CN104582476A - Methods for making fully human bispecific antibodies using a common light chain - Google Patents

Methods for making fully human bispecific antibodies using a common light chain Download PDF

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Publication number
CN104582476A
CN104582476A CN201380030014.1A CN201380030014A CN104582476A CN 104582476 A CN104582476 A CN 104582476A CN 201380030014 A CN201380030014 A CN 201380030014A CN 104582476 A CN104582476 A CN 104582476A
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people
light chain
mouse
heavy chain
antigen
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CN104582476B (en
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R·巴布
J·麦克维尔特
L·麦克唐纳
S·史蒂文斯
S·戴维斯
D·R·巴克勒
K·A·霍西阿瓦
A·J·墨菲
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Priority claimed from US13/488,628 external-priority patent/US20130045492A1/en
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Abstract

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that express just one or a few immunoglobulin light chain variable domains from a limited repertoire in their germline. Methods for making bispecific antibodies having universal light chains using mice as described herein, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, and host cells are provided. Bispecific antibodies capable of binding first and second antigens are provided, wherein the first and second antigens are separate epitopes of a single protein or separate epitopes on two different proteins are provided.

Description

Common light chain is used to prepare the method for complete people's bi-specific antibody
The application submitted to as pct international patent application on June 5th, 2013, and required the U.S. Patent application No.13/488 that on June 5th, 2012 submits to, and the right of priority of 628, the disclosure of described U.S. Patent application is incorporated herein by reference in their entirety.
Technical field
Provide the mouse of genetic modification, it expresses the antibody with the common people variable region/murine constant region light chain be combined with diversified people variable region/murine constant region heavy chain.Provide the method for preparation from people's bi-specific antibody of people's variable region gene sequence of mouse B cell.
Background technology
Antibody generally includes homodimer heavy chain component, and wherein every bar heavy chain monomer combines with identical light chain.Expect that the antibody (such as bi-specific antibody) with heterodimer heavy chain component is as therapeutic antibodies.But preparation has can prove existing problems in conjunction with the bi-specific antibody of the suitable light chain component of every bar heavy chain of bi-specific antibody satisfactorily.
In one approach, can the most frequently used light chain and in vitro this light chain and two not homospecific heavy chains are carried out pairing to select light chain in the service condition Statistical information of all light variable domains, identifier's antibody by inquiry.
In another approach, can such as, by the sequence of light chain in observation phage display library (such as comprising the phage display library of people's light-chain variable sequence, people scFv library) and select the variable region of light chain the most often used to select light chain from this library.Light chain can be tested subsequently on two different object heavy chains.
In another approach, light chain can be selected by using the variable heavy chain sequence of the heavy chain of two entries to carry out mensuration as probe to the phage display library of light chain variable sequence.The light chain that light chain in conjunction with two variable heavy chain sequence can be used as described heavy chain is selected.
In another approach, candidate's light chain can be compared with the same endogenous light chain of heavy chain, and modifies to mate more closely the common sequence signature of the same endogenous light chain of two heavy chains to light chain.If immunogenic possibility needs to be minimized, modify and preferably cause being present in the sequence in known people's sequence of light chain, like this based on the parameter for assessment of immunogenic possibility known in the art and method (namely, detect at silicon chip (in silico) and wet method), proteolysis process unlikely produces t cell epitope.
Above-mentioned all methods depend on the in vitro method comprising many prior-constrained (such as sequence iden, the ability etc. that is combined with specific previously selected heavy chain).This area exists for the operation do not relied under conditions in vitro, and uses the rational method of biology more to prepare on the contrary to comprise the protein-bonded composition of people's epi-position of common light chain and the needs of method.
Invention summary
Provide the mouse of genetic modification, it expresses human immunoglobulin heavy chain and light variable domains, and wherein said mouse has limited light chain variable storehouse.Provide and be combined and the biosystem of people's light variable domains of expressing for generation of with people's heavy-chain variable domains storehouse of diversified affinity maturation.Provide the protein-bonded method for the preparation of comprising immunoglobulin variable domain territory, comprise the mouse with object antigen immune with limited light chain immunoglobulin storehouse, and the immune globulin variable region gene sequence of mouse is used for the associated proteins of specific binding object antigen by specific binding.Method comprises the method that preparation is suitable for preparing human immunoglobulin heavy chain's variable domains of polyspecific antigen-binding proteins.
Provide genetic engineering mouse, it selects the suitable human immunoglobulin heavy chain's variable domains deriving from the affinity maturation without people's heavy chain variable region gene section storehouse of resetting, and wherein people's heavy-chain variable domains of affinity maturation is combined with the single people's light variable domains deriving from a kind of people's chain variable region gene section and expresses.Additionally provide the genetic engineering mouse of the selection presenting two kinds of people's chain variable region gene sections.In many aspects, one or both constant gene segment Cs comprise people V κ 1-39 and people V κ 3-20.
Provide genetic engineering mouse, it is expressed from limited people's light variable domains storehouse in limited people's chain variable region gene section storehouse or single people's light variable domains.By mouse genetic through engineering approaches to comprise single people's chain variable region gene section (or two kinds of people's chain variable region gene sections) without resetting, described constant gene segment C is reset with the people's chain variable region gene forming the rearrangement expressing single light chain (or any one or two kinds of expression in two kinds of light chains) (or two kinds of chain variable region genes reset).The people's light variable domains reset can match with people's heavy chain of multiple affinity maturation selected through mouse, and wherein variable region of heavy chain is specifically in conjunction with different epi-positions.
Provide genetic engineering mouse, it is expressed from limited people's light variable domains storehouse in limited people's light-chain variable sequence storehouse or single people's light variable domains.By mouse genetic through engineering approaches to comprise single V/J people's sequence of light chain (or two kinds of V/J sequences), described sequence expresses single variable region of light chain (or any one or two kinds of expression in two kinds of variable regions).The light chain comprising variable sequence can match with people's heavy chain of multiple affinity maturation selected through cloned mouse, and wherein variable region of heavy chain is specifically in conjunction with different epi-positions.
On the one hand, provide the mouse of genetic modification, it comprises single human normal immunoglobulin light chain variable (V l) district's constant gene segment C, described constant gene segment C (can be selected from one or more J with people J constant gene segment C lsection) reset and the V of encoding human immunoglobulin's light chain lstructural domain.On the other hand, mouse comprises the people V of no more than two kinds lconstant gene segment C, wherein each (can be selected from one or more J with people J constant gene segment C lsection) reset and the V of encoding human immunoglobulin's light chain lstructural domain.
In one embodiment, single people V lconstant gene segment C may be operably coupled to the people J being selected from J κ 1, J κ 2, J κ 3, J κ 4 and J κ 5 lconstant gene segment C, wherein said single people V lconstant gene segment C can with the people J of any one or more lconstant gene segment C is reset with the sequence forming encoded light chain variable region gene.
In one embodiment, the mouse of genetic modification comprises not containing the endogenous mouse V that can reset to be formed light chain immunoglobulin gene lthe light chain immunoglobulin gene seat of constant gene segment C, wherein V llocus comprises the V that can reset with encoded light chain gene lthe single people V in district lconstant gene segment C.In a particular embodiment, people V lconstant gene segment C is people V κ 1-39J κ 5 constant gene segment C or people V κ 3-20J κ 1 constant gene segment C.In one embodiment, the mouse of genetic modification comprises not containing the endogenous mouse V that can reset to be formed light chain immunoglobulin gene lthe V of constant gene segment C llocus, wherein V llocus comprises no more than two can reset with the V of encoded light chain gene lthe people V in district lconstant gene segment C.In a particular embodiment, the people V of no more than 2 lconstant gene segment C is people V κ 1-39J κ 5 constant gene segment C and people V κ 3-20J κ 1 constant gene segment C.
On the one hand, provide and comprise encoding human immunoglobulin's light chain V lsingle (V/J) human normal immunoglobulin light chain variable (V through resetting of structural domain l) district (that is, V l/ J ldistrict) the mouse of genetic modification.On the other hand, mouse comprise no more than two can encoding human immunoglobulin's light chain V lthe people V of the rearrangement of structural domain ldistrict.
In one embodiment, V ldistrict is the people V κ 3-20/J sequence of people V κ 1-39/J sequence or the rearrangement of resetting.In one embodiment, the V of rearrangement l/ J lthe people J of sequence lsection is selected from J κ 1, J κ 2, J κ 3, J κ 4 and J κ 5.In a particular embodiment, V ldistrict is people V κ 1-39J κ 5 sequence or people V κ 3-20J κ 1 sequence.In a particular embodiment, mouse has people V κ 1-39J κ 5 sequence and people V κ 3-20J κ 1 sequence.
In one embodiment, people V lconstant gene segment C may be operably coupled to people or mouse leader sequence.In one embodiment, leader sequence is mouse leader sequence.In a specific embodiment, mouse leader sequence is mouse V κ 3-7 leader sequence.In a particular embodiment, leader sequence may be operably coupled to the people V without resetting lconstant gene segment C.In a particular embodiment, leader sequence may be operably coupled to the people V of rearrangement l/ J lsequence.
In one embodiment, V lconstant gene segment C is operably connected to immunoglobulin promoter sequence.In one embodiment, promoter sequence is people's promoter sequence.In a particular embodiment, humen immunoglobulin storter is people V κ 3-15 promotor.In a particular embodiment, promotor may be operably coupled to the people V without resetting lconstant gene segment C.In a particular embodiment, promotor may be operably coupled to the people V of rearrangement l/ J lsequence.
In one embodiment, light chain gene seat comprises leader sequence, described leader sequence 5 ' (relative to V lthe transcriptional orientation of constant gene segment C) side joint humen immunoglobulin storter, and 3 ' side joint people V lconstant gene segment C, described people V lconstant gene segment C and people J section are reset and encoded packets contains endogenous mouse constant region of light chain (C l) the V of reverse imbedding light chain lstructural domain.In a particular embodiment, V lconstant gene segment C is on mouse V kappa gene seat, and mouse C lmouse C κ.
In one embodiment, light chain gene seat comprises leader sequence, described leader sequence 5 ' (relative to V lthe transcriptional orientation of constant gene segment C) side joint humen immunoglobulin storter, and the people V that 3 ' side joint is reset ldistrict (V l/ J lsequence) and encoded packets contains endogenous mouse constant region of light chain (C l) the V of reverse imbedding light chain lstructural domain.In a particular embodiment, the people V of rearrangement l/ J lsequence is at mouse kappa (κ) locus, and mouse C lmouse C κ.
In one embodiment, the V of the mouse of modification llocus is κ light chain gene seat, and κ light chain gene seat comprises mouse κ intron enhanser, mouse κ 3 ' enhanser or intron enhanser and 3 ' enhanser.
In one embodiment, mouse comprises non-functional immunoglobulin (Ig) lambda (λ) light chain gene seat.In a particular embodiment, lambda light chain gene seat comprises the deletion of one or more sequences of locus, and wherein one or more deletions cause lambda light chain gene seat can not reset to form light chain gene.In another embodiment, all or substantially all V of lambda light chain gene seat lconstant gene segment C is deleted.
In one embodiment, mouse produces to comprise and derives from people V lthe V through somatic mutation of constant gene segment C lthe light chain of structural domain.In one embodiment, light chain comprises and derives from people V lthe V through somatic mutation of constant gene segment C lstructural domain and mouse C κ district.In one embodiment, mouse does not express lambda light chain.
In one embodiment, the mouse of genetic modification can to people V lregion sequence carries out somatic hypermutation.In a particular embodiment, mouse comprises cell, and described cell comprises deriving from can be reset and the V that encodes lthe people V of structural domain lthe light chain immunoglobulin gene of the rearrangement of constant gene segment C, and the V of the light chain immunoglobulin gene occlusion body cell mutation reset lstructural domain.
In one embodiment, mouse comprises expression containing the people V through somatic mutation being connected to mouse C κ lthe cell of the light chain of structural domain, wherein light chain derives from people V with comprising hthe V through somatic mutation of constant gene segment C hthe heavy chain of structural domain combines, and wherein heavy chain comprises murine heavy chain constant region (C h).In a particular embodiment, heavy chain comprises mouse C h1, mouse hinge, mouse C h2 and mouse C h3.In a particular embodiment, heavy chain comprises people C h1, hinge, mouse C h2 and mouse C h3.
In one embodiment, mouse comprises with one or more people V hconstant gene segment C is to endogenous mouse V hthe displacement of constant gene segment C, wherein people V hconstant gene segment C may be operably coupled to mouse C hdistrict's gene, so that mouse resets people V hconstant gene segment C and express comprise people V hstructural domain and mouse C hreverse imbedding heavy chain immunoglobulin.In one embodiment, the mouse V without rearrangement of 90-100% hconstant gene segment C by least one without reset people V hconstant gene segment C is replaced.In a particular embodiment, all or substantially all endogenous mouse V hconstant gene segment C by least one without reset people V hconstant gene segment C is replaced.In one embodiment, with at least 19, at least 39 or at least 80 or 81 people V without rearrangement hconstant gene segment C is replaced.In one embodiment, with at least 12 functional people V without resetting hconstant gene segment C, at least 25 functional people V without resetting hconstant gene segment C or at least 43 functional people V without resetting hconstant gene segment C is replaced.In one embodiment, mouse comprises with at least one without the people D reset hsection and at least one people J without rearrangement hsection is to all mouse D hand J hthe displacement of section.In one embodiment, at least one is without rearrangement people D hsection is selected from 1-1, D 1-7,1-26,2-8,2-15,3-3,3-10,3-16,3-22,5-5,5-12,6-6,6-13,7-27 and combination thereof.In one embodiment, at least one is without rearrangement people J hsection is selected from 1,2,3,4,5,6 and combination.In a particular embodiment, one or more people V hconstant gene segment C is selected from 1-2,1-8,1-24,1-69,2-5,3-7,3-9,3-11,3-13,3-15,3-20,3-23,3-30,3-33,3-48,3-53,4-31,4-39,4-59,5-51,6-1 people V hconstant gene segment C and combination thereof.
In one embodiment, mouse comprises the protein-bonded B cell of expression specificity binding purposes antigen, wherein associated proteins comprise derive from people V κ 1-39/J κ 5 reset or people V κ 3-20/J κ 1 reset light chain, and wherein cell comprises the immunoglobulin heavy chain gene of rearrangement, it derives from the people V being selected from 1-69,2-5,3-13,3-23,3-30,3-33,3-53,4-39,4-59 and 5-51 constant gene segment C hthe rearrangement of constant gene segment C.In one embodiment, one or more people V hconstant gene segment C and the people's heavy chain J being selected from 1,2,3,4,5 and 6 hconstant gene segment C is reset.In one embodiment, one or more people V hand J hconstant gene segment C and the people D being selected from 1-1,1-7,1-26,2-8,2-15,3-3,3-10,3-16,3-22,5-5,5-12,6-6,6-13 and 7-27 hconstant gene segment C is reset.In a particular embodiment, light chain gene has the somatic hypermutation of 1,2,3,4 or 5 or more.
In one embodiment, mouse comprises B cell, described B cell comprises containing being selected from 2-5/6-6/1, 2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4, 3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/3, 3-30/3-3/4, 3-30/3-22/5, 3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3, 3-30/6-6/4, 3-30/6-6/5, 3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5, 3-30/7-27/6, 3-33/1-7/4, 3-33/2-15/4, 4-39/1-26/3, 4-59/3-16/3, 4-59/3-16/4, 4-59/3-22/3, 5-51/3-16/6, 5-51/5-5/3, 5-51/6-13/5, 3-53/1-1/4, the V of 1-69/6-6/5 and 1-69/6-13/4 h/ D h/ J hthe immunoglobulin heavy chain variable region gene order of the rearrangement in district.In a particular embodiment, B cell expresses the associated proteins comprised with the human immunoglobulin heavy chain variable region of murine heavy chain constant domain and the human normal immunoglobulin variable region of light chain with mouse light chain constant domain.
In one embodiment, the people V of rearrangement ldistrict is people V κ 1-39J κ 5 sequence, and mouse is expressed and comprises (i) and derive from people V l/ J lthe V of sequence lstructural domain and (ii) mouse C lreverse imbedding light chain; Wherein light chain is combined with reverse imbedding heavy chain, and described reverse imbedding heavy chain comprises (i) mouse C h(ii) derive from and be selected from 1-2,1-8,1-24,1-69,2-5,3-7,3-9,3-11,3-13,3-15,3-20,3-23,3-30,3-33,3-48,3-53,4-31,4-39,4-59,5-51,6-1 people V hthe people V of constant gene segment C and combination thereof hthe people V through somatic mutation of constant gene segment C hstructural domain.In one embodiment, mouse expresses the light chain through somatic mutation.In one embodiment, C lmouse C κ.In a particular embodiment, people V hconstant gene segment C is selected from 2-5,3-13,3-23,3-30,4-59,5-51 and 1-69 constant gene segment C.In a particular embodiment, the people V of somatic mutation hstructural domain comprises the D deriving from and be selected from 1-1,1-7,2-8,3-3,3-10,3-16,3-22,5-5,5-12,6-6,6-13 and 7-27 hthe sequence of section.In a particular embodiment, the people V of somatic mutation hstructural domain comprises the J deriving from and be selected from 1,2,3,4,5 and 6 hthe sequence of section.In a particular embodiment, the people V of somatic mutation hstructural domain is selected from 2-5/6-6/1, 2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4, 3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/4, 3-30/3-22/5, 3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3, 3-30/6-6/4, 3-30/6-6/5, 3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5, 3-30/7-27/6, 4-59/3-16/3, 4-59/3-16/4, 4-59/3-22/3, 5-51/5-5/3, the people V of the rearrangement of 1-69/6-6/5 and 1-69/6-13/4 h/ D h/ J hsequence encoding.
In one embodiment, the people V of rearrangement ldistrict is people V κ 3-20J κ 1 sequence, and mouse expression comprises the people V that (i) derives from rearrangement l/ J lthe V of sequence lstructural domain and (ii) mouse C lreverse imbedding light chain; Wherein light chain is combined with reverse imbedding heavy chain, and described reverse imbedding heavy chain comprises (i) mouse C h(ii) derive from and be selected from 1-2,1-8,1-24,1-69,2-5,3-7,3-9,3-11,3-13,3-15,3-20,3-23,3-30,3-33,3-48,3-53,4-31,4-39,4-59,5-51,6-1 people V hthe people V of constant gene segment C and combination thereof hthe people V through somatic mutation of constant gene segment C h.In one embodiment, mouse expresses the light chain through somatic mutation.In one embodiment, C lmouse C κ.In a particular embodiment, people V hconstant gene segment C is selected from 3-30,3-33,3-53,4-39 and 5-51 constant gene segment C.In a particular embodiment, the people V of somatic mutation hstructural domain comprises the D deriving from and be selected from 1-1,1-7,1-26,2-15,3-3,3-16 and 6-13 hthe sequence of section.In a particular embodiment, the people V of somatic mutation hstructural domain comprises the J deriving from and be selected from 3,4,5 and 6 hthe sequence of section.In a particular embodiment, the people V of somatic mutation hstructural domain is selected from the people V of the rearrangement of 3-30/1-1/4,3-30/3-3/3,3-33/1-7/4,3-33/2-15/4,4-39/1-26/3,5-51/3-16/6,5-51/6-13/5 and 3-53/1-1/4 h/ D h/ J hsequence encoding.
In one embodiment, mouse comprises people V κ 1-39J κ 5 sequence of rearrangement and people V κ 3-20J κ 1 sequence of rearrangement, and mouse expression comprises the V that (i) derives from people V κ 1-39J κ 5 sequence or people V κ 3-20J κ 1 sequence lstructural domain and (ii) mouse C lreverse imbedding light chain; Wherein light chain is combined with reverse imbedding heavy chain, and described reverse imbedding heavy chain comprises (i) mouse C h(ii) derive from and be selected from 1-2,1-8,1-24,1-69,2-5,3-7,3-9,3-11,3-13,3-15,3-20,3-23,3-30,3-33,3-48,3-53,4-31,4-39,4-59,5-51,6-1 people V hthe people V of constant gene segment C and combination thereof hthe people V through somatic mutation of constant gene segment C h.In one embodiment, mouse expresses the light chain through somatic mutation.In one embodiment, C lmouse C κ.
In one embodiment, the endogenous mouse V without resetting of 90-100% hconstant gene segment C by least one without reset people V hconstant gene segment C is replaced.In a particular embodiment, all or substantially all endogenous mouse V without resetting hconstant gene segment C by least one without reset people V hconstant gene segment C is replaced.In one embodiment, with at least 18, at least 39, at least 80 or 81 people V without rearrangement hconstant gene segment C is replaced.In one embodiment, with at least 12 functional people V without resetting hconstant gene segment C, at least 25 functional people V without resetting hconstant gene segment C or at least 43 people V without rearrangement hconstant gene segment C is replaced.
In one embodiment, the mouse of genetic modification is C57BL strain, and it is selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola in a particular embodiment.In a particular embodiment, the mouse of genetic modification is the mixing of aforesaid 129 strains and aforesaid C57BL/6 strain.In another particular embodiment of the invention, mouse is the mixing of aforementioned 129 strains, or the mixing of aforementioned BL/6 strain.In a particular embodiment, 129 strains of mixing are 129S6 (129/SvEvTac) strains.
