctatggaga ccgacacact cctcctctgg 960
gtgctgctcc tctgggtgcc aggatctaca ggcgatatcg tgatgaccca gactccactc 1020
tccctgcccg tcacccctgg agagcccgcc agcattagct gcaggtctag caagagcctc 1080
ttgcacagca atggcatcac ttatttgtat tggtacctgc aaaagccagg gcagtctcca 1140
cagctcctga tttatcaaat gtccaacctt gtctctggcg tccctgaccg gttctcagga 1200
tccgggtcag gcactgattt cacactgaaa atcagcaggg tggaggctga ggatgttgga 1260
gtttattact gcgctcagaa tctagaactt ccttacacct tcggcggagg gaccaaggtg 1320
gagatcaaag gcggcggaag cggaggagga ggacaagtcc agcttgtgca gtcaggtgct 1380
gaggttaaaa aaccaggaga aagtctgaag gtatcatgta aggcctcagg ttatacgttt 1440
acttcatatt acatgcactg ggtgcgacag gctcctggtc aggggttgga gtggatggga 1500
atcatcaatc catccggtgg tagcaccagc tacgcacaaa agtttcaggg tcgcgtgaca 1560
atgacaagag acacgtccac gtccacggtc tacatggaat tgtcatccct gagatcagag 1620
gacaccgccg tatattattg tgcacgcggc agtgcttact actacgattt cgcagattat 1680
tgggggcagg ggactttggt tacagtctcc tccgccagca ccaaaggcga agtggcagct 1740
tgcgagaagg aagtggccgc tctggagaag gaagtggccg ctctggaaaa ggaagtggca 1800
gccctggaga agcatcatca ccatcatcat tga 1833
<210> 18
<211> 3213
<212> DNA
<213>encoding gene of anti-CD20/CD16-hmBTEC (DART-Fc)
<400> 18
atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60
tcctacgttc tcactcagcc cagtagtgtc tcagttgctc cagggcaaac ggccacgatt 120
agttgcggag gtcacaacat aggcagtaag aatgtacatt ggtaccaaca gcgaccaggc 180
cagagccccg ttttggtcat ctatcaggat aataagcggc caagtggaat accggagcgg 240
ttcagcggta gtaacagtgg gaacaccgcc actctgacta tatccggtac gcaagctatg 300
gacgaagcag actactattg ccaggtgtgg gataactaca gcgtactgtt cggaggcggg 360
acgaaactta cagtcttggg aggaggaagc ggaggaggag gacaggtgca attggtgcag 420
tctggcgctg aagttaagaa gcctgggagt tcagtgaagg tctcctgcaa ggcttcagga 480
tacgccttca gctattcttg gatcaattgg gtgcggcagg cgcctggaca agggctcgag 540
tggatgggac ggatctttcc cggcgatggg gatactgact acaatgggaa attcaagggc 600
agagtcacaa ttaccgccga caaatccact agcacagcct atatggagct gagcagcctg 660
agatctgagg acacggccgt gtattactgt gcaagaaatg tctttgatgg ttactggctt 720
gtttactggg gccagggaac cctggtcacc gtctcctccg ctagcaccaa gggcaaggtg 780
gcagcttgca aggagaaggt ggccgctctg aaggagaaag tggccgctct gaaggagaaa 840
gtggccgccc tgaaggaggg aggcggcgat aagacacaca cttgccctcc ttgtccagct 900
ccagaagcag caggaggacc tagcgtgttc ctgttccctc ccaagcctaa ggacaccctg 960
atgatcagcc ggaccccaga agtgacttgc gtggtggtgg acgtgtccca cgaagacccc 1020
gaggtcaagt tcaattggta cgtggacgga gtggaggtgc acaacgctaa gaccaagccc 1080
agggaggagc agtacaacag cacctacagg gtggtgtccg tgctgacagt gctgcaccag 1140
gattggctga acggcaagga gtacaagtgc aaggtgtcca acaaggccct gccagcccct 1200
atcgagaaga ccatcagcaa ggccaagggc cagcctagag aacctcaggt gtacaccctg 1260
ccccctagca gagaggagat gaccaagaac caggtctccc tctggtgcct ggtgaagggc 1320
ttctacccta gcgacatcgc cgtggagtgg gaatctaacg gtcagccaga gaacaactac 1380
aagaccaccc ccccagtgct ggacagcgac ggcagcttct tcctgtacag caagctgacc 1440
gtggacaaaa gccgctggca gcagggcaac gtgttctctt gcagcgtgat gcacgaggcc 1500
ctgcacaacc actacaccca gaagagcctg agcctgagcc caggaaagag aagaaagaga 1560
ggcagcggcg agggaagagg atctctgctg acttgcggcg acgtggaaga gaatcccggc 1620
cctatggaga ccgacacact cctcctctgg gtgctgctcc tctgggtgcc aggatctaca 1680
ggcgatatcg tgatgaccca gactccactc tccctgcccg tcacccctgg agagcccgcc 1740
agcattagct gcaggtctag caagagcctc ttgcacagca atggcatcac ttatttgtat 1800