In one embodiment, mouse expresses reverse imbedding antibody, and described antibody comprises containing mouse C κ and the people V through somatic mutation deriving from people V κ 1-39J κ 5 sequence of rearrangement or people V κ 3-20J κ 1 sequence of rearrangement lthe light chain of structural domain, and comprise containing mouse C h1-2,1-8,1-24,1-69,2-5,3-7,3-9,3-11,3-13,3-15,3-20,3-23,3-30,3-33,3-48,3-53,4-31,4-39,4-59,5-51 and 6-1 people V is selected from deriving from hthe people V of constant gene segment C hthe people V through somatic mutation of constant gene segment C hthe heavy chain of structural domain, its small mouse does not express mouse antibodies and do not express people's antibody completely completely.In one embodiment, mouse comprises κ light chain gene seat, described locus comprises the displacement by people V κ 1-39J κ 5 sequence of rearrangement or the people V κ 3-20J κ 1 sequence pair endogenous mouse κ light chain gene segments of rearrangement, and comprises with complete or substantially complete people V hgene regions phase library is to all or substantially all endogenous mouse V hthe displacement of constant gene segment C.
An aspect, provide the antigen-specific antibodies group being derived from mouse described herein, wherein antibody comprises and derives from the light chain gene that people V κ 1-39/J κ 5 resets or people V κ 3-20/J κ 1 resets, and wherein antibody comprises deriving from and is selected from 1-2,1-3,1-8,1-18,1-24,1-46,1-58,1-69,2-5,2-26,2-70,3-7,3-9,3-11,3-13,3-15,3-16,3-20,3-21,3-23,3-30,3-33,3-43,3-48,3-53,3-64,3-72,3-73,4-31,4-34,4-39,4-59,5-51 and 6-1 people V hthe people V of constant gene segment C hthe immunoglobulin heavy chain gene through resetting of the rearrangement of constant gene segment C.In one embodiment, one or more people V hconstant gene segment C and the people's heavy chain J being selected from 1,2,3,4,5 and 6 hconstant gene segment C is reset.In a particular embodiment, light chain has the somatic hypermutation of 1,2,3,4 or 5 or more.
In one embodiment, light chain has 1,2,3 or the change of 4 individual cells height.In one embodiment, light chain gene has 1 or 2 sudden changes.In multiple embodiment, light chain gene can bear multiple sudden change in its sequence.
In one embodiment, light chain derive from people V κ 1-39/J κ 5 reset and light chain there is at least one or be no more than 4 individual cells height become.In one embodiment, light chain comprises at least 2 individual cells height changes.In one embodiment, light chain comprises at least 3 individual cells height changes.In one embodiment, light chain comprises at least 4 individual cells height changes.In a particular embodiment, sudden change is present in one or more framework regions (FW) of light chain.In a particular embodiment, sudden change is present in one or more complementary determining regions (CDR) of light chain.In a particular embodiment, suddenly change in the one or more FW being present in light chain and/or one or more CDR.In multiple embodiment, framework region is selected from framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or its combination.In multiple embodiment, CDR is selected from CDR1, CDR2, CDR3 and/or its combination.
In one embodiment, heavy chain comprises at least 1 sudden change in one or more FW or one or more CDR.In one embodiment, heavy chain comprises at least 1 sudden change in one or more FW and one or more CDR.In one embodiment, heavy chain comprises at least 2 sudden changes in one or more FW and one or more CDR.In one embodiment, heavy chain comprises at least 3 sudden changes in one or more FW and one or more CDR.In one embodiment, heavy chain comprises at least 4 sudden changes in one or more FW and one or more CDR.In one embodiment, heavy chain comprises at least 5 or more than 5 sudden changes in one or more FW and one or more CDR; In a particular embodiment, heavy chain comprises at least 5 or more than 5 sudden changes in two FW; In a particular embodiment, heavy chain comprises at least 5 or more than 5 sudden changes in a FW and CDR.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the light chain that the V κ 1-39/J κ 5 of about 9% originates has the sudden change that at least 1 is positioned at FW1; In one embodiment, the light chain of at least 9% comprises the sudden change that 1 is positioned at FW1.In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the light chain that the V κ 1-39/J κ 5 of about 25% originates has the sudden change being positioned at CDR1 of at least 1 or no more than 2; In one embodiment, the light chain of at least 19% has the sudden change that 1 is positioned at CDR1; In one embodiment, the light chain of at least 5% has the sudden change that 2 are positioned at CDR1.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the light chain that the V κ 1-39/J κ 5 of about 20% originates has the sudden change that at least 1 or no more than 3 are positioned at FW2; In one embodiment, the light chain of at least 17% has the sudden change that 1 is positioned at FW2; In one embodiment, the light chain of at least 1% has the sudden change that 2 are positioned at FW2; In one embodiment, the light chain of at least 1% has the sudden change that 3 are positioned at FW2.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the light chain that the V κ 1-39/J κ 5 of about 10% originates has the sudden change that at least 1 or no more than 2 are positioned at CDR2; In one embodiment, the light chain of at least 10% has the sudden change that 1 is positioned at CDR2; In one embodiment, the light chain of at least 1% has the sudden change that 2 are positioned at CDR2.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the light chain that the V κ 1-39/J κ 5 of about 29% originates has the sudden change that at least 1 or no more than 4 are positioned at FW3; In one embodiment, the light chain of at least 21% has the sudden change that 1 is positioned at FW3; In one embodiment, the light chain of at least 5% has the sudden change that 2 are positioned at FW3; In one embodiment, the light chain of at least 2% has the sudden change that 3 are positioned at FW3; In one embodiment, the light chain of at least 2% has the sudden change that 4 are positioned at FW3.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the light chain that the V κ 1-39/J κ 5 of about 37% originates has the sudden change that at least 1 or no more than 4 are positioned at CDR3; In one embodiment, the light chain of at least 27% has the sudden change that 1 is positioned at CDR3; In one embodiment, the light chain of at least 8% has the sudden change that 2 are positioned at CDR3; In one embodiment, the light chain of at least 1% has the sudden change that 3 are positioned at CDR3; In one embodiment, the light chain of at least 1% has the sudden change that 4 are positioned at CDR3.
In one embodiment, provide the antigen-specific antibodies group being derived from mouse described herein, wherein antibody comprises the light chain deriving from people V κ 1-39/J κ 5 and reset, and the light chain that the V κ 1-39/J κ 5 of about 9% originates has the sudden change being positioned at FW1 of 1 or more, the light chain that the V κ 1-39/J κ 5 of about 25% originates has the sudden change being positioned at CDR1 of 1 or more, the light chain that the V κ 1-39/J κ 5 of about 20% originates has the sudden change being positioned at FW2 of 1 or more, the light chain that the V κ 1-39/J κ 5 of about 10% originates has the sudden change being positioned at CDR2 of 1 or more, the light chain that the V κ 1-39/J κ 5 of about 29% originates has the sudden change being positioned at FW3 of 1 or more, and the light chain that the V κ 1-39/J κ 5 of about 37% originates has the sudden change being positioned at CDR3 of 1 or more.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the heavy chain of about 35% has the sudden change that at least 1 is positioned at FW1; In one embodiment, the heavy chain of at least 25% has the sudden change that 1 is positioned at FW1; In one embodiment, the heavy chain of at least 9% has the sudden change that 2 are positioned at FW1; In one embodiment, the heavy chain of at least 1% has the sudden change that 3 are positioned at FW1; In one embodiment, the heavy chain of at least 1% has the sudden change being positioned at FW1 more than 5.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the heavy chain of about 92% has the sudden change that at least 1 or no more than 4 are positioned at CDR1; In one embodiment, the heavy chain of at least 92% has the sudden change that at least 1, at least 2, at least 3 or at least 4 are positioned at CDR1; In one embodiment, the heavy chain of at least 26% has the sudden change that 1 is positioned at CDR1; In one embodiment, the heavy chain of at least 44% has the sudden change that 2 are positioned at CDR1; In one embodiment, the heavy chain of at least 19% has the sudden change that 3 are positioned at CDR1; In one embodiment, the heavy chain of at least 3% has the sudden change that 4 are positioned at CDR1.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the heavy chain of about 66% has the sudden change that at least 1 or no more than 3 are positioned at FW2; In one embodiment, the heavy chain of at least 66% has the sudden change that at least 1, at least 2 or at least 3 are positioned at FW2; In one embodiment, the heavy chain of at least 35% has the sudden change that 1 is positioned at FW2; In one embodiment, the heavy chain of at least 23% has the sudden change that 2 are positioned at FW2; In one embodiment, the heavy chain of at least 8% has the sudden change that 3 are positioned at FW2.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the heavy chain of about 70% has the sudden change that at least 1 or no more than 4 are positioned at CDR2; In one embodiment, the heavy chain of at least 70% has the sudden change that at least 2, at least 3 or at least 4 are positioned at CDR2; In one embodiment, the heavy chain of at least 34% has the sudden change that 1 is positioned at CDR2; In one embodiment, the heavy chain of at least 20% has the sudden change that 2 are positioned at CDR2; In one embodiment, the heavy chain of at least 12% has the sudden change that 3 are positioned at CDR2; In one embodiment, the heavy chain of at least 5% has the sudden change that 4 are positioned at CDR2.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the heavy chain of about 91% has the sudden change being positioned at FW3 of at least 1 or as many as 5 or more; In one embodiment, the heavy chain of at least 91% has the sudden change that at least 2, at least 3, at least 4 or at least 5 or more are positioned at FW3; In one embodiment, the heavy chain of at least 19% has the sudden change that 1 is positioned at FW3; In one embodiment, the heavy chain of at least 33% has the sudden change that 2 are positioned at FW3; In one embodiment, the heavy chain of at least 22% has the sudden change that 3 are positioned at FW3; In one embodiment, the heavy chain of at least 11% has the sudden change that 4 are positioned at FW3; In one embodiment, the heavy chain of at least 7% has the sudden change that 5 or more are positioned at FW3.
In one embodiment, light chain derives from people V κ 1-39/J κ 5 and resets, and the heavy chain of about 63% has the sudden change that at least 1 or no more than 2 are positioned at CDR3; In one embodiment, the heavy chain of at least 63% has the sudden change that at least 1 is positioned at CDR3; In one embodiment, the heavy chain of at least 54% has the sudden change that 1 is positioned at CDR3; In one embodiment, the heavy chain of at least 9% has the sudden change that 2 are positioned at CDR3.
In one embodiment, provide the antigen-specific antibodies group being derived from mouse described herein, wherein antibody comprises and derives from light chain that people V κ 1-39/J κ 5 resets and the heavy chain of at least 35% has the sudden change that 1 or more is positioned at FW1, the heavy chain of about 92% has the sudden change that 1 or more is positioned at CDR1, the heavy chain of about 66% has the sudden change that 1 or more is positioned at FW2, the heavy chain of about 70% has the sudden change that 1 or more is positioned at CDR2, the heavy chain of about 91% has the sudden change that 1 or more is positioned at FW3, the heavy chain of about 63% has the sudden change that 1 or more is positioned at CDR3.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and light chain gene has at least 1 or no more than 2 individual cells height changes; In one embodiment, light chain gene has at least 2, at least 3, at least 4 or more individual cells height and becomes.In a particular embodiment, sudden change is present in one or more framework regions of light chain.In a particular embodiment, sudden change is present in one or more CDR districts of light chain.In a particular embodiment, suddenly change in the one or more framework region being present in light chain and/or one or more CDR district.In multiple embodiment, framework region is selected from framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or its combination.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the light chain that the V κ 3-20/J κ 1 of about 10% originates has the sudden change that at least one is positioned at FW1; In one embodiment, the light chain of at least 10% has the sudden change that is positioned at FW1.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the light chain that the V κ 3-20/J κ 1 of about 53% originates has the sudden change that at least 1 or no more than 2 are positioned at CDR1; In one embodiment, the light chain of at least 27% has the sudden change that one or more are positioned at CDR1; In one embodiment, the light chain of about 54% has the sudden change that 1 or 2 is positioned at CDR1.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the light chain that the V κ 3-20/J κ 1 of about 6% originates has the sudden change that at least 1 or no more than 2 are positioned at FW2; In one embodiment, the light chain of at least 6% has the sudden change that at least 1 is positioned at FW2; In one embodiment, the light chain of at least 3% has the sudden change that 1 is positioned at FW2; In one embodiment, the light chain of at least 3% has the sudden change that 2 are positioned at FW2.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and has at least about the light chain in V κ 3-20/J κ 1 source of 3% sudden change that at least 1 is positioned at CDR2; In one embodiment, the light chain of at least 3% has the sudden change that 1 is positioned at CDR2.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the light chain that the V κ 3-20/J κ 1 of about 17% or more originates has the sudden change that at least 1 or no more than 2 are positioned at FW3; In one embodiment, the light chain of at least 20% has the sudden change that 1 is positioned at FW3; In one embodiment, the light chain of at least 17% has the sudden change that 2 are positioned at FW3.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the light chain that the V κ 3-20/J κ 1 of at least 43% originates has the sudden change that at least 1 is positioned at CDR3; In one embodiment, the light chain of at least 43% has the sudden change that 1 is positioned at CDR3.
In one embodiment, provide the antigen-specific antibodies group being derived from mouse described herein, wherein antibody comprises the light chain deriving from people V κ 3-20/J κ 1 and reset, and the light chain that the V κ 3-20/J κ 1 of about 10% originates has the sudden change being positioned at FW1 of 1 or more, the light chain that the V κ 3-20/J κ 1 of about 53% originates has the sudden change being positioned at CDR1 of 1 or more, the light chain that the V κ 3-20/J κ 1 of about 6% originates has the sudden change being positioned at FW2 of 1 or more, the light chain that the V κ 3-20/J κ 1 of about 3% originates has the sudden change being positioned at CDR2 of 1 or more, the light chain that the V κ 3-20/J κ 1 of about 37% originates has the sudden change being positioned at FW3 of 1 or more, and the light chain that the V κ 3-20/J κ 1 of about 43% originates has the sudden change being positioned at CDR3 of 1 or more.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the heavy chain of about 43% has the sudden change that at least 1 or no more than 2 are positioned at FW1; In one embodiment, the heavy chain of at least 41% has the sudden change that at least 1 is positioned at FW1; In one embodiment, the heavy chain of about 41% has the sudden change that 1 is positioned at FW1; In one embodiment, the heavy chain of about 2% has the sudden change that 2 are positioned at FW1.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the heavy chain of about 92% has the sudden change that at least 1 or no more than 4 are positioned at CDR1; In one embodiment, the heavy chain of at least 43% has the sudden change that at least 1 is positioned at CDR1; In one embodiment, the heavy chain of at least 25% has the sudden change that at least 2 are positioned at CDR1; In one embodiment, the heavy chain of at least 15% has the sudden change that at least 3 are positioned at CDR1; In one embodiment, the heavy chain of at least 10% has the sudden change being positioned at CDR1 of 4 or more.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the heavy chain of about 46% has the sudden change that at least 1 or no more than 3 are positioned at FW2; In one embodiment, the heavy chain of at least 34% has the sudden change that at least 1 is positioned at FW2; In one embodiment, the heavy chain of at least 10% has the sudden change being positioned at FW2 of 2 or more; In one embodiment, the heavy chain of at least 2% has the sudden change being positioned at FW2 of 3 or more.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the heavy chain of about 84% has at least 1 or as many as 5 or is positioned at the sudden change of CDR2 more than 5; In one embodiment, the heavy chain of at least 39% has the sudden change being positioned at CDR2 of 1 or more; In one embodiment, the heavy chain of at least 18% has the sudden change being positioned at CDR2 of 2 or more; In one embodiment, the heavy chain of at least 21% has the sudden change being positioned at CDR2 of 3 or more; In one embodiment, the heavy chain of at least 3% has the sudden change being positioned at CDR2 of 4 or more; In one embodiment, the heavy chain of at least 2% has the sudden change being positioned at CDR2 of 5 or more.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the heavy chain of about 92% has at least 1 or as many as 5 or is positioned at the sudden change of FW3 more than 5; In one embodiment, the heavy chain of at least 21% has the sudden change that at least 1 is positioned at FW3; In one embodiment, the heavy chain of at least 20% has the sudden change that at least 2 are positioned at FW3; In one embodiment, the heavy chain of at least 13% has the sudden change that at least 3 are positioned at FW3; In one embodiment, the heavy chain of at least 20% has the sudden change that at least 4 are positioned at FW3; In one embodiment, the heavy chain of at least 18% has the sudden change that at least 5 are positioned at FW3.
In one embodiment, light chain derives from people V κ 3-20/J κ 1 and resets, and the heavy chain of about 7% has the sudden change that at least 1 is positioned at CDR3; In one embodiment, the heavy chain of about 7% has the sudden change that 1 is positioned at CDR3.
In one embodiment, provide the antigen-specific antibodies group being derived from mouse described herein, wherein antibody comprises and derives from light chain that people V κ 3-20/J κ 1 resets and the heavy chain of about 43% has the sudden change that 1 or more is positioned at FW1, the heavy chain of about 92% has the sudden change that 1 or more is positioned at CDR1, the heavy chain of about 46% has the sudden change that 1 or more is positioned at FW2, the heavy chain of about 84% has the sudden change that 1 or more is positioned at CDR2, the heavy chain of about 92% has the sudden change that 1 or more is positioned at FW3, the heavy chain of about 7% has the sudden change that 1 or more is positioned at CDR3.
In one aspect, provide the mouse of the light chain immunoglobulin of expressing from the light chain immunoglobulin sequence of resetting, the light chain immunoglobulin sequence of wherein resetting is present in the germline of mouse, and wherein light chain immunoglobulin comprises people's variable sequence.In one embodiment, the germline of mouse comprises and derives from the V section identical with all non-proxy sequence of light chain in each B cell of the mouse being present in the sequence of light chain comprising rearrangement and the light chain immunoglobulin sequence of the rearrangement of identical J section.
In one embodiment, the germline of mouse lacks the functional light chain immunoglobulin V constant gene segment C without resetting.In one embodiment, the germline of mouse lacks the functional light chain immunoglobulin J constant gene segment C without resetting.
In one embodiment, the germline of mouse comprises no more than 1, no more than 2 or no more than 3 (V/J) sequence of light chain through rearrangement.
In one embodiment, the V/J sequence of rearrangement comprises κ sequence of light chain.In a particular embodiment, κ sequence of light chain is human kappa light chain sequence.In a particular embodiment, κ sequence of light chain is selected from people V κ 1-39/J sequence, people V κ 3-20/J sequence and combination thereof.In a particular embodiment, κ sequence of light chain is people V κ 1-39/J κ 5 sequence.In a particular embodiment, κ sequence of light chain is people V κ 3-20/J κ 1 sequence.
In one embodiment, mouse also comprises the sequence be selected from relative to the mouse κ intron enhanser of 5 ' side of the light chain immunoglobulin sequence of resetting, mouse κ 3 ' enhanser and combination thereof in its germline.
In one embodiment, mouse comprises the people V without resetting hconstant gene segment C, without reset people D hconstant gene segment C and the people J without rearrangement hconstant gene segment C, wherein V h, D hand J hconstant gene segment C can reset to be formed the immunoglobulin heavy chain variable gene order that may be operably coupled to light chain constant gene order.In one embodiment, mouse comprises multiple people V h, D hand J hconstant gene segment C.In a particular embodiment, people V h, D hand J hconstant gene segment C replaces endogenous mouse V on endogenous mouse immunoglobulin heavy chain gene seat h, D hand J hconstant gene segment C.In a particular embodiment, mouse comprises with all or substantially all functional human V h, D hand J hconstant gene segment C is to all or substantially all functional mouse V h, D hand J hthe displacement of constant gene segment C.
In one embodiment, mouse expresses the light chain immunoglobulin comprising mouse constant sequence.In one embodiment, mouse expresses the light chain immunoglobulin comprising people's constant series.
In one embodiment, mouse is expressed to comprise and is selected from C h1 sequence, hinge sequence, C h2 sequences, C hthe heavy chain immunoglobulin of the mouse sequence of 3 sequences and combination thereof.
In one embodiment, mouse is expressed to comprise and is selected from C h1 sequence, hinge sequence, C h2 sequences, C hthe heavy chain immunoglobulin of the human sequence of 3 sequences and combination thereof.
In one embodiment, the light chain immunoglobulin sequence of resetting in mouse germline is positioned at endogenous mouse light chain immunoglobulin gene seat.In a particular embodiment, the light chain immunoglobulin sequence of resetting in mouse germline replaces all or substantially all mouse light chain V and J sequence on endogenous mouse light chain immunoglobulin gene seat.
In one aspect, provide the mouse comprising B cell group, each B cell that the feature of described B cell group is to comprise non-proxy sequence of light chain comprises the light chain gene through resetting deriving from single people V section and single people J section, unique light chain variable sequence in its small mouse germline is the retracing sequence deriving from single people V section and single people J section, and each B cell of the light chain gene wherein comprising rearrangement also comprises the gene of encoding homologous people heavy-chain variable domains, and the light chain gene wherein reset comprises at least 1, at least 2, at least 3 or the change of at least 4 individual cells height.
In one aspect, the versatility, inducing pluripotent or the totipotent cell that are derived from mouse as herein described is provided.In a particular embodiment, cell is mouse embryonic stem (ES) cell.
In one aspect, the tissue being derived from mouse as herein described is provided.In one embodiment, tissue source is from the spleen of mouse as herein described, lymphoglandula or marrow.
In one aspect, the nucleus being derived from mouse described herein is provided.In one embodiment, nucleus is from the diploid cell of non-B cell.
In one aspect, the mouse cell be separated from mouse as herein described is provided.In one embodiment, cell is ES cell.In one embodiment, cell is lymphocyte.In one embodiment, lymphocyte is B cell.In one embodiment, B cell expresses the chimeric heavy chain comprising the variable domains deriving from people's gene section; With derive from the people V κ 1-39/J sequence of rearrangement, the people V κ 3-20/J sequence of rearrangement or its combination light chain; Wherein heavy-chain variable domains merge to murine constant region and light variable domains merge to mouse or human constant region.
In one aspect, provide hybridoma, prepared by the wherein said hybridoma B cell of mouse as herein described.In a particular embodiment, B cell is controlled oneself with the mouse as herein described of the antigen immune comprising object epi-position, and B cell expresses the associated proteins of binding purposes epi-position, and associated proteins has the people V through somatic mutation hstructural domain and mouse C h, and there is the people V of the people V κ 1-39J κ 5 deriving from rearrangement or the people V κ 3-20J κ 1 reset lstructural domain and mouse C l.
In one aspect, provide mice embryonic, wherein said embryo comprises the contributing ES cells being derived from mouse described herein.
In one aspect, provide targeting vector, its relative to carrier 5 ' and 3 ' mouse homology arm sequence transcriptional orientation on from 5 ' to 3 ', comprise the 5 ' mouse homology arm, people or mouse immuning ball protein promotor, people or mouse leader sequence, and be selected from the people V of the people V κ 1-39J κ 5 of rearrangement or the people V κ 3-20J κ 1 of rearrangement ldistrict, and the 3 ' mouse homology arm.In one embodiment, 5 ' and 3 ' homology arm by carrier target to relative to being present in mouse C κ5 ' side of gene and be adjacent to the sequence of 5 ' of its enhancer sequence.In one embodiment, promotor is human normal immunoglobulin variable gene segment promotor.In a particular embodiment, promotor is people V κ 3-15 promotor.In one embodiment, leader sequence is mouse leader sequence.In a particular embodiment, mouse leader sequence is mouse V κ 3-7 leader sequence.
In one aspect, provide targeting vector as described above, but 5 ' side joint of people or mouse promoter Site-specific recombinase recognition site (SRRS) is replacing the 5 ' mouse homology arm, and people V l3 ' the side joint in district SRRS is to replace the 3 ' mouse homology arm.
In one aspect, the reverse imbedding antibody that mouse as described herein produces, wherein said reverse imbedding antibody comprises containing people V lwith mouse C llight chain, with containing people V hwith mouse C hheavy chain.
In one aspect, provide the method for Dispersal risk, described method be included in single cell express (a) mouse through immunity as herein described with people C hthe V that gene order merges hgene order; (b) mouse through immunity as herein described with people C lthe V that gene order merges lgene order; And (c) maintains cell under the condition enough expressing fully human antibodies, and separation antibody.In one embodiment, cell contain the second immune mouse as herein described with people C hthe 2nd V that gene order merges hgene order, a V hthe V of gene sequences encode identification first epi-position hstructural domain, and the 2nd V hthe V of gene sequences encode identification second epi-position hstructural domain, wherein the first epi-position and the second epi-position are different.
In one aspect, provide the protein-bonded method of preparation epi-position, described method comprises mouse described herein is exposed to the immunogen comprising object epi-position, mouse is maintained, with the immunoglobulin molecules being separated specific binding object epi-position under being enough to make mouse produce the condition of the immunoglobulin molecules of specific binding object epi-position; Wherein epi-position associated proteins comprises containing the people V through somatic mutation hwith mouse C hheavy chain, described heavy chain with containing mouse C lwith the people V of the people V κ 3-20J κ 1 of the people V κ 1-39J κ 5 or rearrangement that are derived from rearrangement llight chain combine.
In one aspect, provide and express the protein-bonded cell of epi-position, wherein said cell contains: (a) coding derives from the people V of the people V κ 1-39J κ 5 of rearrangement or the people V κ 3-20J κ 1 of rearrangement lthe human nucleotide sequence of structural domain, wherein said human nucleotide sequence merges (directly or pass through joint) to human normal immunoglobulin light chain constant domain cDNA sequence (such as people κ constant domain DNA sequence dna); And (b) coding derives from the first V hthe people V of nucleotide sequence hthe first V of structural domain hnucleotide sequence, wherein said the first V hnucleotide sequence merges (directly or pass through joint) to human immunoglobulin heavy chain's constant domain cDNA sequence; Wherein epi-position associated proteins identification first epi-position.In one embodiment, epi-position associated proteins is with lower than 10 -6m, lower than 10 -8m, lower than 10 -9m, lower than 10 -10m, lower than 10 -11m or lower than 10 -12the dissociation constant of M is in conjunction with the first epi-position.
In one embodiment, cell comprises coding second people V hsecond human nucleotide sequence of structural domain, wherein the second human sequence merges (directly or pass through joint) to human immunoglobulin heavy chain's constant domain cDNA sequence, and wherein the second people V hstructural domain not specific recognition first epi-position (such as, indication example is as 10 -6m, 10 -5m, 10 -4the dissociation constant of M or higher), and wherein epi-position associated proteins identification first epi-position and the second epi-position, and wherein the first and second heavy chain immunoglobulins combine with the light chain of identical (a) separately.
In one embodiment, the 2nd V hstructural domain is with lower than 10 -6m, lower than 10 -7m, lower than 10 -8m, lower than 10 -9m, lower than 10 -10m, lower than 10 -11m or lower than 10 -12the dissociation constant of M is in conjunction with the second epi-position.
In one embodiment, epi-position associated proteins comprises the first heavy chain immunoglobulin and the second heavy chain immunoglobulin, every bar heavy chain and the people V deriving from the rearrangement being selected from people V κ 1-39J κ 5 or people V κ 3-20J κ 1 lthe identical light chain in district combines, wherein the first heavy chain immunoglobulin is to receive the dissociation constant of rubbing and rubbing in scope to skin in conjunction with the first epi-position, the dissociation constant that second heavy chain immunoglobulin rubs in scope with nmole to skin is in conjunction with the second epi-position, first epi-position and the second epi-position are different, first heavy chain immunoglobulin not in conjunction with the second epi-position or with the dissociation constant being weaker than micro-scope of rubbing (such as milli rub scope) in conjunction with the second epi-position, second heavy chain immunoglobulin not in conjunction with the first epi-position or with the dissociation constant being weaker than micro-scope of rubbing (such as milli rub scope) in conjunction with the first epi-position, and V l, the first heavy chain immunoglobulin V hwith the V of the second heavy chain immunoglobulin hin one or more through somatic mutation.
In one embodiment, the first heavy chain immunoglobulin comprises albumin A in conjunction with residue, and the second heavy chain immunoglobulin lacks albumin A in conjunction with residue.
In one embodiment, cell be selected from CHO, COS, 293, the retina cell of HeLa and expression nucleic acid sequence is (as routine PERC.6 tMcell).
In one aspect, provide reverse imbedding antibody, it comprises people V hwith murine heavy chain constant domain, people V lwith mouse light chain constant domain, wherein antibody is prepared by the method for the immunogen immune comprising epi-position mouse as herein described by comprising, and antibodies specific combines and is used for the immunogenic epi-position of immune mouse.In one embodiment, V lstructural domain is through somatic mutation.In one embodiment, V hstructural domain is through somatic mutation.In one embodiment, V lstructural domain and V hstructural domain is all through somatic mutation.In one embodiment, V lbe connected to mouse C κ structural domain.
In one aspect, provide mouse, it is included on endogenous mouse heavy chain locus and replaces all or substantially all mouse V hthe people V of constant gene segment C hconstant gene segment C; Replace the people's light chain V being selected from the rearrangement of the V κ 1-39/J of rearrangement and the V κ 3-20/J of rearrangement or its combination of no more than 1 or 2 of all mouse light chain constant gene segment Cs l/ J lsequence; Wherein people's heavy chain variable gene section is connected to mouse constant gene, and people's light chain gene sequence of resetting is connected to people or mouse constant gene.
In one aspect, mouse ES cells comprises the displacement of employment heavy chain variable gene section to all or substantially all murine heavy chain variable gene segments, and people's light chain V of the rearrangement of no more than 1 or 2 l/ J lsequence, wherein people's heavy chain variable gene section is connected to the constant gene of mouse immunoglobulin heavy, and the people's light chain V reset l/ J lsequence is connected to mouse or human normal immunoglobulin chain constant gene.In a particular embodiment, chain constant gene is mouse constant gene.
In one aspect, the antigen-binding proteins prepared by mouse described herein is provided.In a particular embodiment, antigen-binding proteins comprises the human immunoglobulin heavy chain variable region of merging with murine constant region, with the human normal immunoglobulin variable region of light chain deriving from V κ 1-39 constant gene segment C or V κ 3-20 constant gene segment C, wherein constant region of light chain is murine constant region.
In one aspect, provide the complete human antigen's associated proteins prepared from the immune globulin variable region gene sequence from mouse described herein, wherein antigen-binding proteins comprises complete people's heavy chain of the people variable region containing the sequence being derived from mouse described herein, and comprises the complete people's light chain containing V κ 1-39 or V κ 3-20.In one embodiment, variable region of light chain comprises 1 to 5 individual cells sudden change.In one embodiment, variable region of light chain is the homology variable region of light chain of matching with variable region of heavy chain in mouse B cell.
In one embodiment, complete human antigen's associated proteins comprises the first heavy chain and the second heavy chain, wherein the first heavy chain and the second heavy chain comprise the different variable regions being derived from mouse described herein independently, and each of wherein the first and second heavy chains is combined with the people's light chain deriving from V κ 1-39 constant gene segment C or V κ 3-20 constant gene segment C and expresses in host cell.In one embodiment, the first heavy chain comprises the first variable region of heavy chain of the first epi-position of specific binding first antigen, and the second heavy chain comprises the second variable region of heavy chain of the second epi-position of specific binding second antigen.In a particular embodiment, the first antigen and the second antigen are different.In a particular embodiment, the first antigen and the second antigen are identical, and the first epi-position and the second epi-position are different; In a particular embodiment, protein-bonded first molecule can not hinder protein-bonded second molecule to the combination of the second epi-position to the combination of the first epi-position.
In one aspect, the complete human conjugated protein being derived from the human normal immunoglobulin sequence of mouse described herein comprises the first heavy chain immunoglobulin and the second heavy chain immunoglobulin, wherein the first heavy chain immunoglobulin comprises the first variable region of the variable region being different from the second heavy chain immunoglobulin, and wherein the first heavy chain immunoglobulin comprises wild-type protein A in conjunction with determinant, and the second heavy chain lacks wild-type protein A in conjunction with determinant.In one embodiment, first heavy chain immunoglobulin is associated proteins A under separation condition, and the second heavy chain immunoglobulin not associated proteins A or be weaker than the ability associated proteins A of the first immunoglobulin heavy chain binding protein A with at least 10 times, hundred times or thousand times under separation condition.In a particular embodiment, the first and second heavy chains are IgG1 isotypes, and wherein the second heavy chain comprises the modification being selected from 95R (EU 435R), 96F (EU 436F) and combination thereof, and wherein the first heavy chain lacks such modification.
In one aspect, provide the method preparing Bispecific antigen binding proteins, comprise the first object antigen being exposed to by the first mouse described herein and comprising the first epi-position, second mouse described herein is exposed to the second object antigen comprising the second epi-position, allow the immunne response that the first and second mouse carry out for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, derive from cells first heavy chain of single complete people's light chain of people V κ 1-39 or people V κ 3-20 constant gene segment C and the second heavy chain to form Bispecific antigen binding proteins expressing, and be separated described Bispecific antigen binding proteins.
In one embodiment, the first antigen and the second antigen are different.
In one embodiment, the first antigen and the second antigen are identical, and the first epi-position and the second epi-position are different.In one embodiment, the combination of the first variable region of heavy chain and the first epi-position can not hinder the combination of the second variable region of heavy chain and the second epi-position.
In one embodiment, the first epi-position of people's light chain specific binding first antigen when matching with the first heavy chain, and when matching with the second heavy chain the second epi-position of specific binding second antigen.
In one embodiment, the first antigen is selected from soluble antigen and cell-surface antigens (such as tumour antigen), and the second antigen comprises cell surface receptor.In a particular embodiment, cell surface receptor is immunoglobulin receptor.In a particular embodiment, immunoglobulin receptor is Fc acceptor.In one embodiment, the first antigen and the second antigen are identical cell surface receptors, and the first heavy chain is bonded to the first epi-position does not hinder the second heavy chain to be bonded to the second epi-position.
In one embodiment, the light variable domains of light chain comprises 2 to 5 individual cells sudden changes.In one embodiment, light variable domains is the same endogenous light chain through somatic mutation of expressing with the first or second weight chain variable binding domain in the B cell of the mouse of the first or second immunity.In one embodiment, the light chain of cell comprises Germline sequences.
In one embodiment, first complete people's heavy chain has the amino acid modified of the avidity reducing albumin A at that time, and second complete people's heavy chain do not comprise reduce its to the modification of the avidity of albumin A.
In one aspect, provide the method for the bi-specific antibody preparing specific binding first and second antigen, wherein said method comprises the first weight chain variable (V that (a) identification code is specific to the first antigen h) the first nucleotide sequence of structural domain; B () identification code is specific to second people's weight chain variable (V of the second antigen h) the second nucleotide sequence of structural domain; C () provides encoding human light chain variable (V l) the 3rd nucleotide sequence in district, described people's light chain variable (V l) V of Qu Dangyu (a) hspecific binding first antigen during district's timing, and as the V with (b) hspecific binding second antigen during district's pairing; D the host cell of () cultivation containing first, second, and third nucleotide sequence is to allow the first and second people V hdistrict and people V ldistrict expresses to form bi-specific antibody; (d) described bi-specific antibody is reclaimed.In many aspects, the first and second antigens are different from each other.In many aspects, the first and second nucleotide sequences are separated the human normal immunoglobulin V from expressing from the light chain immunoglobulin sequence of resetting lthe mouse through immunity in district, the immunoglobulin sequences wherein reset is in the germline of mouse.
In one embodiment, people V ldistrict derives from people's sequence of light chain of the rearrangement comprising people V κ 1-39 constant gene segment C or people V κ 3-20 constant gene segment C.In a particular embodiment, people's sequence of light chain of rearrangement is Germline sequences (namely not comprising somatic hypermutation in V gene segment sequences).
In one embodiment, the 3rd nucleotide sequence is separated the human normal immunoglobulin V from expressing from the light chain immunoglobulin sequence of the rearrangement in mouse germline lthe mouse in district.In one embodiment, the light chain immunoglobulin sequence of rearrangement comprises people V κ 1-39 or people V κ 3-20 constant gene segment C.In a particular embodiment, the light chain immunoglobulin sequence of rearrangement comprises people V κ 1-39 constant gene segment C.In one embodiment, human normal immunoglobulin V ldistrict expresses from modified endogenous immunoglobulin light chain gene seat.
In one embodiment, the first and second antigens are present on a molecule.In one embodiment, the first and second antigens are present on different molecules.In multiple embodiment, the first or second nucleotide sequence comprises the heavy chain of reduction coding to the modification of the avidity of albumin A.
In one embodiment, the first or second nucleotide sequence comprises people's weight chain variabl area sequence of rearrangement, and described variable region sequences comprises and is selected from V hl-2, V hl-3, V hl-8, V h1-18, V hl-24, V hl-46, V hl-58, V h1-69, V h2-5, V h2-26, V h2-70, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-21, V h3-23, V h3-30, V h3-33, V h3-43, V h3-48, V h3-53, V h3-64, V h3-72, V h3-73, V h4-31, V h4-34, V h4-39, V h4-59, V h5-51 and V hpeople's heavy chain gene section of 6-1.In a particular embodiment, heavy chain gene section is V h2-5, V h3-23 or V h3-30.
In one aspect, provide the method for the bi-specific antibody preparing specific binding first and second antigen, wherein said method comprises the first weight chain variable (V that (a) identification code is specific to the first antigen h) the first nucleotide sequence of structural domain; B () identification code is specific to second people's weight chain variable (V of the second antigen h) the second nucleotide sequence of structural domain; C () provides coding to derive from people's light chain variable (V of people V κ 1-39 or people V κ 3-20 constant gene segment C l) the 3rd nucleotide sequence in district, described people's light chain variable (V l) V of Qu Dangyu (a) hspecific binding first antigen during district's pairing, and as the V with (b) hspecific binding second antigen during district's pairing; D the host cell of () cultivation containing first, second, and third nucleotide sequence is to allow the first and second people V hdistrict and people V ldistrict expresses to form bi-specific antibody; (d) described bi-specific antibody is reclaimed.In many aspects, the first and second antigens are different from each other.In many aspects, the first and second nucleotide sequences are separated the mouse of immunity of hanging oneself, and described mouse expresses the human normal immunoglobulin V of the immunoglobulin sequences from the rearrangement deriving from people V κ 1-39 or people V κ 3-20 constant gene segment C ldistrict, the people V κ 1-39 wherein reset or people V κ 3-20 constant gene segment C are in the germline of mouse.
In one embodiment, the 3rd nucleotide sequence is Germline sequences (namely not comprising somatic hypermutation in V gene segment sequences).In one embodiment, the 3rd nucleotide sequence is separated from expressing human normal immunoglobulin V lthe mouse in district, described human normal immunoglobulin V ldistrict derives from people V κ 1-39 from the light chain immunoglobulin sequence of the rearrangement in mouse germline or people V κ 3-20 constant gene segment C.In a particular embodiment, the 3rd nucleotide sequence comprises 2 to 5 individual cells height changes in complementary determining region (CDR) and/or framework region (FWR).In one embodiment, human normal immunoglobulin V ldistrict expresses from modified endogenous immunoglobulin light chain gene seat.
In one embodiment, the first and second antigens are present on a molecule.In one embodiment, the first and second antigens are present on different molecules.In one embodiment, the first or second nucleotide sequence comprises the heavy chain of reduction coding to the modification of the avidity of albumin A.
In one embodiment, the first or second nucleotide sequence comprises people's weight chain variabl area sequence of rearrangement, and described variable region sequences comprises and is selected from V hl-2, V hl-3, V hl-8, V h1-18, V hl-24, V hl-46, V hl-58, V h1-69, V h2-5, V h2-26, V h2-70, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-21, V h3-23, V h3-30, V h3-33, V h3-43, V h3-48, V h3-53, V h3-64, V h3-72, V h3-73, V h4-31, V h4-34, V h4-39, V h4-59, V h5-51 and V hpeople's heavy chain gene section of 6-1.In a particular embodiment, heavy chain gene section is V h2-5, V h3-23 or V h3-30.
In one aspect, provide the method preparing bi-specific antibody, comprise and mouse described herein is exposed to object antigen, permission mouse carries out the immunne response for object antigen, the first variable region of heavy chain of the first epi-position of qualification binding purposes antigen, second people variable region of heavy chain of the second epi-position of qualification binding purposes antigen, first complete people's heavy chain gene of the first heavy chain of the first epi-position of preparation coding binding purposes antigen, second complete people's heavy chain gene of the second heavy chain of the second epi-position of preparation coding binding purposes antigen, derive from cells first heavy chain of single complete people's light chain of people V κ 1-39 or people V κ 3-20 constant gene segment C and the second heavy chain to form bi-specific antibody expressing, and separation Bispecific antigen binding proteins.
In one embodiment, the first epi-position and the second epi-position are different.In one embodiment, the combination of the first variable region of heavy chain and the first epi-position can not hinder the combination of the second variable region of heavy chain and the second epi-position.In one embodiment, the first and second heavy chains can simultaneously in conjunction with the first and second epi-positions.
In one embodiment, bi-specific antibody is simultaneously in conjunction with the first and second epi-positions.In one embodiment, bi-specific antibody is independently in conjunction with the first and second epi-positions.
In one embodiment, the association reaction of bi-specific antibody to antigen about 2 times higher than the association reaction of the first variable region of heavy chain to antigen.In one embodiment, the association reaction of bi-specific antibody to antigen about 2 times higher than the association reaction of the second variable region of heavy chain to antigen.In one embodiment, bi-specific antibody is approximately identical or approximate greatly the first variable region of heavy chain and/or the second variable region of heavy chain to the association reaction of antigen to the association reaction of antigen.
In one embodiment, antigen is selected from soluble antigen, cell-surface antigens (such as tumour antigen) and cell surface receptor.In a particular embodiment, cell surface receptor is immunoglobulin receptor.In a particular embodiment, immunoglobulin receptor is Fc acceptor.
In one embodiment, the light variable domains of light chain comprises 2 to 5 individual cells sudden changes.In one embodiment, light variable domains is the same endogenous light chain through somatic mutation of expressing with the first or second weight chain variable binding domain in the B cell of the mouse through immunity.
In one embodiment, first complete people's heavy chain has and reduces its amino acid modified to the avidity of albumin A, and second complete people's heavy chain do not comprise reduction its to the modification of the avidity of albumin A.
In multiple embodiment, the method preparing bi-specific antibody is strengthened by adopting common light chain to come each variable region of heavy chain of timing bi-specific antibody.In multiple embodiment, adopt common light chain described herein, compared to the original same endogenous light chain of use, reduce the unsuitable number lacking the kind of the immunoglobulin (Ig) of dual specific.In multiple embodiment, identify the variable region of heavy chain of bi-specific antibody from the Mono-specific antibodies comprising common light chain.In multiple embodiment, the variable region of heavy chain of bi-specific antibody is included in the people's heavy chain variable gene section through resetting in the inherent mouse B cell of body, it resets in vivo in mouse B cell, described B cell before by through engineering approaches to express and limited people's light chain storehouse of people's heavy chain homology or single people's light chain, and as being exposed to the response of object antigen, produce containing multiple chimeric antibody storehouse with the people variable region of heavy chain of the possible people variable region of light chain homology of in one or two, wherein said chimeric antibody is specific to object antigen.
In many aspects, provide the method preparing bi-specific antibody, described bi-specific antibody comprises 1) the first polypeptide and the second polypeptide, wherein the first and second polypeptide comprise multimerization domain (such as immunoglobulin Fc domain) separately thus allow the first and second polypeptide to form dimer, and multimerization domain promotes interaction stable between the first and second polypeptide, and wherein one of multimerization domain has and reduces it to the amino acid modified of the avidity of albumin A and another multimerization domain lacks described modification, 2) binding domains in each of the first and second polypeptide, each binding domains comprises variable heavy chain and variable light, wherein the variable region chain of the first polypeptide and the variable light of the second polypeptide have common aminoacid sequence, described common sequence has the original light chain at least 80% being equal to every bar polypeptide, at least 85%, preferred at least 90%, more preferably at least 95% and the aminoacid sequence of most preferably 100% sequence iden.In multiple embodiment, variable light derives from people V κ 1-39 or people V κ 3-20 constant gene segment C.In multiple embodiment, variable light is the people's sequence of light chain reset.In multiple embodiment, variable light is separated from mouse described herein.