tggtacctgc aaaagccagg gcagtctcca cagctcctga tttatcaaat gtccaacctt 1860
gtctctggcg tccctgaccg gttctcagga tccgggtcag gcactgattt cacactgaaa 1920
atcagcaggg tggaggctga ggatgttgga gtttattact gcgctcagaa tctagaactt 1980
ccttacacct tcggcggagg gaccaaggtg gagatcaaag gcggcggaag cggaggagga 2040
ggacaagtcc agcttgtgca gtcaggtgct gaggttaaaa aaccaggaga aagtctgaag 2100
gtatcatgta aggcctcagg ttatacgttt acttcatatt acatgcactg ggtgcgacag 2160
gctcctggtc aggggttgga gtggatggga atcatcaatc catccggtgg tagcaccagc 2220
tacgcacaaa agtttcaggg tcgcgtgaca atgacaagag acacgtccac gtccacggtc 2280
tacatggaat tgtcatccct gagatcagag gacaccgccg tatattattg tgcacgcggc 2340
agtgcttact actacgattt cgcagattat tgggggcagg ggactttggt tacagtctcc 2400
tccgccagca ccaaaggcga agtggcagct tgcgagaagg aagtggccgc tctggagaag 2460
gaagtggccg ctctggaaaa ggaagtggca gccctggaga agggaggcgg cgataagaca 2520
cacacttgcc ctccttgtcc agctccagaa gcagcaggag gacctagcgt gttcctgttc 2580
cctcccaagc ctaaggacac cctgatgatc agccggaccc cagaagtgac ttgcgtggtg 2640
gtggacgtgt cccacgaaga ccccgaggtc aagttcaatt ggtacgtgga cggagtggag 2700
gtgcacaacg ctaagaccaa gcccagggag gagcagtaca acagcaccta cagggtggtg 2760
tccgtgctga cagtgctgca ccaggattgg ctgaacggca aggagtacaa gtgcaaggtg 2820
tccaacaagg ccctgccagc ccctatcgag aagaccatca gcaaggccaa gggccagcct 2880
agagaacctc aggtgtacac cctgccccct agcagagagg agatgaccaa gaaccaggtg 2940
tccctgtctt gcgccgtgaa gggcttctac cctagcgaca tcgccgtgga gtgggaatct 3000
aacggtcagc cagagaacaa ctacaagacc accccccccg tgctggatag cgacggcagc 3060
ttcttcctgg tgtccaagct gaccgtggac aaaagccgct ggcagcaggg caacgtgttc 3120
tcttgcagcg tgatgcacga ggccctgcat aacagataca cccagaagag cctgagcctg 3180
agcccaggaa agcatcatca ccatcatcat tga 3213

Claims (10)

1. a kind of expression people-monkey cross reactivity target cell-effector cell's bridging molecules (Bridge between human- Monkey cross-reactive target cells and effector cells, hmBTEC) recombination carrier, It is characterized in that, the bridging molecules include: the part A with the specific binding of target cell action target spot, and make with effector cell The part B specifically bound with target spot；Preferably, the recombination carrier is selected from non-viral gene vector, such as standard plasmid Or other circular expression cassette；It is furthermore preferred that the recombination carrier is selected from minicircle dna carrier；Further preferably, the bridge joint Molecule is selected from albumen or polypeptide；It is furthermore preferred that the bridging molecules are selected from bispecific antibody；It is particularly preferred, the part A Protein molecular or peptide molecule are respectively selected from the part B；Most preferably, the 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 other.
2. recombination carrier as described in claim 1, which is characterized in that the target cell is selected from B cell or B system cancer is thin Born of the same parents；The effector cell is selected from T cell, NK cell or other；Preferably, the action target spot choosing of the part A specific binding From: CD20；Further preferably, the action target spot of part B specific binding is selected from: CD3, CD16, CD28,4-1BB, OX40, TCR, CD56, NKG2D, NCR or other.