In multiple embodiment, method comprises step (i) and cultivates the host cell comprising the nucleic acid of coding first polypeptide, the second polypeptide and common light chain, and wherein said nucleic acid is expressed; (ii) bi-specific antibody is reclaimed from host cell cultures; In one embodiment, the nucleic acid of the nucleic acid of the first polypeptide of encoding or coding the second polypeptide, has and reduces its amino acid modified to the avidity of albumin A.In one embodiment, encode the first polypeptide, the second polypeptide and the nucleic acid of common light chain is present in single carrier or the carrier that is separated.In one embodiment, according to aforementioned paragraphs by host cell for the preparation of bi-specific antibody.
In one aspect, provide the method preparing bi-specific antibody, described method comprises (a) and selects to be separated the first nucleic acid from the first variable region of heavy chain of the coding of mouse described herein; B () is selected to be separated the second nucleic acid from the coding second people variable region of heavy chain of same or different mouse described herein; C () provides and is separated from mouse described herein or the 3rd nucleic acid of encoding human variable region of light chain of people variable region of light chain deriving from rearrangement described herein; C first, second, and third nucleic acid is introduced host cell by (), and cultivate host cell and occur to make the expression of first, second, and third nucleic acid; And (d) reclaims the bi-specific antibody formed from cell culture.
In one embodiment, the first and second people variable region of heavy chain are through somatic mutation.In a particular embodiment, the first and second people variable region of heavy chain derive from independently and are selected from 1-2,1-3,1-8,1-18,1 -24,1-46,1-58,1-69,2-5,2-26,2-70,3-7,3-9,3-11,3-13,3-15,3-16,3 -20,3-21,3-23,3-30,3-33,3-43,3-48,3-53,3-64,3-72,3-73,4-31,4-34,4-39,4-59,5-51 and 6-1 people V hthe people V of the rearrangement of constant gene segment C hconstant gene segment C.In one embodiment, the first and second people variable region of heavy chain derive from the people V of the rearrangement being selected from 2-5,3-30 and 3-23 independently hconstant gene segment C.In one embodiment, the first variable region of heavy chain derives from people V h2-5 constant gene segment C and the second people variable region of heavy chain derives from people V h3-30 constant gene segment C.In one embodiment, the first variable region of heavy chain derives from people V h3-30 constant gene segment C and the second people variable region of heavy chain derives from people V h3-23 constant gene segment C.In one embodiment, the first variable region of heavy chain derives from people V h3-23 constant gene segment C and the second people variable region of heavy chain derives from people V h3-30 constant gene segment C.
In one embodiment, the first or second nucleic acid was modified before step (c), and wherein the first or second nucleic acid is modified so that it has the avidity to albumin A of reduction.
In one embodiment, the 3rd separate nucleic acid is from mouse described herein.In one embodiment, the 3rd nucleic acid comprises 2 to 5 individual cells sudden changes.In one embodiment, the 3rd nucleic acid encoding derives from the people variable region of light chain of people V κ 1-39 constant gene segment C.In one embodiment, the 3rd nucleic acid encoding derives from the people variable region of light chain of people V κ 3-20 constant gene segment C.
In one embodiment, the 3rd nucleic acid source is in the people variable region of light chain of resetting.In one embodiment, the people variable region of light chain of rearrangement comprises the sequence deriving from people V κ 1-39 constant gene segment C or people V κ 3-20 constant gene segment C.In one embodiment, the people variable region of light chain of rearrangement comprises germline people V κ 1-39 sequence (that is, not comprising somatic hypermutation in V gene segment sequences).In one embodiment, the people variable region of light chain of rearrangement comprises germline people V κ 3-20 sequence
In multiple embodiment, provide the method that preparation comprises the bi-specific antibody of the first heavy chain of the variable domains comprising the modified mouse deriving from the people's sequence of light chain lacking rearrangement in its germline, wherein the first heavy chain matches with the homology people light chain of the people variable region of light chain comprising the rearrangement deriving from people V κ 1-39 or people V κ 3-20 constant gene segment C.In multiple embodiment, from the qualification of the mouse through immunity described herein, there is the specific second people heavy chain different from the first heavy chain.The nucleic acid of two heavy chains and common light chain of encoding is introduced in the host cell that aforementioned paragraphs describes, so that the expression of all three chains occurs and reclaim bi-specific antibody from cell culture.
In one embodiment, the same antigen immune mouse for generation of the first heavy-chain variable domains is used.In one embodiment, the different mice immunized with antigen for generation of the first heavy-chain variable domains are used.
On the one hand, provide and select match the method for the people's heavy chain to prepare bi-specific antibody with single people's light chain, comprise the nucleic acid of encoding bispecific antibody and contain the host cell of described nucleic acid.
In one aspect, provide the bi-specific antibody that increases expectation in the cell culture method relative to the quantity of unexpected product such as Mono-specific antibodies, modified to reduce the avidity of albumin A at that time for one in the heavy chain of wherein bi-specific antibody.
In one aspect, provide the host cell of separation, wherein said host cell comprises first nucleotide sequence of (a) coding in conjunction with the first variable region of heavy chain of the first antigen, wherein the first nucleotide sequence is separated the mouse of personal first antigen immune, and described mouse expresses the human normal immunoglobulin V from the light chain immunoglobulin sequence of the rearrangement in mouse germline ldistrict; B () coding is in conjunction with the second nucleotide sequence of the second people variable region of heavy chain of the second antigen, wherein the second nucleotide sequence is separated the mouse of personal second antigen immune, and described mouse expresses the human normal immunoglobulin V from the light chain immunoglobulin sequence of the rearrangement in mouse germline ldistrict; 3rd nucleotide sequence of (c) encoding human variable region of light chain, described people variable region of light chain is specific binding first antigen when the variable region of heavy chain with (a) is matched, and when the variable region of heavy chain with (b) is matched specific binding second antigen.
In many aspects, the first and second antigens are different each other.In many aspects, the expression of first, second, and third nucleotide sequence causes the formation of the bi-specific antibody of specific binding first and second antigen.
In one embodiment, people V ldistrict derives from people's sequence of light chain of the rearrangement comprising people V κ 1-39 constant gene segment C or people V κ 3-20 constant gene segment C.In a particular embodiment, people's sequence of light chain of rearrangement is Germline sequences (namely not comprising somatic hypermutation in variable domains).In one embodiment, the 3rd nucleotide sequence is separated the light chain immunoglobulin sequence table intelligent immunoglobulin (Ig) V since resetting lthe mouse in district, the people's sequence of light chain wherein reset is present in the germline of mouse.In one embodiment, the light chain immunoglobulin sequence of rearrangement comprises people V κ 1-39 constant gene segment C or people V κ 3-20 constant gene segment C.In a particular embodiment, people V κ 1-39 constant gene segment C or people V κ 3-20 constant gene segment C comprise at least one somatic hypermutation in complementary determining region (CDR) or framework region (FWR).In a particular embodiment, first, second, and third nucleotide sequence is separated from expressing human normal immunoglobulin V lthe mouse in district, described human normal immunoglobulin V ldistrict derives from the people V κ 1-39 constant gene segment C of light chain immunoglobulin sequence from resetting or people V κ 3-20 constant gene segment C, and the light chain immunoglobulin sequence of wherein resetting is present in the germline of mouse.
In multiple embodiment, mouse is containing resetting the endogenous light chain variable gene segment forming light chain immunoglobulin.
In one embodiment, human normal immunoglobulin V ldistrict expresses from the endogenous immunoglobulin light chain gene seat modified.In one embodiment, the first and second antigens are present on a molecule.In one embodiment, the first and second antigens are present on different molecules.In one embodiment, the first or second nucleotide sequence comprises the heavy chain of reduction coding to the modification of the avidity of albumin A.
In one embodiment, the first or second nucleotide sequence comprises people's weight chain variabl area sequence of rearrangement, and described variable region sequences comprises and is selected from V hl-2, V hl-3, V hl-8, V h1-18, V hl-24, V hl-46, V hl-58, V h1-69, V h2-5, V h2-26, V h2-70, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-21, V h3-23, V h3-30, V h3-33, V h3-43, V h3-48, V h3-53, V h3-64, V h3-72, V h3-73, V h4-31, V h4-34, V h4-39, V h4-59, V h5-51 and V hpeople's heavy chain gene section of 6-1.In a particular embodiment, heavy chain gene section is V h2-5, V h3-23 or V h3-30.
In one aspect, the antibody or the bi-specific antibody that contain people's heavy-chain variable domains prepared in accordance with the present invention is provided.In yet another aspect, the purposes that mouse described herein prepares fully human antibodies or complete people's bi-specific antibody is provided.
In one aspect, genetically modified mouse described herein, embryo or cell comprise the κ light chain gene seat retaining endogenous adjustment or controlling elements such as mouse κ intron enhanser, mouse κ 3 ' enhanser or intron enhanser and 3 ' enhanser, wherein regulate or controlling elements promotes somatic mutation and the affinity maturation of the expressed sequence of κ light chain gene seat.
In one aspect, provide the mouse comprising B cell group, the feature of described B cell group be to have the rearrangement deriving from no more than 1 or no more than 2 or without the light chain immunoglobulin of the light chain immunoglobulin V reset and J constant gene segment C, wherein said mouse demonstrates the κ roughly the same with the mouse of the wild-type complement of J constant gene segment C with comprising light chain immunoglobulin V: lambda light chain ratio.
In one embodiment, light chain immunoglobulin derives from light chain immunoglobulin V and the J constant gene segment C of the rearrangement of no more than 1 or no more than 2.In a particular embodiment, light chain derives from light chain immunoglobulin V and the J constant gene segment C of the rearrangement of no more than 1.
In one aspect, provide mouse described herein, it expresses the light chain immunoglobulin of the people V κ/J κ sequence deriving from no more than 1 or no more than 2, wherein said mouse comprises with one or more people's heavy chain variable region gene section to the displacement of all or substantially all endogenous mouse heavy variable gene segment, and (a) that described mouse demonstrates about 1 to about 20 expresses the CD19 with the immunoglobulin (Ig) of lambda light chain +b cell expresses the CD19 with the immunoglobulin (Ig) of κ light chain to (b) +the ratio of B cell.
In one embodiment, mouse expresses the single κ light chain deriving from people V κ 1-39J κ 5 sequence, and expresses the CD19 with the immunoglobulin (Ig) of lambda light chain +b cell has the CD19 of the immunoglobulin (Ig) of κ light chain to expressing +the ratio of B cell is about 1 to about 20; In one embodiment, ratio is about 1 at least about 66; In a particular embodiment, ratio is about 1 to 66.
In one embodiment, mouse expresses the single κ light chain deriving from people V κ 3-20J κ 5 sequence, and expresses the CD19 with the immunoglobulin (Ig) of lambda light chain +the CD19 with the immunoglobulin (Ig) of κ light chain is expressed during B cell +the ratio of B cell is about 1 to about 20; In one embodiment, ratio is about 1 to about 21.In a particular embodiment, ratio is 1 to 20 or 1 to 21.
In one aspect, provide the genetically modified mouse of expressing the single κ light chain through resetting, wherein said mouse comprises functional lambda light chain gene seat, and the expression of wherein said mouse comprises Ig κ +the B cell group of cell, described Ig κ +cell expressing derives from the κ light chain of the identical single κ light chain through resetting.In one embodiment, Ig κ in mouse +ig λ +identical approximately with wild-type mice of the per-cent of B cell.In a particular embodiment, Ig κ in mouse +ig λ +the per-cent of B cell is about 2% to about 6%.In a particular embodiment, the wherein single κ light chain through resetting derives from the Ig κ in the mouse of V κ 1-39J κ 5 sequence +ig λ +the per-cent of B cell is about 2 to about 3; In a particular embodiment, per-cent is about 2.6.In a particular embodiment, the wherein single κ light chain through resetting derives from the Ig κ in the mouse of V κ 3-20J κ 1 sequence +ig λ +the per-cent of B cell is about 4 to about 8; In a particular embodiment, per-cent is about 6.
In one aspect, provide the mouse of genetic modification, wherein said mouse expresses the single κ light chain through resetting deriving from people V κ and J kappa gene section, wherein said mouse expresses the B cell group comprising the single κ light chain deriving from the single κ sequence of light chain through resetting, and the mouse of wherein said genetic modification is not endowed the resistance to somatic hypermutation.In one embodiment, the κ light chain of the B cell of mouse is expressed at least 90% shows the somatic hypermutation from least 1 to about 5.
In one aspect, provide the mouse of genetic modification, it is modified to express the single κ light chain of the κ sequence of light chain of the rearrangement deriving from no more than 1 or no more than 2, and wherein said mouse shows and to use higher than the κ light chain shown by wild-type mice or to use about 2 times or more higher than the κ light chain that the mouse of same strain by the wild-type storehouse comprising κ light chain gene segments shows, at least about 3 times or more or use at least about the κ light chain of 4 times or more.In a particular embodiment, mouse expresses the single κ light chain from no more than 1 κ sequence of light chain reset.In embodiment more specifically, the κ sequence of light chain of rearrangement is selected from V κ 1-39J κ 5 and V κ 3-20J κ 1 sequence.In one embodiment, the κ sequence of light chain of rearrangement is V κ 1-39J κ 5 sequence.In one embodiment, the κ sequence of light chain of rearrangement is V κ 3-20J κ 1 sequence.
In one aspect, provide the mouse of genetic modification, it expresses the single κ light chain of the κ sequence of light chain of the rearrangement deriving from no more than 1 or no more than 2, wherein said mouse shows higher than identical about 100 times of κ light chain use shown by the mouse with complete or substantially complete human kappa light chain locus or more, at least about 200 times or more, at least about 300 times or more, at least about 400 times or more, at least about 500 times or more, at least about 600 times or more, at least about 700 times or more, at least about 800 times or more, at least about 900 times or more, use at least about the κ light chains of 1000 times or more.In a particular embodiment, the mouse with complete or substantially complete human kappa light chain locus lacks the functional mouse kappa light chain sequence without resetting.In a particular embodiment, mouse expresses the single κ light chain from no more than 1 κ sequence of light chain reset.In a particular embodiment, mouse comprises the κ sequence of light chain (such as heterozygote) of the rearrangement of a copy.In a particular embodiment, mouse comprises the κ sequence of light chain (such as homozygote) of the rearrangement of two copies.In embodiment more specifically, the κ sequence of light chain of rearrangement is selected from V κ 1-39J κ 5 and V κ 3-20J κ 1 sequence.In one embodiment, the κ sequence of light chain of rearrangement is V κ 1-39J κ 5 sequence.In one embodiment, the κ sequence of light chain of rearrangement is V κ 3-20J κ 1 sequence.
In one aspect, provide the mouse of genetic modification, it expresses the single light chain of the sequence of light chain of the rearrangement deriving from no more than 1 or no more than 2, light chain in the mouse of wherein genetic modification demonstrates expression at least 10 times higher than the light chain of the identical rearrangement shown by the mouse with complete or substantially complete light chain gene seat to about 1000 times, 100 times to about 1000 times, 200 times to about 1000 times, 300 times to about 1000 times, 400 times to about 1000 times, 500 times to about 1000 times, 600 times to about 1000 times, 700 times to about 1000 times, 800 times to about 1000 times, or the expression level of 900 times to about 1000 times.In one embodiment, light chain comprises human sequence.In a particular embodiment, human sequence is κ sequence.In one embodiment, human sequence is λ sequence.In one embodiment, light chain is complete people's light chain.
In one embodiment, expression level is characterized by the mRNA of sequence of light chain that quantitatively transcribes and it being compared with the sequence of light chain of transcribing of the mouse with complete or substantially complete light chain gene seat.
In one aspect, provide the mouse of genetic modification, it expresses the single κ light chain of the κ sequence of light chain of the rearrangement deriving from no more than 1 or no more than 2, and wherein said mouse shows after with antigen immune can serum titer compared with using the wild-type mice of same antigen immunity.In a particular embodiment, mouse expression is from the single κ light chain of the κ sequence of light chain of the rearrangement of no more than 1.In one embodiment, serum titer is characterized as being total immunoglobulin (Ig).In a particular embodiment, serum titer is characterized as being IgM than titre (specific titer).In a particular embodiment, serum titer is characterized as being IgG and compares titre.In embodiment more specifically, the κ sequence of light chain of rearrangement is selected from V κ 1-39J κ 5 and V κ 3-20J κ 1 sequence.In one embodiment, the κ sequence of light chain of rearrangement is V κ 1-39J κ 5 sequence.In one embodiment, the κ sequence of light chain of rearrangement is V κ 3-20J κ 1 sequence.
In one aspect, provide the cell of genetic modification, its antigen expressed specific antibody group, wherein all light chain immunoglobulins of antigen-specific antibodies group comprise and derive from identical single people V lpeople's light chain variable (V of constant gene segment C l) district, and heavy chain immunoglobulin comprises and derives from various human V ha kind of people's weight chain variable (V in constant gene segment C h) district.
In multiple embodiment, people V hconstant gene segment C is selected from V h1-2, V h1-3, V h1-8, V h1-18, V h1-24, V h1-46, V h1-58, V h1-69, V h2-5, V h2-26, V h2-70, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-21, V h3-23, V h3-30, V h3-33, V h3-43, V h3-48, V h3-53, V h3-64, V h3-72, V h3-73, V h4-31, V h4-34, V h4-39, V h4-59, V h5-51 and V h6-1.
In multiple embodiment, identical single people V lconstant gene segment C is selected from people V κ 1-39 constant gene segment C and people V κ 3-20 constant gene segment C.In multiple embodiment, all light chain immunoglobulins comprise the people's light chain J (J being selected from J κ and J λ constant gene segment C l) constant gene segment C.In a particular embodiment, people J lconstant gene segment C is selected from people J κ 1 and J κ 5 constant gene segment C.In multiple embodiment, mouse lacks and is selected from mouse immuning ball protein V lconstant gene segment C, mouse immuning ball protein J lthe sequence of constant gene segment C and combination thereof.In multiple embodiment, people V ldistrict is operably connected to people, mouse or rat immunoglobulin chain constant (C l) district.In a particular embodiment, people V ldistrict is operably connected to mouse C κ district.In a particular embodiment, people V ldistrict is operably connected to rat C κ district.
In multiple embodiment, people V ldistrict expresses from endogenous immunoglobulin light chain gene seat.In multiple embodiment, people V hdistrict is operably connected to the constant (C of people, mouse or rat immunoglobulin heavy chain h) district.In multiple embodiment, (C h) district comprises and be selected from C h1, hinge, C h2, C h3, C h4 and/or its combination human sequence.In multiple embodiment, people V hdistrict is expressed by endogenous immunoglobulin heavy chain gene seat.
In one aspect, provide the mouse of genetic modification, it expresses the heavy chain immunoglobulin that multiple and single light chain is combined.In one embodiment, heavy chain comprises human sequence.In multiple embodiment, human sequence is selected from variable sequence, C h1, hinge, C h2, C h3 and combination.In one embodiment, single light chain comprises human sequence.In multiple embodiment, human sequence is selected from variable sequence, constant series and combination thereof.In one embodiment, mouse comprises the endogenous immunoglobulin locus that loses function and expresses heavy chain and/or light chain from transgenosis or extrachromosomal episome.In one embodiment, mouse comprises with one or more human normal immunoglobulin sequence pair part or all of endogenous mouse heavy constant gene segment C (i.e. V, D, J) and/or part or all of endogenous mouse heavy constant series (such as, C on endogenous mouse locus h1, hinge, C h2, C h3 or its combination) and/or part or all of endogenous mouse sequence of light chain (such as, V, J, constant region or its combination) displacement.
In one aspect, provide the mouse being suitable for preparing the antibody with identical light chain, all or substantially all antibody expressions that wherein said mouse produces have identical light chain.In one embodiment, light chain is expressed by endogenous light chain genes seat.
In one aspect, provide the method for the light chain of preparation people antibody, it comprises obtain sequence of light chain and sequence of heavy chain from mouse described herein, and by sequence of light chain and sequence of heavy chain for the preparation of people's antibody.In one embodiment, people's antibody is bi-specific antibody.
In one aspect, provide qualification can together with through engineering approaches light variable domains described herein the method for people's heavy-chain variable domains of binding purposes antigen, wherein said method comprises providing source from can the heavy-chain variable domains of first antibody of conjugated antigen, heavy-chain variable domains and germline light chain sequence are matched and transfectional cell again, to be expressed separately to form second antibody, second antibody is exposed to antigen, and measures the combination of second antibody and antigen.
In one embodiment, the light chain of first antibody comprises people V κ 1-39 sequence.In one embodiment, the light chain of first antibody comprises people V κ 3-20 sequence.In one embodiment, germline light chain sequence comprises people V κ 1-39 or V κ 3-20 sequence.In multiple embodiment, second antibody and antigen in combination with first antibody with determine the comparing of combination of antigen.
In some embodiments, provide the method preparing Bispecific antigen binding proteins, wherein said method comprises and is exposed to expressing the first mouse of single human normal immunoglobulin light chain the first object antigen comprising the first epi-position, second mouse of expressing single human normal immunoglobulin light chain is exposed to the second object antigen comprising the second epi-position, the first and second mouse are allowed to produce immunne response for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, expressing cells first heavy chain of single complete people's light chain and the second heavy chain to form Bispecific antigen binding proteins, with be separated Bispecific antigen binding proteins.
In some embodiments, provide the method preparing Bispecific antigen binding proteins, wherein said method comprises and is exposed to expressing the first mouse of single human normal immunoglobulin light chain the first object antigen comprising the first epi-position, second mouse of expressing single human normal immunoglobulin light chain is exposed to the second object antigen comprising the second epi-position, the first and second mouse are allowed to produce immunne response for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, expressing cells first heavy chain of single complete people's light chain and the second heavy chain to form Bispecific antigen binding proteins, with be separated Bispecific antigen binding proteins, and wherein said single complete people's light chain derives from people V κ 1-39 or the people V κ 3-20 constant gene segment C of rearrangement.
In some embodiments, provide the method preparing Bispecific antigen binding proteins, wherein said method comprises and is exposed to expressing the first mouse of single human normal immunoglobulin light chain the first object antigen comprising the first epi-position, second mouse of expressing single human normal immunoglobulin light chain is exposed to the second object antigen comprising the second epi-position, the first and second mouse are allowed to produce immunne response for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, expressing cells first heavy chain of single complete people's light chain and the second heavy chain to form Bispecific antigen binding proteins, with be separated Bispecific antigen binding proteins, and wherein (a) first and second antigen be different, or (b) first and second antigen be identical, and the first epi-position and the second epi-position are different.
In some embodiments, provide the method preparing Bispecific antigen binding proteins, wherein said method comprises and is exposed to expressing the first mouse of single human normal immunoglobulin light chain the first object antigen comprising the first epi-position, second mouse of expressing single human normal immunoglobulin light chain is exposed to the second object antigen comprising the second epi-position, the first and second mouse are allowed to produce immunne response for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, expressing cells first heavy chain of single complete people's light chain and the second heavy chain to form Bispecific antigen binding proteins, with be separated Bispecific antigen binding proteins, and wherein people's light chain specific binding first antigen when matching with the first heavy chain the first epi-position and when matching with the second heavy chain the second epi-position of specific binding second antigen.
In some embodiments, provide the method preparing Bispecific antigen binding proteins, wherein said method comprises and is exposed to expressing the first mouse of single human normal immunoglobulin light chain the first object antigen comprising the first epi-position, second mouse of expressing single human normal immunoglobulin light chain is exposed to the second object antigen comprising the second epi-position, the first and second mouse are allowed to produce immunne response for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, expressing cells first heavy chain of single complete people's light chain and the second heavy chain to form Bispecific antigen binding proteins, with be separated Bispecific antigen binding proteins, and wherein said first complete people's heavy chain gene has and reduces its amino acid modified to the avidity of albumin A, and second complete people's heavy chain do not comprise reduction its to the modification of the avidity of albumin A.In some embodiments, reduce to the modification of the avidity of albumin A be selected from 95R (EUR 435R), 96F (EUR 436F) and combination.
In some embodiments, provide the method preparing Bispecific antigen binding proteins, wherein said method comprises and is exposed to expressing the first mouse of single human normal immunoglobulin light chain the first object antigen comprising the first epi-position, second mouse of expressing single human normal immunoglobulin light chain is exposed to the second object antigen comprising the second epi-position, the first and second mouse are allowed to produce immunne response for object antigen separately, in the first mouse, qualification is in conjunction with the first variable region of heavy chain of the first epi-position of the first object antigen, in the second mouse, qualification is in conjunction with the second people variable region of heavy chain of the second epi-position of the second object antigen, preparation coding is in conjunction with first complete people's heavy chain gene of the first heavy chain of the first epi-position of the first object antigen, preparation coding is in conjunction with second complete people's heavy chain gene of the second heavy chain of the second epi-position of the second object antigen, expressing cells first heavy chain of single complete people's light chain and the second heavy chain to form Bispecific antigen binding proteins, with be separated Bispecific antigen binding proteins, and wherein people's light chain of cell comprises Germline sequences.
In some embodiments, provide the method preparing people's bi-specific antibody, it comprises the step two people's weight chain variabl area sequences of two kinds of the mouse of expressing single people's light variable domains different B cell being used for bi-specific antibody.
In some embodiments, provide the method preparing people's bi-specific antibody, it comprises the step two people's weight chain variabl area sequences of two kinds of the mouse of expressing single people's light variable domains different B cell being used for bi-specific antibody, and wherein said single people's light variable domains comprises people V κ 1-39 or the people V κ 3-20 constant gene segment C of rearrangement.
In some embodiments, provide the method preparing people's bi-specific antibody, it comprises the step two people's weight chain variabl area sequences of two kinds of the mouse of expressing single people's light variable domains different B cell being used for bi-specific antibody, and wherein single people's light variable domains also comprises people J κ 5 or people J κ 1 constant gene segment C of rearrangement.
In some embodiments, provide the method preparing people's bi-specific antibody, it comprises the step two people's weight chain variabl area sequences of two kinds of the mouse of expressing single people's light variable domains different B cell being used for bi-specific antibody, and wherein said mouse also comprises the one or more people V without resetting containing may be operably coupled to one or more non-human heavy chain's constant region gene hconstant gene segment C, one or more people D without resetting hconstant gene segment C and one or more people J without resetting hhumanized heavy chain gene's seat of constant gene segment C.In some embodiments, humanized heavy chain gene's seat comprises 80 the people V without rearrangement that may be operably coupled to one or more murine heavy chain constant region gene hconstant gene segment C, 27 people D without rearrangement hconstant gene segment C and 6 people J without rearrangement hconstant gene segment C.In some embodiments, method comprises wherein one or more people V hconstant gene segment C comprises people V h1-2, V h1-8, V h1-24, V h1-69, V h2-5, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-23, V h3-30, V h3-33, V h3-48, V h3-53, V h4-31, V h4-39, V h4-59, V h5-51, V h6-1 or its combination.
In some embodiments, provide the method preparing people's bi-specific antibody, it comprises the step two people's weight chain variabl area sequences of two kinds of the mouse of expressing single people's light variable domains different B cell being used for bi-specific antibody, and wherein said mouse does not conform to the endogenous light chain variable gene segment having and can reset to be formed light chain immunoglobulin.
In some embodiments, provide the method selected for two people's immunoglobulin heavy chain variable structural domains of bi-specific antibody, it comprises uses object mice immunized with antigen, wherein said mouse expresses single people's light variable domains, and wherein said two people's immunoglobulin heavy chain variable structural domains are combined with single people's light variable domains with binding purposes antigen independently.
In some embodiments, provide the method preparing people's bi-specific antibody, it comprises the step two people's weight chain variabl area sequences of two kinds of the mouse of expressing single people's light variable domains different B cell being used for bi-specific antibody, and wherein said single people's light variable domains derives from the people V κ 1-39 constant gene segment C of rearrangement.In some embodiments, single people's light variable domains also comprises people J κ 5 constant gene segment C of rearrangement.In some embodiments, single people's light variable domains derives from the people V κ 3-20 constant gene segment C of rearrangement.In some embodiments, single people's light variable domains also comprises people J κ 1 constant gene segment C of rearrangement.
In some embodiments, provide the method selected for two people's immunoglobulin heavy chain variable structural domains of bi-specific antibody, it comprises uses object mice immunized with antigen, wherein said mouse expresses single people's light variable domains, and wherein said two people's immunoglobulin heavy chain variable structural domains are combined with binding purposes antigen with single people's light variable domains independently, wherein said mouse is containing resetting the endogenous light chain variable gene segment forming light chain immunoglobulin.
Any embodiment described herein and aspect, except as otherwise noted or apparent from context, all can be combined with each other.Other embodiment is obvious to those skilled in the art from hereafter describe.
Accompanying drawing
Fig. 1 demonstrates the target strategy for end user V κ 1-39J κ 5 gene regions displacement endogenous mouse immunoglobulin light chain variable region constant gene segment C.
Fig. 2 demonstrates the target strategy for end user V κ 3-20J κ 1 gene regions displacement endogenous mouse immunoglobulin light chain variable region constant gene segment C.
Fig. 3 demonstrates the target strategy for end user VpreB/J λ 5 gene regions displacement endogenous mouse immunoglobulin light chain variable region constant gene segment C.
The V κ 1-39J κ 5 light chain district that Fig. 4 shows wild-type mice (WT), reset for the people of through engineering approaches is the mouse (V κ 1-39J κ 5HO) of isozygotying and is the CD19 from peripheral blood of the mouse (V κ 3-20J κ 1HO) of isozygotying for the V κ 3-20J κ 1 light chain district that the people of through engineering approaches resets +the per-cent of B cell (y-axis).
Fig. 5 A shows and is being the mouse (H κ), the wild-type mice (WT) that isozygoty for the displacement utilizing people V κ and J kappa gene section to endogenous V κ and J kappa gene section and is being in the mouse (V κ 1-39J κ 5HET) of heterozygosis for the V κ 1-39J κ 5 light chain district that through engineering approaches people resets, the relative mrna expression (y-axis) of the light chain in the V κ 1-39 source that the junction surface (V κ 1-39J κ 5 junction surface probe) in the V κ 1-39J κ 5 light chain district using the people being specific to through engineering approaches to reset and the probe of people V κ 1-39 constant gene segment C (V κ 1-39 probe) measure in quantitative PCR detection.Stdn is carried out in the expression of signal pin to mouse C κ.N.D.: do not detect.
Fig. 5 B shows and is being the mouse (H κ), the wild-type mice (WT) that isozygoty for the displacement utilizing people V κ and J kappa gene section to endogenous V κ and J kappa gene section and is being in the mouse (V κ 1-39J κ 5HO) of isozygotying for the V κ 1-39J κ 5 light chain district that through engineering approaches people resets, the relative mrna expression (y-axis) of the light chain in the V κ 1-39 source that the junction surface (V κ 1-39J κ 5 junction surface probe) in the V κ 1-39J κ 5 light chain district using the people being specific to through engineering approaches to reset and the probe of people V κ 1-39 constant gene segment C (V κ 1-39 probe) measure in quantitative PCR detection.Stdn is carried out in the expression of signal pin to mouse C κ.
Fig. 5 C shows for utilizing people V κ and the displacement of J kappa gene section to endogenous V κ and J kappa gene section to be the mouse (H κ) of isozygotying, wild-type mice (WT) and be in heterozygosis (HET) and (HO) mouse of isozygotying for the V κ 3-20J κ 1 light chain district that through engineering approaches people resets, the relative mrna expression (y-axis) of the light chain using the junction surface (V κ 3-20J κ 1 junction surface probe) being specific to the V κ 3-20J κ 1 light chain district that through engineering approaches people resets to originate with the V κ 3-20 that the probe of people V κ 3-20 constant gene segment C (V κ 3-20 probe) measures in quantitative PCR detection, .Stdn is carried out in the expression of signal pin to mouse C κ.
Fig. 6 A shows at the wild-type (WT with beta-galactosidase enzymes immunity; And be (the V κ 1-39J κ 5HO isozygotied for the V κ 1-39J κ 5 light chain district that the people of through engineering approaches resets N=2); N=2) titre of IgM (left side) and IgG (right side) in mouse.
Fig. 6 B shows at the wild-type (WT with beta-galactosidase enzymes immunity; And be (the V κ 3-20J κ 1HO isozygotied for the V κ 3-20J κ 1 light chain district that the people of through engineering approaches resets N=5); N=5) titre of total immunoglobulin (Ig) (IgM, IgG, IgA) in mouse.
The diagram of bi-specific antibody (dual specific) that Fig. 7 A shows Mono-specific antibodies (parent 1 and parent 2) and builds from the variable region of heavy chain of each parent's Mono-specific antibodies.Common variable region of light chain (black) is shown in bi-specific antibody.
Fig. 7 B shows two parental monoclonal antibody (parent 1 and parent 2) to the binding characteristic of object antigen and the diagram of the binding characteristic of bi-specific antibody that built by the variable region of heavy chain of matching each monospecific parental antibody with common light chain.Illustrate the ability of bi-specific antibody (lower-left) or side by side two different epi-positions of (bottom right) binding purposes antigen respectively.
Fig. 8 shows 300nM dual specific (black post) and monospecific (striped and grey post) antibody to the histogram of the combination on the monomeric antigen E surface through catching, and uses BIACORE tMunit (RU).Show mono-clonal parent 1 antibody (P1Ab), mono-clonal parent 2 antibody (P2Ab) and bi-specific antibody (BsAb).
Detailed Description Of The Invention
These methods and condition the invention is not restricted to the experiment condition of specific method and description, because can change.It is also understood that term used herein only for describing the object of particular, and be not intended to limit, because scope of the present invention is defined by the claims.
Unless otherwise defined, all terms used herein and phrase comprise the implication of term and the phrase obtained in the art, except where explicitly so indicated or have the contrary instruction clearly shown in context that term or phrase use.Although any similar or be equal to the method for method described herein and material and material can use in practice of the present invention with in detecting, specific method and material now be described.All publications mentioned all are incorporated herein by reference in their entirety.
Term used herein " antibody " comprises the immunoglobulin molecules containing 4 polypeptide chains (by interconnective two weight (H) chains of disulfide linkage and two light (L) chains).Every bar heavy chain comprises weight chain variable (V h) district and light chain constant (C h) district.CH comprises 3 structural domains, C h1, C h2 and C h3.Every bar light chain comprises light chain variable (V l) district and constant region of light chain (C l).V hdistrict and V ldistrict can be further subdivided into the hypervariable region being named as complementary determining region (CDR), is wherein studded with the region of more conservative called after framework region (FR).Each V hand V lcomprise 3 CDR and 4 FR, it arranges in the following order from N-terminal to C-terminal: (heavy chain CDR can referred to as HCDR1, HCDR2 and HCDR3 for FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4; Light chain CDR can referred to as LCDR1, LCDR2 and LCDR3).Term " high-affinity " antibody refers to have relative to its target epi-position about 10 -9m or lower (such as, about 1 × 10 -9m, 1 × 10 -10m, 1 × 10 -11m or about 1 × 10 -12m) K dantibody.In one embodiment, by surface plasma body resonant vibration such as BIACORE tMmeasure K d; In another embodiment, K is measured by ELISA d.
Phrase " bi-specific antibody " comprises can the antibody of two or more epi-positions of selective binding.Bi-specific antibody comprises the fragment of two different monoclonal antibodies (Fig. 7 A) and generally includes from two of two different monoclonal antibodies not identical heavy chains, the epi-position that wherein every bar heavy chain specific binding is different---(the different epi-positions such as in two different immunogens on two different molecules; See Fig. 7 B lower-left) or on same molecule (the different epi-positions such as in same immunogen; See Fig. 7 B bottom right).If bi-specific antibody can the different epi-position (the first epi-position and the second epi-position) of selective binding two, so usual first heavy chain to the avidity of the first epi-position by lower than the first heavy chain to the avidity at least 1 of the second epi-position to 2 or 3 or 4 an or more order of magnitude, vice versa.The epi-position of bi-specific antibody specific binding can on same or different targets (such as on same or different albumen; See Fig. 7 B).Exemplary bi-specific antibody comprises and having the first special heavy chain of tumour antigen and the antibody to special the second heavy chain of cytotoxic markers such as Fc acceptor (such as Fc γ RI, Fc γ RII, Fc γ RIII etc.) or T cell marker (such as CD3, CD28 etc.).In addition, the second variable region of heavy chain can be had the specific variable region of heavy chain of different expectations and replaced.Such as, have and can be paired the bi-specific antibody of the first special heavy chain of tumour antigen and special the second heavy chain of contratoxin thus send toxin (such as saponin, vinca alkaloids etc.) to tumour cell.Other exemplary bi-specific antibody comprises and having activated receptor the first special heavy chain of (such as B-cell receptor, Fc γ RI, Fc γ RIIA, Fc γ RIIIA, Fc α RI, φt cell receptor etc.) and the antibody to special the second heavy chain of Inhibitory receptor (such as Fc γ RIIB, CD5, CD22, CD72, CD300a etc.).Such bi-specific antibody can be fabricated for the therapeutic patient's condition (such as irritated and asthma) relevant to cell activation.Bi-specific antibody can identify prepared by the heavy chain of same or different immunogenic difference table (Fig. 7 B) position by such as combination.Such as, the nucleotide sequence of the variable heavy chain sequence of the same or different immunogenic different epi-position of code identification can merge to nucleotide sequence that is same or different heavy chains constant region of encoding, and these sequences can at the cells of expressing light chain immunoglobulin.Typical bi-specific antibody has two heavy chains, and every bar heavy chain has 3 heavy chain CDR, is (N-terminal is to C-terminal) C subsequently h1 structural domain, hinge, C h2 structural domains and C h3 structural domains, and light chain immunoglobulin, described light chain is not given epitope binding specificity but can be combined with every bar heavy chain, or it can be combined with every bar heavy chain and can in conjunction with one or more epi-position combined by heavy chain epitope binding region, or it can be combined with every bar heavy chain and makes it possible to or make one of heavy chain or two can be bonded to one or two epi-position.
Term " cell " comprises any cell being suitable for expressing recombinant nucleic acid sequence.Cell comprise the cell of prokaryotic organism and eukaryote (unicellular or many cells), bacterial cell (such as the bacterial strain of intestinal bacteria (E.coli), genus bacillus (Bacillus spp.), streptomycete (StreptomycES spp.) etc.), mycobacterial cells, fungal cell, yeast cell (such as yeast saccharomyces cerevisiae (S.cerevisiae), wine fission yeast(S.pombe), pichia pastoris phaff (P.pastoris), pichia methanolica (P.methanolica) etc.), vegetable cell, insect cell (insect cell, cabbage looper (Trichoplusia ni) etc. of such as SF-9, SF-21, baculovirus infection), non-human animal cell, people's cell or cell fusion such as hybridoma or four source hybridomas.In some embodiments, cell is people, monkey, ape, hamster, rat or mouse cell.In some embodiments, cell is eucaryon and is selected from following cell: CHO (such as CHOKl, DXB-11 CHO, Veggie-CHO), COS (such as COS-7), retina cell, Vero, CV1, nephrocyte (such as HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (such as BHK21), Jurkat, Daudi, A431 (epidermis), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumour cell and the clone deriving from aforementioned cells.In some embodiments, cell comprises one or more virogene, such as, express retina cell (the such as PER.C6 of virogene tMcell).
Phrase " complementary determining region " or term " CDR " comprise by the aminoacid sequence of the nucleic acid sequence encoding of the immunoglobulin gene of organism, and its usual (namely in wild type animal) appears between two framework regions of the light chain of immunoglobulin molecules (such as antibody or φt cell receptor) or the variable region of heavy chain.CDR can be by, such as Germline sequences or rearrangement or without the sequence of resetting with such as encoded by initial or mature B cell or T cell.CDR by somatic mutation (such as different from the sequence of encoding in animal germline), humanization and/or can modify with amino-acid substitution, interpolation or delete.In some cases (such as CDR3), CDR can be encoded by two or more sequences (such as Germline sequences), described sequence is discontinuous (such as in the nucleotide sequence without rearrangement) but is continuous print in B cell nucleotide sequence, such as, produce because of montage or catenation sequence (such as V-D-J restructuring is to form heavy chain CDR3).
Term " conservative ", when amino-acid substitution for describing either conservative, comprise contain the side chain R group with similar chemical character (such as electric charge or hydrophobicity) with another amino-acid residue to the replacement of amino-acid residue.Usually, conservative aminoacid replacement can not the functional property of material alterations target protein, and such as variable region is with the ability of the avidity specific binding target epi-position expected.The example of amino acid whose group containing the side chain with similar chemical character comprises aliphatic lateral chain such as glycine, L-Ala, α-amino-isovaleric acid, leucine and Isoleucine; Aliphatic-hydroxy side chains such as Serine and Threonine; Amide containing side chain such as l-asparagine and glutamine; Beta-branched side is phenylalanine, tyrosine and tryptophane such as; Basic side chain is Methionin, arginine and Histidine such as; Acid side-chain such as aspartic acid and L-glutamic acid; And sulfur-containing side chain such as halfcystine and methionine(Met).Conservative aminoacid replacement group comprises, such as, and α-amino-isovaleric acid/leucine/Isoleucine, phenylalanine/tyrosine, Methionin/arginine, L-Ala/α-amino-isovaleric acid, L-glutamic acid/aspartic acid and l-asparagine/glutamine.In some embodiments, conservative aminoacid replacement can be any natural residue replaced with L-Ala in albumen, as used in such as alanine scanning mutagenesis.In some embodiments, in the conservative PAM250 of the being substituted in log-likelihood matrix carried out, there is positive, described matrix is disclosed in the people such as Gonnet (1992) Exhaustive Matching of the Entire Protein Sequence Database, in Science 256:1443-45, it is incorporated to herein by reference.In some embodiments, replacement is the conservative replacement of moderate, has the value of nonnegative number in the wherein said PAM250 of being substituted in log-likelihood matrix.
In some embodiments, the resi-dues in light chain immunoglobulin or heavy chain is different from one or more conservative aminoacid replacement.In some embodiments, resi-dues in light chain immunoglobulin or its functional fragment (such as allowing the fragment of expression and secretion from such as B cell) is not identical with the light chain that its aminoacid sequence is listed herein, but is different from one or more conservative aminoacid replacement.
Phrase " epi-position associated proteins " comprises the albumen with at least one CDR and it can Selective recognition epi-position, such as, micro-ly can to rub or lower K with about one d(such as K dabout 1 × 10 -6m, 1 × 10 -7m, 1 × 10 -8m, 1 × 10 -9m, 1 × 10 -10m, 1 × 10 -11m or about 1 × 10 -12m) in conjunction with epi-position.Therapeutic epi-position associated proteins (such as therapeutic antibodies) needs the K rubbed in scope at Na Mo or skin usually d.
Phrase " functional fragment " comprise can express, secrete and micro-ly to rub, the K that rubs in scope of Na Mo or skin dthe protein-bonded fragment of epi-position of specific binding epitope.Specific recognition comprises and having at least at micro-scope of rubbing, the K that the scope of rubbing or skin rub in scope that receives d.