3. recombination carrier as claimed in claim 1 or 2, which is characterized in that
(1) the part A specifically binds CD20 molecule, and the part B specifically binds CD3 molecule；Preferably, the part A includes: heavy chain variable region, and amino acid sequence is as shown in SEQ ID NO:1 and light chain variable region, amino acid sequence such as SEQ Shown in ID NO:3；And the part B includes: heavy chain variable region, amino acid sequence are and light as shown in SEQ ID NO:5 Chain variable region, amino acid sequence is as shown in SEQ ID NO:7；Or,
(2) the part A specifically binds CD20 molecule, and the part B specifically binds CD16 molecule；Preferably, the portion Point A includes: heavy chain variable region, and amino acid sequence is as shown in SEQ ID NO:1 and light chain variable region, and amino acid sequence is such as Shown in SEQ ID NO:3；And the part B includes: heavy chain variable region, amino acid sequence as shown in SEQ ID NO:9, And light chain variable region, amino acid sequence is as shown in SEQ ID NO:11.
4. recombination carrier as claimed in any one of claims 1-3, which is characterized in that the recombination carrier packet Contain: the encoding gene of part A, and/or the encoding gene comprising part B；Preferably, the encoding gene of part A includes: heavy chain can Become the encoding gene in area, nucleotide sequence is as shown in SEQ ID NO:2 and/or the encoding gene of light chain variable region, nucleosides Acid sequence is as shown in SEQ ID NO:4；Preferably, the encoding gene of part B includes: the encoding gene of heavy chain variable region, core Nucleotide sequence is as shown in SEQ ID NO:6 or 10 and/or the encoding gene of light chain variable region, nucleotide sequence such as SEQ ID Shown in NO:8 or 12.
5. such as recombination carrier of any of claims 1-4, which is characterized in that the recombination carrier includes The encoding gene of the bridging molecules, nucleotide sequence is as shown in any one of SEQ ID NO:13-18.
6. bridging molecules expressed by recombination carrier according to any one of claims 1 to 5.
7. the preparation method of recombination carrier according to any one of claims 1 to 5, which is characterized in that specific steps packet It includes:
(1) people-monkey cross reactivity anti-CD 20 antibodies, anti-cd 3 antibodies and anti-CD16 antibody are obtained respectively from the prior art Heavy chain variable region (VH) sequence, light chain variable region (VL) sequence；
(2) according to above-mentioned VH, VL sequence design at polymorphic bridging molecules, and the expression bridging molecules are constructed Recombination carrier；
Optional, (3) identify the expression of the bridging molecules in vivo and in vitro, and detect expression product mediate T cell or NK Fragmentation effect of the cell to CD20 positive cell；
Preferably, the bridging molecules are selected from bispecific antibody；Preferably, it is double special to be selected from expression for the recombination carrier The minicircle dna carrier (MC.BsAb) of property antibody；Preferably, the monkey is selected from machin.
8. a kind of host cell, which is characterized in that it include recombination carrier according to any one of claims 1 to 5, or By the obtained recombination carrier of preparation method as claimed in claim 7；Preferably, the host cell includes that bacterium is thin Born of the same parents, yeast cell, insect cell or mammalian cell.
9. a kind of pharmaceutical composition, which is characterized in that it include recombination carrier according to any one of claims 1 to 5, Or bridging molecules as claimed in claim 6, or by the obtained recombination carrier of preparation method as claimed in claim 7, And pharmaceutically acceptable carrier.
10. recombination carrier according to any one of claims 1 to 5, bridging molecules as claimed in claim 6 are such as weighed Benefit require 8 described in host cell, or pharmaceutical composition as claimed in claim 9 is used for treating cancer or other diseases in preparation Purposes in the drug of disease.
CN201711013584.6A 2017-10-26 2017-10-26 The bridging molecules and its application of minicircle dna expression connection people and monkey CD20 and effector cell's antigen Pending CN109706164A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015018331A1 (en) * 2013-08-06 2015-02-12 深圳先进技术研究院 Minicircle dna recombinant parental plasmid having genetically engineered antibody gene expression cassette, a minicircle dna having the expression cassette, and applications
WO2016177846A1 (en) * 2015-05-04 2016-11-10 Affimed Gmbh Combination of a cd30xcd16 antibody with a pd-1 antagonist for therapy
CN106661119A (en) * 2014-07-01 2017-05-10 辉瑞公司 Bispecific heterodimeric diabodies and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015018331A1 (en) * 2013-08-06 2015-02-12 深圳先进技术研究院 Minicircle dna recombinant parental plasmid having genetically engineered antibody gene expression cassette, a minicircle dna having the expression cassette, and applications
CN106661119A (en) * 2014-07-01 2017-05-10 辉瑞公司 Bispecific heterodimeric diabodies and uses thereof
WO2016177846A1 (en) * 2015-05-04 2016-11-10 Affimed Gmbh Combination of a cd30xcd16 antibody with a pd-1 antagonist for therapy

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Application publication date: 20190503