Term " germline " comprises with reference to the immunoglobulin nucleic acid sequence in the cell (B cell of such as non-somatic mutation or precursor B cells or hematopoietic cell) of non-somatic mutation.
Phrase " heavy chain " or " heavy chain immunoglobulin " comprise the immunoglobulin heavy chain constant region sequence from any organism.Unless otherwise prescribed, heavy-chain variable domains comprises 3 heavy chain CDR and 4 FR district.The fragment of heavy chain comprises CDR, CDR and FR and combination thereof.Typical heavy chain has, after variable domains (from N-terminal to C-terminal): C h1 structural domain, hinge, C h2 structural domains and C h3 structural domains.The functional fragment of heavy chain comprise can specific recognition epi-position (such as micro-ly to rub, the K that rubs in scope of Na Mo or skin didentify epi-position), can secrete from cells and comprise the fragment of at least one CDR.
Term " identity ", when being combined with sequence, comprises the identity measured by the multiple different algorithm that can be used in measuring Nucleotide and/or amino acid sequence identity as known in the art.In embodiments more described herein, identity by using ClustalW v.1.83 (slowly) comparison of the open Gap Penalty of employing 10.0, the expansion Gap Penalty of 0.1, and uses Gonnet similarity matrix (MACVECTOR tM10.0.2, MacVector Inc., 2008) measure.The length of the sequence compared relative to the identity of sequence will depend on concrete sequence, but when light chain constant domain, length should comprise the sequence of the length being enough to be folded into light chain constant domain, it can from combining with the light chain constant domain forming standard, such as can form two β lamellas comprising β chain and can with at least one C of people or mouse h1 domain interaction.At C hwhen 1 structural domain, sequence length should be folded into C containing being enough to hthe sequence of the length of 1 structural domain, it can form two and comprises the β lamella of β chain and can interact with at least one light chain constant domain of mouse or people.
Phrase " immunoglobulin molecules " comprises two heavy chain immunoglobulins and two light chain immunoglobulins.Heavy chain can be identical or different, and light chain can be identical or different.
Phrase " light chain " comprises the light chain immunoglobulin sequence from any organism, and except as otherwise noted, comprises people κ and lambda light chain and VpreB, and alternative light chain.Except as otherwise noted, light chain variable (V l) structural domain generally includes 3 light chain CDR and 4 framework (FR) districts.Normally, full-length light chains comprises, and from N-terminal to C-terminal, comprises the V of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 lstructural domain and light chain constant domain.Light chain comprises the light chain of the first or second epi-position that such as non preference combination is combined by epi-position associated proteins (wherein occurring described light chain).Light chain also comprise combine and identify or assist heavy chain combine or identify one or more by the light chain of the epi-position of epi-position associated proteins (wherein occurring described light chain) selective binding.Common light chain is the light chain deriving from people V κ 1-39J κ 5 sequence of rearrangement or people V κ 3-20J κ 1 sequence of rearrangement, and comprises (the such as affinity maturation) form through somatic mutation.
Phrase " micro-scope of rubbing " refers to that 1-999 is micro-and rubs; Phrase " receive the scope of rubbing " refers to that 1-999 receives and rubs; Phrase " skin rub scope " refers to that 1-999 skin rubs.
Phrase " somatic mutation " comprises reference from the nucleotide sequence experiencing classification conversion B cell, the nucleotide sequence of the immune globulin variable region (such as heavy-chain variable domains or comprise heavy chain CDR or FR sequence) wherein in the B cell of classification conversion is different from the nucleotide sequence in the B cell before classification conversion, such as, the CDR between the B cell not experiencing classification conversion and the B cell having experienced classification conversion or the difference in framework nucleotide sequence." somatic mutation " comprises with reference to the nucleotide sequence from the B cell of affinity maturation, and it is different from the corresponding immunoglobulin variable domain sequence (sequence such as in germ line cell genome) in the B cell of non-affinity maturation.Phrase " somatic mutation " also comprises with reference to from immune globulin variable region nucleotide sequence B cell being exposed to the B cell after object epi-position, and wherein said nucleotide sequence is different from corresponding nucleotide sequence B cell be exposed to before object epi-position.Phrase " somatic mutation " refers to control oneself and to produce in response to immunogenic stimulation in the animal such as mouse with human normal immunoglobulin variable region nucleic acid sequence, and the sequence of the antibody produced because of the chosen process of operation intrinsic in such animal.
Term " without what reset " about nucleotide sequence comprises the nucleotide sequence be present in the germline of zooblast.
Phrase " variable domains " comprises light chain immunoglobulin or heavy chain (by what expect to modify) aminoacid sequence, it comprises following amino acid region, the order (except as otherwise noted) with from N-terminal to C-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
Universal light chain
Prepare the effort of useful Multispecific epitope binding proteins such as bi-specific antibody before this, hindered by various problem, these problems have common pattern usually: the external selection of sequence or operation are with reasonably through engineering approaches or to the appropriate form of dual specific human normal immunoglobulin being used for timing heterodimer by trial and error through engineering approaches.Unfortunately, the engineered in vitro method of great majority (if not all) provides the terms of settlement being suitable for (if any) individual molecular special in a large number.On the other hand, also unrealized being used for by complex biological body selects method in the suitable body that can cause the pairing of human therapy agent.
Usually, native mouse sequence is not often the high-quality source of human therapy sequence.At least for this reason, produce the murine heavy chain immune globulin variable region of matching with common people's light chain and there is limited practical value.In trial and error process, more engineered in vitro work can expend and expect that reservation table position specificity and avidity maintain the ability with common people's light chain coupling attempting humanization murine heavy chain variable sequence simultaneously simultaneously, and this is with uncertain result.Last in such process, final product can maintain some specificitys and avidity and combine with common light chain, but its immunogenicity finally in people may still have very dark risk.
Therefore, people's heavy chain variable region gene section storehouse of suitably a large amount of replacement endogenous mouse heavy variable gene segment should be comprised for the preparation of the suitable mouse of human therapy agent.People's heavy chain variable region gene section should be able to be reset and recombinate to form reverse imbedding heavy chain (namely heavy chain comprises people's variable domains and murine constant region) with endogenous mouse heavy constant domain.Heavy chain should be able to type conversion and somatic hypermutation, so that suitably a large amount of heavy-chain variable domains storehouses can be used for mouse to select a heavy-chain variable domains that can be combined with limited storehouse, people variable region of light chain.
The mouse of the common light chain being used for multiple heavy chain is selected to have practical value.In multiple embodiment, can combine having with identical or substantially the same light chain and the heavy chain of expressing the antibody of expressing in the mouse of common light chain only can be expressed.This is useful especially preparing in bi-specific antibody.Such as, such mouse can by the first immunogen immune to produce the B cell of expression specificity in conjunction with the antibody of the first epi-position.Mouse (or the upper identical mouse of heredity) can by the second immunogen immune to produce the B cell of expression specificity in conjunction with the antibody of the second epi-position.Weight variable sequence can be cloned from B cell, and with same CH and same light chain expression, and at cells to prepare bi-specific antibody, wherein the light chain component of bi-specific antibody is selected to be combined with light chain component and to express by mouse.
The present inventor by mouse through engineering approaches to produce light chain immunoglobulin, described light chain will suitably with quite diversified heavy chain families timing, comprise its variable region and Germline sequences deviates from the heavy chain of (such as affinity maturation or the variable region through somatic mutation).In numerous embodiments, design mouse people's light variable domains and the people's heavy-chain variable domains comprising somatic mutation to be matched, thus makes it possible to the protein-bonded approach of high-affinity that obtains being suitable as human therapy agent.
By the very long in vivo and antibody chosen process of complexity, genetically engineered mouse produces the biologically appropriate selection in being selected in diversified people's heavy-chain variable domains storehouse and a limited number of people's light chain to match.In order to realize this, mouse is selected a limited number of people light variable domains that combine to select to present with extensively various people's heavy-chain variable domains by through engineering approaches.After stimulating by immunogen, the number that mouse maximizes solution in its storehouse is to produce for immunogenic antibody, and this mainly or is only subject to the restriction that number in its storehouse or light chain are selected.In numerous embodiments, this comprises and allows mouse to obtain the suitable and compatible somatic mutation of light variable domains, and however it is by compatible for the people's heavy-chain variable domains comprised especially through somatic mutation with relative a large amount of people's heavy-chain variable domains.
For obtaining the limited storehouse that light chain is selected, mouse by through engineering approaches to make its preparation or to reset the ability nonfunctional of native mouse light variable domains or essentially no function.This can such as be realized by the chain variable region gene section deleting mouse.Subsequently can by the external source of the selection that may be operably coupled to endogenous mouse constant region of light chain suitable people's chain variable region gene section, by this way so that external source people variable gene segment can combine with endogenous mouse light chain constant region gene and form the reverse imbedding light chain gene (people variable region, murine constant region) reset modify endogenous mouse locus.In numerous embodiments, somatic mutation can be carried out in variable region of light chain.In numerous embodiments, obtaining the ability of somatic mutation in order to maximize variable region of light chain, in mouse, retaining suitable enhanser.Such as, at modification mouse kappa light chain locus with in employment κ light chain gene segments displacement endogenous mouse κ light chain gene segments, mouse κ intron enhanser and mouse κ 3 ' enhanser obtain functional maintenance or not by broken ring.
The mouse of the genetic modification provided, it expresses limited reverse imbedding (people variable region, murine constant region) the light chain storehouse combined with diversified reverse imbedding (people variable region, murine constant region) heavy chain.In numerous embodiments, endogenous mouse κ light chain gene segments deleted and that be operably connected to endogenous mouse C kappa gene single (or two) reset people's light chain district displacement.In the embodiment of somatic hypermutation maximizing people's light chain district of resetting, retain mouse κ intron enhanser and mouse κ 3 ' enhanser.In numerous embodiments, mouse also contains lambda light chain gene seat or its deletion of non-functional or causes locus can not prepare the deletion of lambda light chain.
In numerous embodiments, provide the mouse of genetic modification, it comprises and lacks endogenous mouse light chain V land J lconstant gene segment C also comprises people variable region of light chain (in one embodiment, the people V of rearrangement of the rearrangement being operably connected to murine constant region l/ J lsequence) chain variable region gene seat, wherein said locus can carry out somatic hypermutation, and wherein said locus express comprise the people V being connected to murine constant region l/ J lthe light chain of sequence.Therefore, in numerous embodiments, locus comprises the mouse κ 3 ' enhanser relevant to there being the somatic hypermutation of normal or wild-type levels.
In numerous embodiments, when with object antigen immune, genetically engineered mouse produces B cell, described B cell demonstrates with one or two light chain expression reset and the human immunoglobulin heavy chain variable region the playing function diversity of resetting, and comprises the embodiment that wherein one or two light chain comprises the people variable region of light chain containing such as 1 to 5 individual cells sudden change.In numerous embodiments, anyone immunoglobulin heavy chain variable region that people's light chain of so expressing can be expressed in mouse is combined and expresses.
In conjunction with the epi-position associated proteins of more than one epi-position
Composition described herein and method can be used to preparation with the associated proteins of high-affinity in conjunction with more than one epi-position, such as bi-specific antibody.Favourable aspect of the present invention comprises the ability selecting suitable high associativity (such as affinity maturation) the heavy chain immunoglobulin chain be combined with single light chain separately.
Report several technology preparing bispecific antibody fragment from the cell culture of restructuring.But the synthesis of bispecific binding protein and express existing problems, is partly due to and differentiates suitable can combination with two different heavy chains and the relevant problem of the light chain of expressing, being partly due to the problem of separation.In numerous embodiments, composition described herein and method provide does not need the specific favourable aspect modified to be maintained the total length bi-specific antibody (Fig. 7 A) of conventional immunoglobulin structure by the stability/interaction of increase component.In numerous embodiments, such modification be proved to be loaded down with trivial details and development bi-specific antibody technology and be used for the treatment of human diseases potential use in serve as obstruction.Therefore, in numerous embodiments, by providing the native immunoglobulin structure (i.e. total length) of the polyspecific character with interpolation, the effector function of key that bi-specific antibody before total length bi-specific antibody maintains lacks, and the therapeutical agent of important pharmacokinetic parameter providing the display longer transformation period.
Composition described herein and method allow the mouse of genetic modification by suitable can the combining and the light chain of expressing with more than one heavy chain (comprising (the such as affinity maturation) heavy chain through somatic mutation) of strategy and suggestion natural in other respects.The people V of the suitable B cell from expression with the mouse through immunity described herein of the antibody of reverse imbedding heavy chain (i.e. people variable region and murine constant region) affinity maturation can be identified hand V lsequence is also cloned in expression vector in frame with suitable human constant region gene order (such as human IgG1).Can prepare two such constructs, wherein each construct coding is in conjunction with people's heavy-chain variable domains of different epi-position.Can by Germline sequences or from wherein sequence by the people V of the B cell of somatic mutation lone (such as people V κ 1-39J κ 5 or people V κ 3-20J κ 1), merge in frame to suitable human constant region gene (such as the constant gene of people κ).These 3 complete people's heavy chains and light chain construct can be placed in the suitable cell for expressing.Cell is by kind main for expression two: the homodimer heavy chain with same light chain, and the heterodimer heavy chain with same light chain.In order to allow easily to be separated these main kinds, being modified to delete albumin A in conjunction with determinant, being caused the avidity that homologous dimerization binding protein precursor is different from heterodimeric binding protein precursor for one of heavy chain.The composition addressed this problem and method are described in June 25 2010 applying date, name is called " Readily Isolated Bispecific AntibodiES with Native Immunoglobulin Format ", as USSN 12/832 disclosed in US 2010/0331527A1, in 838, it is incorporated to by reference herein.
In one aspect, epi-position associated proteins described herein is provided, wherein people V hand V lsequence derives from the mouse described herein of the antigen immune of involved object epi-position.
In one embodiment, provide epi-position associated proteins, it comprises the first and second polypeptide, the first polypeptide comprise from N-terminal to C-terminal selective binding first epi-position the first epitope binding region, be the C comprising the human IgG being selected from IgG1, IgG2, IgG4 and combination thereof subsequently hthe constant region in 3rd district; And the second polypeptide comprise from N-terminal to C-terminal selective binding second epi-position the second epitope binding region, be comprise the 2nd C being selected from IgG1, IgG2, IgG4 and the thin human IgG closed thereof subsequently hthe constant region in 3rd district, wherein the 2nd C h3rd district comprise reduction or eliminate described 2nd C hthe modification of the combination of 3rd district and albumin A.
In one embodiment, the 2nd C h3rd district comprise H95R modification and (number according to IMGT exon; H435R is numbered) according to EU.In another embodiment, the 2nd C h3rd district also comprise Y96F and modify (IMGT; Be Y436F according to EU).
In one embodiment, the 2nd C h3rd district from the human IgG1 modified, and comprise and are selected from D16E, L18M, N44S, K52N, V57M and V82I (IMGT; Be D356E, L358M, N384S, K392N, V397M and V422I according to EU) modification.
In one embodiment, the 2nd C h3rd district from the human IgG2 modified, and comprise and are selected from N44S, K52N and V82I (IMGT; Be N384S, K392N and V422I according to EU) modification.
In one embodiment, the 2nd C h3rd district from the human IgG 4 modified, and comprise and are selected from 015R, N44S, K52N, V57M, R69K, E79Q and V82I (IMGT; Be Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) modification.
Preparing in conjunction with the protein-bonded method of epi-position of more than one epi-position is use the antigen immune comprising the first object epi-position according to the first mouse of the present invention, and wherein said mouse comprises not containing can resetting and forming the endogenous mouse V of light chain lendogenous immunoglobulin chain variable region gene seat, is the single people V through resetting that may be operably coupled to endogenous mouse light chain constant region gene wherein on the locus of endogenous mouse immunoglobulin light chain variable region ldistrict, and the people V reset ldistrict is selected from people V κ 1-39J κ 5 and people V κ 3-20J κ 1, and endogenous mouse V hconstant gene segment C is by people V hconstant gene segment C is replaced whole or in part, so that heavy chain immunoglobulin prepared by mouse is completely or the substantial heavy chain comprising people's variable domains and mouse constant structural domain.When by immunity, such mouse will produce the reverse imbedding antibody of (in such as people V κ 1-39J κ 5 or people V κ 3-20J κ 1) only comprised in two kinds of people's light variable domains.Identifying the V of coding binding purposes epi-position hb cell after, recyclable (such as passing through PCR) V h(alternatively and V l) nucleotide sequence and suitable human normal immunoglobulin constant domain in frame, be cloned into expression construct.This process can by the 2nd V repeating to identify in conjunction with the second epi-position hstructural domain, and recyclable 2nd V hgene order is also cloned into expression vector with the second suitable immunoglobulin constant domains in frame.First and second immunoglobulin constant domains can be identical or different isotypes, and can as ground one (but not another) of modified immunoglobulin constant domain herein or described in US 2010/0331527A1, and epi-position associated proteins can compare the different avidity of albumin A based on itself and homodimer epi-position associated proteins be separated at suitable cells, such as, described in US 2010/0331527A1.
In one embodiment, provide and prepare the protein-bonded method of dual specific epi-position, it comprises (such as comprising one or more somatic hypermutations) the people V identifying the first affinity maturation from mouse described herein hnucleotide sequence (V h1) from mouse described herein, identify (such as comprising one or more somatic hypermutations) people V of the second affinity maturation hnucleotide sequence (V h2), by V h1 be described in people's heavy chain that the shortage albumin A determinant in US 2010/0331527A1 modifies and clone to form heavy chain 1 (HC1), by V in frame h2 clone to form heavy chain 2 (HC2) with the people's heavy chain comprising albumin A determinant be described in US 2010/0331527A1 in frame, the expression vector and the same or different expression vectors comprising HC2 that comprise HC1 are introduced cell, wherein said cell is also expressed to comprise and is merged to the people V κ 1-39/ people J κ 5 of people's light chain constant domain or the human normal immunoglobulin light chain of people V κ 3-20/ people J κ 1, allows cell expressing to comprise by V hthe V of 1 coding hstructural domain and by V hthe V of 2 codings hthe dual specific epi-position associated proteins of structural domain, is separated dual specific epi-position associated proteins with the ability based on its associated proteins A different compared with monospecific homodimer epi-position associated proteins.In a particular embodiment, HC1 is IgG1, and HC2 comprises H95R (IMGT; Be H435R according to EU) modify and also comprise Y96F (IMGT; Be Y436F according to EU) IgG1 that modifies.In one embodiment, by V hthe V of 1 coding hstructural domain, by V hthe V of 2 codings hstructural domain or its both through somatic mutation.
With common people V lthe people V expressed hgene
From for four kinds of not various human variable regions of antibody of affinity maturation of producing of synantigen, with its with endogenous light chain or be selected from people V κ 1-39/J κ 5, people V κ 3-20/J κ 1 or people VpreB/J λ 5 people's light chain at least one together with express (see embodiment 1).For the antibody of each antigen, the high-affinity heavy chain through somatic mutation from different genes family successfully matches with the people germline V κ 1-39J κ 5 reset and V κ 3-20J κ 1 district and secretes from the cell of expressing heavy chain and light chain.For V κ 1-39J κ 5 and V κ 3-20J κ 1, derive from following people V hthe V of gene family hstructural domain preferred expression: 1-2,1-8,1-24,2-5,3-7,3-9,3-11,3-13,3-15,3-20,3-23,3-30,3-33,3-48,4-31,4-39,4-59,5-51 and 6-1.Therefore, by through engineering approaches to express limited one or two the people V from V κ 1-39J κ 5 and V κ 3-20J κ 1 lthe mouse in structural domain storehouse will from modified with employment V hconstant gene segment C displacement mouse V hthe V of constant gene segment C hlocus produces the diversified people V through somatic mutation hstructural domain group.
When with object antigen immune, through genetically engineered with the mouse of expressing reverse imbedding (people variable region, the murine constant region) heavy chain immunoglobulin combined with the single light chain (such as V κ 1-39/J or V κ 3-20/J) through resetting, its B cell produced comprises diversified people V hreset and express diversified high-affinity antigen specific antibodies, described antibody has and blocks antigen and the ability of combination of their part and the diversified character (see embodiment 5-10) relative to the ability of its conjugated antigen variant relative to it.
Therefore, mouse described herein and method can be used for preparation and select human immunoglobulin heavy chain variable domains, comprise produce because of diversified rearrangement, the extensively various avidity of display (comprises and demonstrates Yue Namo or less K d), extensively various specificity (comprising the different epi-positions being bonded to same antigen) and combining and the people's heavy-chain variable domains through somatic mutation of expressing with identical or substantially the same human normal immunoglobulin variable region of light chain.
There is complete people's bi-specific antibody of common light chain
As the first step in different embodiment, the first and second nucleotide sequences of respective encoding human heavy-chain variable domains (with other the nucleotide sequence any forming bi-specific antibody) from the characteristic with expectation such as can in conjunction with different epi-position (see Fig. 7 A and 7B), there are different avidity etc. parental monoclonal antibody choose.Usually, from the nucleotide sequence being separated encoding human heavy-chain variable domains through the mouse of immunity described herein to allow with people's heavy chain constant domain to be suitable for human administration.By introducing sudden change, sequence is carried out to other modification, it increases attainable in addition functional to bi-specific antibody, comprise, such as, increase the cell-mediated cytotoxicity of serum half-life (for example, see U.S.7,217,797) and/or increase antibody-dependant (for example, see U.S.6,737,056).In antibody constant region, introduce sudden change is as known in the art.In addition, can recombinate in cell culture and prepare the part of bi-specific antibody, and prepare the other parts of molecule by above-mentioned those technology mentioned.
Some technology of producing antibody are described.Such as, in numerous embodiments, in mouse described herein, produce chimeric antibody.The B cell that antibody directly can be separated the mouse of (for example, see U.S.2007/0280945A1) and/or immunity from the B cell of the mouse of immunity can be used to prepare hybridoma (Kohler and Milstein, 1975, Nature 256:495-497).Routine techniques is used easily to be separated and to check order from the DNA of the encoding antibody (people's heavy chain and/or light chain) of mouse described herein.Derive from the preferred source that the hybridoma of mouse described herein and/or B cell are used as such DNA.After being separated, DNA can be placed in expression vector, and it is transfected into the host cell not producing immunoglobulin (Ig) in addition subsequently, to realize the synthesis of monoclonal antibody in recombinant host cell.Also such as can carry out modifying DNA by replacing encoding sequence with the people's heavy chain and light chain constant domain of replacing mouse sequence.
In numerous embodiments, after DNA isolation and selection coding have the first and second nucleotide sequences of first and second people's heavy-chain variable domains of the specificity/avidity of expectation and the 3rd nucleotide sequence of encoding human light chain domain (being separated the sequence of resetting from sequence of light chain or the germline of mouse described herein), use 3 nucleotide sequences of widely used heavy thin technological expression coding molecule in this area to form bi-specific antibody.Usually, the expression system of selection comprises mammalian cell expression vector and host, so that bi-specific antibody is by glycosylation rightly (such as in the bi-specific antibody situation comprising glycosylated antibody domain).But, molecule can also be produced in prokaryotic expression system.Normally, the DNA transformed host cell of identical carrier or the first heavy-chain variable domains of coding independently on carrier, second people's heavy-chain variable domains, people's light chain domain is used in.But, the first heavy-chain variable domains, second people's heavy-chain variable domains and people's light chain domain (component of bi-specific antibody) can be expressed in independently expression system and in vitro in conjunction with the polypeptide of expression.In numerous embodiments, people's light chain domain comprises Germline sequences.In numerous embodiments, people's light chain domain comprises no more than 1, no more than 2, no more than 3, somatic hypermutation in the light chain variable sequence of the light chain domain of no more than 4 or no more than 5.
In numerous embodiments, the nucleic acid (such as cDNA or genomic dna) of coding two heavy chains and single people's light chain is inserted in replicable vector for further clone's (DNA amplification) and/or for expressing.Many carriers be available and generally include perseverance be not limited to following in one or more: signal sequence, replication origin, one or more marker gene, enhancer element, promotor and transcription termination sequence.Can seriatim or select based on host cell or test the other standards determined to select each component.Some examples of each component are well known in the art.
Cloning and expression carrier usually contains by the promotor of host organisms identification and may be operably coupled to each of encoding bispecific antibody or the sequence of all components.A large amount of is known by the promotor of multiple potential host cell identification.Remove promotor by always source DNA by Restriction Enzyme digestion and in the promoter sequence insertion vector of separation, these promotors will be may be operably coupled to the DNA of encoding bispecific antibody.
Expression vector for eukaryotic host cell (yeast, fungi, insect, plant, animal, people or the karyocyte from other multicellular organisms) also can containing the termination of transcribing and the necessary sequence of stable mRNA.Such sequence usually can from eucaryon or viral DNA or 5 ' of cDNA and the non-translational region of occasionally 3 ' obtain.The nucleotide segment of the polyadenylated fragments in the non-translational region of the mRNA being transcribed into encoding bispecific antibody component is contained in these regions.Suitable expression vector for numerous embodiments is included in mammalian cell the carrier of the transient expression of the DNA providing encoding bispecific antibody.Generally speaking, transient expression comprises the expression vector using and effectively can copy in host cell, and such host cell is assembled many copies of expression vector and and then synthesized the high-caliber polypeptide being expressed the expectation of vector encoded.The transient expression system comprising suitable expression vector and host cell allows the polypeptide of the DNA encoding to clone to carry out positive identification easily, and allows rapid screening to have the bi-specific antibody of the binding specificity/avidity of expectation or the gel shift characteristic of expectation relative to the parental antibody of the homodimer with first or second people's heavy-chain variable domains.
In multiple embodiment, after the DNA of encoding bispecific antibody component is assembled in the carrier of expectation described herein, they are introduced in the suitable host cell for expressing and reclaim.The standard technique (fusion of such as electroporation, core microinjection, bacterial protoplast and intact cell or polycation, such as polybrene, poly ornithine etc.) being suitable for selected host cell known in the art can be used to realize transfection host cell.
In numerous embodiments, select the most applicable expression vector containing component and allow the host cell of the production that bi-specific antibody kind is efficient and the most favourable.Exemplary host cells for expressing comprise the cell of prokaryotic organism and eukaryote (unicellular or many cells), bacterial cell (such as the bacterial strain of intestinal bacteria, genus bacillus, streptomycete etc.), mycobacterial cells, fungal cell, yeast cell (such as yeast saccharomyces cerevisiae, wine fission yeast, pichia pastoris phaff, pichia methanolica etc.), vegetable cell, insect cell (insect cell, cabbage looper etc. of such as SF-9, SF-21, baculovirus infection), non-human animal cell, people's cell or cell fusion such as hybridoma or four source hybridomas.In numerous embodiments, cell is people, monkey, ape, hamster, rat or mouse cell.In numerous embodiments, cell is that eukaryotic cell is selected from: CHO (such as CHO K1, DXB-11 CHO, Veggie-CHO), COS (such as COS-7), retina cell, Vero, CV1, nephrocyte (such as HEK293, 293EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB 8065, HL-60, (such as BHK21), Jurkat, Daudi, A431 (epidermis), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, sertoli cell, BRL3A cell, HT1080 cell, myeloma cell, tumour cell and the clone deriving from aforementioned cells.In numerous embodiments, cell comprises one or more virogene, such as, express retina cell (the such as PER.C6 of virogene tMcell).
Mammalian host cell for generation of bi-specific antibody can be cultivated in multiple substratum.Commercially available substratum is Ham ' s F10 (Sigma), Minimal Essential Medium ((MEM) such as, Sigma), RPMI-1640 (Sigma) and Dulbeccols Modified Eagle ' s Medium ((DMEM), Sigma) is suitable for cultivating host cell.Substratum can hormone supplemented and/or other somatomedin (such as Regular Insulin, Transferrins,iron complexes or Urogastron), salt (such as sodium-chlor, calcium, magnesium and phosphoric acid salt), damping fluid (such as HEPES), nucleosides (such as adenosine and thymidine), microbiotic (such as GENTAMYCIN as required tM), the energy of trace element (being defined as usually with the mineral compound that the final concentration of micro-scope of rubbing exists) and glucose or equivalence.Can also well known to a person skilled in the art that suitable concentration comprises other supplement any.In multiple embodiment, culture condition such as temperature, pH etc. are the selected conditions previously used for the host cell of expressing, and it is apparent to those skilled in the art.
In multiple embodiment, reclaimed from cell culture by the polypeptide of bi-specific antibody as secretion, although when directly being produced when not having secretion signal, it also can reclaim from host cell lysats.If bi-specific antibody is membrane-bound, then suitable detergent solution (such as Triton-X100) can be used to discharge it from film.Preferably, bi-specific antibody described herein comprises the first immunoglobulin (Ig) C h3 structural domains and the second immunoglobulin (Ig) C hthe application of 3 structural domains, wherein the first and second immunoglobulin (Ig) C h3 structural domains are not identical at least one amino acid each other, and wherein at least one amino acid whose difference reduces the combination of bi-specific antibody and albumin A (see U.S.2010/0331527A1 compared with lacking the bi-specific antibody of this amino acid difference; Be incorporated to by reference herein).In one embodiment, the first immunoglobulin (Ig) C h3 Domain Binding protein A and the second immunoglobulin (Ig) C h3 structural domains contain the sudden change reducing or abolish albumin A and combine, and such as H95R modifies and (numbers according to IMGT exon; H435R is numbered) according to EU.2nd C h3 also can comprise Y96F modifies (according to IMGT; Be Y436F according to EU).Can at the 2nd C hother modification found in 3 comprises: when IgG1 antibody, D16E, L18M, N44S, K52N, V57M and V82I are (according to IMGT; Be D356E, L358M, N384S, K392N, V397M and V422I according to EU); N44S, K52N and V82I (IMGT when IgG2 antibody; Be N384S, K392N and V422I according to EU); With Q15R, N44S, K52N, V57M, R69K, E79Q and V82I when IgG4 antibody (according to IMGT; Be Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU).The variation of above-mentioned bi-specific antibody form within the scope of the present invention.
Due to the double properties of bi-specific antibody, (the different epi-position that can be specific to a polypeptide maybe can comprise the antigen-binding domains being specific to more than one target polypeptide, see Fig. 7 B; Also can see people such as such as Tutt, 1991, J.Immunol.147:60-69; Kufer etc., 2004, Trends Biotechnol.22:238-244), they provide many favourable aspects for treatment use.Such as, bi-specific antibody may be used for redirected cytotoxicity (such as to kill tumour cell), as vaccine adjuvant, for sending thrombolytic agent to grumeleuse, for the prodrug activated at target site (such as tumour) saccharase, be used for the treatment of and catch, target immunocomplex to cell surface receptor, or for sending immunotoxin to tumour cell.
Bi-specific antibody described herein can also be used for several therapeutic and non-therapeutic and/or diagnostic assays method, such as, immunodiagnosis in the external or body of enzyme immunodetection, dibit point immunodetection, various diseases (such as cancer), competitive binding assay, directly and indirect sandwich measure and immune precipitation determination.Other purposes of bi-specific antibody will be apparent to those skilled in the art.
Provide the following example how to prepare to describe to those of ordinary skill in the art and to use method and composition of the present invention, be not intended to its scope of invention that restriction contriver thinks.Make efforts to guarantee the accuracy for used numeral (such as quantity, temperature etc.), but there is some experimental errors and deviation.Unless otherwise noted, the part that is by weight of part, molecular weight is molecular-weight average, and temperature is expressed as degree Celsius, and pressure is normal atmosphere or close to normal atmosphere.
Embodiment
Provide the following example with to those skilled in the art describe how to prepare and to use method and composition of the present invention, and be not intended to limit contriver regard it as scope of invention.To make an effort the accuracy guaranteed for used numeral (such as quantity, temperature etc.), but some experimental errors and deviation should have been considered.Except as otherwise noted, temperature is with degree Celsius to represent, pressure is normal atmosphere or close to normal atmosphere, the part that part is by weight, and molecular weight is molecular-weight average.
Embodiment 1
With the people V selected lthe people V that district combines hthe qualification in district
Construct vitro expression systems to determine whether that the single people's germline light chain through resetting can with the people's heavy chain coexpression from antigen-specific people antibody.
For produce in the mouse of genetic modification the method for people's antibody be known (see such as US6,596,541, Regeneron Pharmaceuticals, ). technology relates to the genomic genetic modification mouse producing and have people's heavy chain and the variable region of light chain comprising and may be operably coupled to endogenous mouse constant region gene seat, so that mouse produces the antibody comprising people variable region and murine constant region in response to antigenic stimulation.Coding produces certainly the DNA of the heavy chain of the antibody of mouse and the variable region of light chain is complete people.First, the high-affinity chimeric antibody with people variable region and murine constant region is separated.As described below, comprise avidity, selectivity, epi-position etc. for the characteristic expected and characterize and select antibody.The human constant region displacement that murine constant region is supposed to is to produce the fully human antibodies comprising IgG1, IgG2, IgG3 or IgG4 of non-IgM isotype such as wild-type or modification.Although the constant region selected can be different according to specific purposes, high-affinity antigen combines and target-specific feature is positioned at variable region.
With somatomedin (antigens c) immunity promoting vasculogenesis mouse, is separated antigen-specific people antibody, uses the use of standard technique to V gene of this area accreditation to check order.By the antibody cloning of selection to people's heavy chain and constant region of light chain, and select 69 heavy chains to match with one of 3 people's light chains: (1) is connected to the homology κ light chain of people κ constant region, (2) be connected to the people germline V κ 1-39J κ 5 of the rearrangement of people κ constant region, or (3) are connected to the people germline V κ 3-20J κ 1 of the rearrangement of people κ constant region.Use standard technique that each heavy chain and light chain pairing cotransfection are entered CHO-KI cell.Detect the existence of antibody in supernatant liquor by anti-human igg in ELISA measures.Measure the antibody titers (ng/ml) of each heavy chain/light chain pairing, and the titre of the titre of the germline light chain of different rearrangements with the parent antibody molecule (heavy chain such as matched with same endogenous light chain) obtained is compared, calculates the per-cent (table 1) of natural titre (native titier).V h: heavy chain variable gene.ND: expression do not detected under current experiment condition.
Table 1
In similar experiment, with several different antigen immune mouse also detects (as described above) from the ability that people's germline light chain of different rearrangements matches the heavy chain of antigen-specific people antibody selected.The antigen used in this experiment comprises the enzyme (antigen A) relating to cholesterol homeostasis, relate to the serum hormone (antigen B) regulating glucose homeostasis, promote somatomedin (antigens c) and the cell surface receptor (antigen D) of vasculogenesis.Antigen-specific antibodies is separated from the mouse of each immune group, and Cloning and sequencing heavy chain and variable region of light chain.According to the sequence of heavy chain and light chain, determine the use of V gene and people germline V κ 1-39J κ 5 district of the heavy chain of selection and the light chain of its homology or rearrangement is matched.Each heavy chain/light chain pairing cotransfection is entered CHO-K1 cell and in ELISA measures, detects the existence of antibody in supernatant liquor by anti-human igg.Measure the antibody titers (μ g/ml) of each heavy chain/light chain pairing, and the titre of the titre of the germline light chain of different rearrangements with the parent antibody molecule (heavy chain such as matched with same endogenous light chain) obtained is compared, calculates the per-cent (table 2) of natural titre.V h: heavy chain variable gene.V κ: kappa light chain variable gene.ND: expression do not detected under current experiment condition.
Table 2
Test from these high-affinity heavy chain through somatic mutation that the result that obtains demonstrates from different genes family can match with the people germline V κ 1-39J κ 5 reset and V κ 3-20J κ 1 district and secrete from cell as normal antibody molecule.As shown in table 1, with parental antibody with compared with endogenous light chain, when match with people V κ 1-39J κ 5 light chain reset about 61% (42 in 69) heavy chain and when people V κ 3-20J κ 1 light chain with rearrangement matches the antibody titers of the heavy chain of about 29% (20 in 69) add.With parental antibody with compared with endogenous light chain, for the heavy chain of about 20% (14 in 69), two kinds of people's germline light chain reset all give the increase of expression.As shown in table 2, with parental antibody with compared with endogenous light chain, people germline V κ 1-39J κ 5 district of rearrangement gives several increase being specific to the expression of the heavy chain of some different types of antigens.With parental antibody with compared with endogenous light chain, the antibody titers of about heavy chain of 35% (15/43) increases above 2 times.For two heavy chains (315 and 316), compared with parental antibody, increase more than 10 times.In all heavy chains relative to the expression of the same endogenous light chain display increase of parental antibody, compare other heavy chain variable region gene family, family 3 (V h3) heavy chain is showed too much.This shows people V hthe relationship favourable that the people germline V κ 1-39J κ 5 of 3 heavy chains and rearrangement and V κ 3-20J κ 1 light chain match.
Embodiment 2
The generation of the people's germline light chain gene seat reset
Use technology (see, such as, US patent No.6,586,251 and Valenzuela etc., (2003) the people's germline light chain targeting vector preparing multiple rearrangement clones 302g12 and 254m04 (Invitrogen) to modify mouse genome bacterial artificial chromosome (BAC) High-throughput engineering of the mouse genome coupled with high-rESolution exprESsion analysis, Nature Biotech.21 (6): 652-659).Use this two BAC clone, genome construct by through engineering approaches to be inserted into modified to delete the variable endogenous κ light chain gene seat with being connected constant gene segment C of endogenous κ in advance containing the single people's germline light chain district through rearrangement.
Build the people's germline light chain targeting vector reset.Art-recognized standard molecular biological technology is used to prepare people's germline light chain district of 3 different rearrangements.People's variable gene segment for building these 3 districts comprises people V κ 1-39J κ 5 sequence of rearrangement, people V κ 3-20J κ 1 sequence of rearrangement and people VpreBJ λ 5 sequence of rearrangement.
The region of DNA section of exons 1 (encoding leader peptide) containing mouse V κ 3-7 gene and introne 1 is prepared by from the beginning DNA synthesis (Integrated DNA Technologies).Include the part of 5 ' non-translational region to naturally occurring BlpI restriction enzyme site.From the exon of human genome BAC Library PCR amplification people V κ 1-39 and V κ 3-20 gene.Forward primer has 5 ' epitaxial part of the acceptor splicing site of the introne 1 containing mouse V κ 3-7 gene.Reverse primer for the PCR of people V κ 1-39 sequence comprises the epitaxial part of encoding human J κ 5, and comprises the epitaxial part of encoding human J κ 1 for the reverse primer of the PCR of people V κ 3-20 sequence.People VpreBJ λ 5 sequence is prepared by from the beginning DNA synthesis (Integrated DNA Technologies).The people J κ-C κ intron part of donor splicing site is comprised from plasmid pBS-296-HA18-PISceI pcr amplification.Forward PCR primer comprises the epitaxial part of the part of encoding human J κ 5, J κ 1 or J λ 5 sequence.Reverse primer comprises the PI-SceI site being engineered into intron in advance.
Mouse V κ 3-7 exons 1/introne 1, people's variable light exon and people J κ-C κ intron fragment are linked together by Overlap extension PCR, digest with BlpI and PI-SceI, and connecting into plasmid pBS-296-HA18-PISceI, described plasmid contains the promotor from people V κ 3-15 variable gene segment.Loxed Totomycin box in plasmid pBS-296-HA18-PISceI is had the FRTed Totomycin box in NotI and AscI site to replace by side joint.The NotI/PI-SceI fragment of this plasmid is connected in the mouse BAC254m04 of modification, the mouse BAC 254m04 of described modification contains the genome sequence that mouse J κ-C κ intron partly, mouse C κ exon and mouse kappa gene seat downstream are about 75kb, which provides the 3 ' homology arm for the homologous recombination in mouse ES cells.The NotI/AscI fragment of this BAC is connected in the mouse BAC 302g12 of modification subsequently, and the mouse BAC 302g12 of described modification contains the genome sequence of FRTed Liu Suanyan NEOMYCIN SULPHATE box and the about 23kb for the endogenous kappa gene seat upstream of homologous recombination in mouse ES cells.
People germline V κ 1-39J κ 5 targeting vector (Fig. 1) reset.Restriction enzyme site be introduced into through engineering approaches light chain Insert Fragment 5 ' and 3 ' end for being cloned into targeting vector: the AscI site of 5 ' end and the PI-SceI site of 3 ' end.In 5 ' AscI site and 3 ' PI-SceI site, target construct from 5 ' to 3 ' comprising: 5 ' homology arm of the sequence of the 5 ' end containing the endogenous mouse κ light chain gene seat available from mouse BAC clone 302g12, FRTed neomycin resistance gene, genome sequence containing people V κ 3-15 promotor, the leader sequence of mouse V κ 3-7 variable gene segment, the intron sequences of mouse V κ 3-7 variable gene segment, the open reading frame in people germline V κ 1-39J κ 5 district of resetting, the genome sequence of the part containing people J κ-C κ intron, and contain 3 ' homology arm (Fig. 1 of the sequence of cloning 3 ' end of endogenous mouse J κ 5 constant gene segment C of 254m04 available from mouse BAC, middle).The gene in the downstream of endogenous mouse κ light chain gene seat upstream and great majority 3 ' J kappa gene section and/or sequence (such as endogenous 3 ' enhanser) are not targeted construct and modify (see Fig. 1).People V κ 1-39J κ 5 locus sequence of through engineering approaches is presented in SEQ ID NO:1.
People germline V κ 1-39J κ 5 district being used the primer of the sequence in the people's germline light chain district being positioned to reset to confirm to reset by polymerase chain reaction (PCR) is inserted to the target in BAC DNA.Briefly, mouse V κ 3-7 leader sequence 3 ' intron sequences primer ULC-m1F (AGGTGAGGGTACAGATAAGT GTTATGAG; SEQ ID NO:2) and ULC-m1R (TGACAAATGCCCTAATTATA GTGATCA; SEQ ID NO:3) confirm.Open reading frame primer 1633-h2F (the GGGCAAGTCA GAGCATTAGC A in people germline V κ 1-39J κ 5 district of resetting; SEQ ID NO:4) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ ID NO:5) confirm.Liu Suanyan NEOMYCIN SULPHATE box primer neoF (GGTGGAGAGG CTATTCGGC; SEQ ID NO:6) and neoR (GAACACGGCG GCATCAG; SEQ ID NO:7) confirm.The BAC DNA of target is used to electroporation mouse ES cells to produce the ES cell of the gomphosis mouse in modified people germline V κ 1-39J κ 5 district for generation of expression rearrangement subsequently.
The probe being specific to the through engineering approaches V κ 1-39J κ 5 light chain district being inserted into endogenous gene locus is used to pass through TAQMAN tMexamination and karyotyping confirm positive ES cell clone.Briefly, probe neoP (TGGGCACAAC AGACAATCGG CTG; SEQ ID NO:8) be combined in neomycin marker gene, probe ULC-m1P (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ ID NO:9) be combined in the intron sequences of 3 ' end of mouse V κ 3-7 leader sequence, and probe 1633h2P (ATCAGCAGAAACCAGGGAAA GCCCCT; SEQ ID NO:10) be combined in people germline V κ 1-39J κ 5 open reading frame of rearrangement.Positive ES cell clone is used to implant female mice to produce a brood of young mouse of expressing germline V κ 1-39J κ 5 light chain district subsequently.
Or, there is the ES cell in the people germline V κ 1-39J κ 5 light chain district of rearrangement, to remove the FRTed Liu Suanyan NEOMYCIN SULPHATE box introduced by target construct with the construct transfection expressing FLP.Optionally, by removing Liu Suanyan NEOMYCIN SULPHATE box (such as US 6,774,279) with the mouse mating expressing FLP recombinase.Optionally, Liu Suanyan NEOMYCIN SULPHATE box is retained in mouse.
People germline V κ 3-20J κ 1 targeting vector (Fig. 2) reset.In a similar fashion, the preparation of target construct is used to express the light chain gene seat of the through engineering approaches in people germline V κ 3-20J κ 1 district of resetting, described target construct from 5 ' to 3 ' comprising: 5 ' homology arm of the sequence of the 5 ' end containing the endogenous mouse κ light chain gene seat available from mouse BAC clone 302g12, FRTed neomycin resistance gene, genome sequence containing people V κ 3-15 promotor, the leader sequence of mouse V κ 3-7 variable gene segment, the intron sequences of mouse V κ 3-7 variable gene segment, the open reading frame in people germline V κ 3-20J κ 1 district of resetting, the genome sequence of the part containing people J κ-C κ intron, and contain 3 ' homology arm (Fig. 2 of the sequence of cloning 3 ' end of endogenous mouse J κ 5 constant gene segment C of 254m04 available from mouse BAC, middle).The sequence of people V κ 3-20J κ 1 locus of through engineering approaches is presented in SEQ ID NO:11.
People germline V κ 3-20J κ 1 district being used the primer of the sequence in the people germline V κ 3-20J κ 1 light chain district being positioned to reset to confirm to reset by polymerase chain reaction (PCR) is inserted to the target in BAC DNA.Briefly, mouse V κ 3-7 leader sequence 3 ' intron sequences primer ULC-m1F (SEQ ID NO:2) and ULC-m1R (SEQ ID NO:3) confirm.Open reading frame primer 1635-h2F (the TCCAGGCACC CTGTCTTTG in people germline V κ 3-20J κ 1 district of resetting; SEQ ID NO:12) and 1635-h2R (AAGTAGCTGC TGCTAACACT CTGACT; SEQ ID NO:13) confirm.Liu Suanyan NEOMYCIN SULPHATE box primer neoF (SEQ ID NO:6) and neoR (SEQ ID NO:7) confirms.The BAC DNA of target is used to electroporation mouse ES cells to create the ES cell that the gomphosis mouse of people germline V κ 3-20J κ 1 light chain reset is expressed in modified generation subsequently.
The probe being specific to the through engineering approaches V κ 3-20J κ 1 light chain district be inserted in endogenous κ light chain gene seat is used to pass through TAQMAN tMexamination and karyotyping confirm positive ES cell clone.Briefly, probe neoP (SEQ ID NO:8) is combined in neomycin marker gene, probe ULC-m1P (SEQ ID NO:9) is combined in mouse V κ 3-7 leader sequence, and probe 1635h2P (AAAGAGCCACCCTCTCCTGC AGGG; SEQ ID NO:14) be combined in people V κ 3-20J κ 1 open reading frame.Positive ES cell clone is used to implant female mice subsequently.A brood of young mouse of expressing people's germline V κ 3-20J κ 1 light chain district.
Or, the ES cell in people germline V κ 3-20J κ 1 light chain district can be had with the construct transfection expressing FLP, to remove the FRTed Liu Suanyan NEOMYCIN SULPHATE box introduced by target construct.Optionally, by removing Liu Suanyan NEOMYCIN SULPHATE box (such as US 6,774,279) with the mouse mating expressing FLP recombinase.Optionally, Liu Suanyan NEOMYCIN SULPHATE box is retained in mouse.
The people germline VpreBJ λ 5 targeting vector (Fig. 3) reset.In a similar fashion, the preparation of target construct is used to express the light chain gene seat of the through engineering approaches in people germline VpreBJ λ 5 district of resetting, described target construct from 5 ' to 3 ' comprising: 5 ' homology arm of the sequence of the 5 ' end containing the endogenous mouse κ light chain gene seat available from mouse BAC clone 302g12, FRTed neomycin resistance gene, the thin sequence of gene containing people V κ 3-15 promotor, the leader sequence of mouse V κ 3-7 variable gene segment, the intron sequences of mouse V κ 3-7 variable gene segment, the open reading frame in people germline VpreBJ λ 5 district of resetting, the genome sequence of the part containing people J κ-C κ intron, and contain 3 ' homology arm (Fig. 3 of the sequence of cloning 3 ' end of endogenous mouse J κ 5 constant gene segment C of 254m04 available from mouse BAC, middle).The sequence of people VpreBJ λ 5 locus of through engineering approaches is presented in SEQ ID NO:15.
People germline VpreBJ λ 5 district being used the primer of the sequence in the light chain district of people germline VpreBJ λ 5 district being positioned to reset to confirm to reset by polymerase chain reaction (PCR) is inserted to the target in BAC DNA.Briefly, the intron sequences of 3 ' of mouse V κ 3-7 leader sequence is confirmed with primer ULC-m1F (SEQ ID NO:2) and ULC-m1R (SEQ ID NO:3).Open reading frame primer 1616-h1F (the TGTCCTCGGC CCTTGGA in people germline VpreBJ λ 5 district of resetting; SEQ ID NO:16) and 1616-h1R (CCGATGTCAT GGTCGTTCCT; SEQ ID NO:17) confirm.Liu Suanyan NEOMYCIN SULPHATE box primer neoF (SEQ ID NO:6) and neoR (SEQ ID NO:7) confirms.The BAC DNA of target is used to electroporation mouse ES cells to create the ES cell that the gomphosis mouse of people germline VpreBJ λ 5 light chain reset is expressed in modified generation subsequently.
The probe being specific to the through engineering approaches VpreBJ λ 5 light chain district being inserted into endogenous κ light chain gene seat is used to pass through TAQMAN tMexamination and karyotyping confirm positive ES cell clone.Briefly, probe neoP (SEQ ID NO:8) is combined in neomycin marker gene, probe ULC-m1P (SEQ ID NO:9) is combined in mouse IgV κ 3-7 leader sequence, and probe 1616h1P (ACAATCCGCCTCACCTGCAC CCT; SEQ ID NO:18) be combined in people VpreBJ λ 5 open reading frame.Positive ES cell clone is used to implant female mice to produce a brood of young mouse of expressing germline light chain district subsequently.
Or, there is the ES cell in the people germline VpreBJ λ 5 light chain district of rearrangement to remove the FRTed Liu Suanyan NEOMYCIN SULPHATE box introduced by target construct with the construct transfection expressing FLP.Optionally, by removing Liu Suanyan NEOMYCIN SULPHATE box (such as US 6,774,279) with the mouse mating expressing FLP recombinase.Optionally, Liu Suanyan NEOMYCIN SULPHATE box is retained in mouse.
Embodiment 3
Express the generation of the mouse of the single people's light chain through resetting
The ES cell of above-described target is used as contributing ES cells and passes through method (see, such as, US patent No.7,294, introduce the mice embryonic of 8 cell stages 754 and Poueymirou etc., (2007) F0 generation mice that are ESsentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech.25 (1): 91-99).The gene type measuring (Valenzuela etc., the same) by the allelotrope of the modification using the existence detecting unique people's germline light chain district through resetting identify there is through engineering approaches independently people germline V κ 1-39J κ 5 light chain district, V κ 3-20J1 light chain district or VpreBJ λ 5 light chain district
Carrying out gene type to young mouse and selecting for unique people's germline light chain district through resetting is that heterozygosis or pure and mild mouse are for the expression characterizing people's germline light chain district of resetting.
Flow cytometry.The expression in people's light chain district of resetting in the normal antibody storehouse of common light chain mouse is verified by the expression analyzing immunoglobulin (Ig) κ and λ in the splenocyte of common light chain mouse and peripheral blood.Use standard method preparation from the spleen of collection of wild-type (n=5), V κ 1-39J κ 5 common light chain heterozygote (n=3), V κ 1-39J κ 5 common light chain homozygote (n=3), V κ 3-20J κ 1 common light chain heterozygote (n=2) and V κ 3-20J κ 1 common light chain homozygote (n=2) mouse and the cell suspending liquid of peripheral blood and use fluorescent-labeled antibody (BD Pharmigen) to carry out CD19 to described cell suspending liquid +, Ig λ +with Ig κ +dyeing.
Briefly, by 1 × 10 6individual cell and anti-mouse CD16/CD32 (clone 2.4G2, BD Pharmigen) incubated on ice 10 minutes, 30 minutes: the APC anti-mouse CD19 puted together (clone 1D3 is marked with following mixtures of antibodies subsequently on ice, BD Pharmigen), PerCP-Cy5.5 put together anti-mouse CD3 (clone 17A2, BioLegend) the anti-mouse Ig κ that, FITC puts together (clones 187.1, BD Pharmigen), the anti-mouse Ig λ (clone RML-42, BioLegend) that puts together of PE.After dyeing, washed cell is also fixed with 2% formaldehyde.LSRII flow cytometer carries out data gathering and analyzes with FlowJo.Gate: total B cell (CD19 +cD3 -), Ig κ +b cell (Ig κ +ig λ -cD19 +cD3 -), Ig λ +b cell (Ig κ -ig λ +cD19 +cD3 -).The similar result from the data acknowledgement of blood and splenocyte sample collection.Table 3 lists from Ig λ +, Ig κ +or Ig λ +ig κ +each group in the positive CD19 of peripheral blood of a representative mouse +the percentage ratio of B cell.The CD19 in the peripheral blood of the mouse of isozygotying from wild-type (WT) with for V κ 1-39J κ 5 or V κ 3-20J κ 1 common light chain +the percentage ratio of B cell is shown in Fig. 4.
Table 3
The expression of common light chain.In heterozygosis with the mouse of isozygotying, use quantitative PCR to measure (such as TAQMAN tM) analyze the expression of often kind of common light chain (V κ 1-39J κ 5 and W κ 3-20J κ 1).
Briefly, use mouse CD19 microballon (Miltenyi Biotec) according to the specification sheets of manufacturer from wild-type, be the mouse (H κ) of isozygotying for the displacement utilizing corresponding people's heavy chain and kappa light chain variable district locus to murine heavy chain and kappa light chain variable district locus and be to isozygoty and the spleen purifying of mouse of heterozygosis obtains CD19 for people's light chain district (V κ 1-39J κ 5 or V κ 3-20J κ 1) of each rearrangement +b cell.Use RNeasy Mini kit (Qiagen) from CD19 according to the specification sheets of manufacturer +b cell purifying obtains total serum IgE, and use removes geneome RNA without the DNase post of RNase processing (Qiagen).Use the first chain cDNA synthetic agent box (Invitrogen) that 200ng mRNA reverse transcription is become cDNA and to increase the cDNA obtained with Taqman Universal PCR Master Mix (Applied Biosystems).Utilize the primer and Taqman MGB probe of crossing over the independent V kappa gene (i.e. V κ 1-39 and V κ 3-20) in the V κ-J κ junction surface of (1) two kind of common light chain, (2) and (3) mouse C κ district, use ABI 7900Sequence Detection System (Applied Biosystems) is carried out responding.Table 4 lists the sequence of primer and the probe used in this detection.Relative expression is carried out stdn for the expression in mouse C κ district.Result is presented in Fig. 5 A, 5B and 5C.
Table 4
Antigen-specific common light chain antibody.There is the common light chain mouse of V κ 1-39J κ 5 or V κ 3-20J κ 1 common light chain on endogenous mouse κ light chain gene seat with beta-galactosidase enzymes immunity and measure antibody titers.
Briefly, according to guidance emulsification beta-galactosidase enzymes (Sigma) in titermax adjuvant (Sigma) of manufacturers.Immune wild-type (n=7), V κ 1-39J κ 5 common light chain homozygote (n=2) and V κ 3-20J κ 1 common light chain homozygote (n=5) is come by subcutaneous injection 100 μ g beta-galactosidase enzymes/Titermax.By within three weeks, strengthening mouse with 50 μ g beta-galactosidase enzymes/Titermax subcutaneous injections twice, interval.After second time strengthens, according to the guidance of manufacturers, after utilizing socket of the eye, the hemorrhage mouse from anaesthetizing, collect blood in blood vessel separator tube (BD BiosciencES).In order to measure IgM or the IgG antibody of anti-beta-galactosidase enzymes, spend the night 4 DEG C of coatings ELISA flat board (Nunc) with the beta-galactosidase enzymes of 1 μ g/mL.First wash excessive antigen off, close 1 hour with the PBS containing 1%BSA in room temperature subsequently.The serum of serial dilution degree is joined flat board and incubation at room temperature 1 hour, washs subsequently.The anti-IgM (Southern Biotech) puted together with HRP subsequently or anti-igg (Southern Biotech) were dull and stereotyped 1 hour of incubation at room temperature.After washing again, with tmb substrate (BD BiosciencES), flat board is developed.Also OD is read with Victor X5 Plate Reader (Perkin Elmer) by the sulfuric acid termination reaction of 1N 450.Be the serum dilution higher than background twice with GraphPad Prism analytical data and by calculated signals.Result is presented in Fig. 6 A and 6B.
As shown in this embodiment, the ratio of the κ/λ B cell in the spleen and periphery compartment of V κ 1-39J κ 5 and V κ 3-20J κ 1 common light chain mouse all shows the pattern (table 3 and Fig. 4) of approximate wild-type.But VpreBJ λ 5 common light chain mouse shows less periphery B cell, wherein approximately 1-2% expresses people's light chain district (data do not show) of through engineering approaches.With containing utilizing people V κ to compare with the endogenous κ light chain gene seat of replacing completely of J kappa gene section mouse V κ with J kappa gene section, the expression level in people's light chain district of resetting from the V κ 1-39J κ 5 of endogenous κ light chain gene seat and V κ 3-20J κ 1 raises (Fig. 5 A, 5B and 5C).The expression level in people's light chain district that VpreBJ λ 5 resets demonstrate in heterozygosis and Mice homozygous from the high expression level (data do not show) that the expression of endogenous κ light chain gene seat is similar.This proves with the direct competitive of mouse λ, κ or two endogenous light chain genes seats, the single people V through resetting l/ J lsequence can produce the expression and the spleen of the normal side of generation and blood B cells frequency that are better than from the wild-type levels of endogenous κ light chain gene seat.In addition, the existence with the κ light chain gene seat of the through engineering approaches of people V κ 1-39J κ 5 or people V κ 3-20J κ 1 sequence is well tolerated by mouse, and is shown by the major portion in light chain storehouse in the body fluid components that represents immunne response and play function (Fig. 6 A and 6B) in the mode of wild-type.
Embodiment 4
Express the breeding of the mouse of the single people's germline light chain through resetting
Present embodiment describes other mouse species of genetic modification several, it with any one mating in common light chain mouse described herein, can modify mouse species to create the multiple genetic with multiple genetic modified immunoglobulin locus.
Endogenous Ig λ knocks out (KO).In order to the use of optimizing project light chain gene seat, will there is a kind of mouse of people's germline light chain district of rearrangement and the another kind of mouse mating containing the deletion of endogenous lambda light chain gene seat.By this way, the offspring obtained will express people's germline light chain district of rearrangement as described in Example 2 as its only light chain.Also bred by business beginner (such as The Jackson Laboratory) alternatively according to standard technique well known in the art.The mouse species screening the deletion with through engineering approaches light chain gene seat and endogenous lambda light chain gene seat is there is not for the existence in unique light chain district and endogenous mouse lambda light chain.
Humanized endogenous heavy chain genes seat.By the mouse with through engineering approaches people germline light chain gene seat with containing the mouse utilizing people's heavy chain variable gene locus to the displacement of endogenous mouse heavy variable gene locus (see US 6,596,541; mouse, Regeneron Pharmaceuticals, Inc.) mating. mouse comprises the genome containing the people variable region of heavy chain that may be operably coupled to endogenous mouse constant region gene seat, so that mouse response antigenic stimulation produces the antibody containing people variable region of heavy chain and murine heavy chain constant region.Be separated the DNA of the variable region of the heavy chain of encoding antibody and may be operably coupled to the DNA of encoding human CH.The cells DNA of complete people's heavy chain of antibody can expressed subsequently.
Acquisition has employment V hlocus is to endogenous mouse V hthe single people germline V through resetting on the displacement of locus and endogenous κ light chain gene seat lthe mouse in district.After with object antigen immune, obtain containing heavy chain (the people V through somatic mutation hwith mouse C h) and single people's light chain (people V lwith mouse C l) reverse imbedding antibody.The V of the B cell of antibody is expressed in qualification hand V lnucleotide sequence, and pass through V in suitable expression system hand V lnucleotide sequence merges to people C hand C lnucleotide sequence prepares fully human antibodies.
Embodiment 5
From the generation of antibody of mouse in people's germline light chain district expressing people's heavy chain and rearrangement
Mouse containing through engineering approaches people light chain district and multiple expectation carried out post-coitum, the mouse selected by available object antigen immune containing the modification of other endogenous Ig loci (as described in Example 4) and the strain of deletion.
Usually, a kind of single people's germline light chain district through resetting is contained with antigenic stimulation mouse, and from the serum of animal, reclaim lymphocyte (such as B cell).Lymphocyte and myeloma cell line are merged to prepare immortal hybridoma cell lines, and carry out Selection and screening to identify the hybridoma cell line of the antibody producing the people's germline light chain containing people variable region of heavy chain and rearrangement to this hybridoma cell line, described antibody is specific to the antigen for immunity.Be separated the DNA of the variable region of encoding heavy chain and light chain, and be connected to the isotype constant region of the expectation of heavy chain and light chain.Due to endogenous mouse sequence and other existence of cis-acting elements any of being present in endogenous gene locus, the single light chain of each antibody can by somatic mutation.This adds extra diversity to the antigen-specific storehouse comprising single light chain and different heavy chains sequence.The clonal antibody sequence generated is expressed subsequently in cell such as Chinese hamster ovary celI.Alternatively, the direct DNA of the variable domains of identification code antigen-specific chimeric antibody or light chain and heavy chain from Antigen-specific lymphocytes.
First, the high-affinity chimeric antibody with people variable region and murine constant region is separated.As mentioned above, comprise avidity, selectivity, epi-position etc. according to the characteristic expected characterize and select antibody.With the displacement murine constant region, human constant region expected with produce containing through somatic mutation people's heavy chain and derive from the fully human antibodies of single light chain in people's germline light chain district of rearrangement of the present invention.Suitable human constant region comprises, IgG1 or IgG4 of such as wild-type or modification.
What employment cell surface receptor protein (antigen E) immunity was different contains employment V h, D hand J hconstant gene segment C to the displacement of endogenous mouse heavy chain locus and with through engineering approaches germline V κ 1-39J κ 5 people's light chain district or through engineering approaches germline V κ 3-20J κ 1 people's light chain district (as mentioned above) to the displacement of endogenous mouse kappa light chain locus mouse group.Antigen E is applied directly to mouse hind leg vola, within every 3-4 days, injects 6 times continuously.Before injection, by CpG ODN (the Cat#tlrl-modn-ODN1826 oligonucleotide of the antigen E of 2 to 3 micrograms and 10 μ g; InVivogen, San Diego, CA) and Adju-Phos (phosphaljel adjuvant, the Cat#H-71639-250 of 25 μ g; Brenntag Biosector, Frederikssund, Denmark) mixing.Altogether give 6 injections last antigen recall (antigen recall) is front, and 3-5 days before execution give described antigen recall.After the 4th and the 6th injection, collect blood and measure the response of monitoring antibody mediated immunity by the antigen specific immune of standard.
When obtaining the immunne response expected, collecting splenocyte and itself and murine myeloma cell are merged keep its viability and form hybridoma cell line.Selection and screening hybridoma cell line produces the clone of antigen E specificity common light chain antibody to identify.This technology is used to obtain some antigen E specificity common light chain antibody (namely having the antibody of people's heavy-chain variable domains, identical people's light variable domains and mouse constant structural domain).
Alternatively, directly never be separated antigen E common light chain antibody in the antigen positive B cell of myeloma cell fusion, as described in U.S.2007/0280945 A1, be incorporated herein by reference in their entirety especially.Use the method, obtain some complete people's antigen E common light chain antibody (namely there is people's heavy-chain variable domains, people V κ 1-39J κ 5 light chain of through engineering approaches or the antibody of through engineering approaches people V κ 3-20J κ 1 light chain district and people's constant domain).
The biological property of the exemplary antigen E common light chain antibody produced according to the method for the present embodiment is described in detail in chapters and sections below.
Embodiment 6
The use of heavy chain gene section in antigen-specific common light chain antibody
In order to analyze the structure of produced people's antigen E common light chain antibody, the nucleic acid of encoding heavy chain antibody variable region is cloned and checks order.According to the nucleotide sequence of antibody and the aminoacid sequence of prediction, the gene identifying the variable region of heavy chain (HCVR) of the common light chain antibody chosen uses, described antibody available from by immunity containing people's antibody V κ 1-39J κ 5 light chain of through engineering approaches or through engineering approaches people V κ 3-20J κ 1 light chain district mouse.Result is presented in table 5 and 6, it proves when adopting the mouse of the light chain of expressing the light chain of originating from unique people V κ 1-39-or people V κ 3-20-, due to various rearrangement, produce the antigen-specific common light chain antibody from various human heavy chain gene section according to mouse of the present invention.2, the people V of 3,4 and 5 families hconstant gene segment C and various human D hsection and people J hsection is reset to produce antigen-specific antibodies.
Table 5
Table 6
Embodiment 7
Pass through LUMINEX tMmeasure the blocking ability determining the antigen special property led common light chain antibody
Based in the mensuration of globule, detect the ability of the native ligand (part Y) of 98 people's common light chain antibody blocking antigen E and the combination of antigen E produced for antigen E.
The extracellular domain (EDC) of antigen E and two myc epitope tags and 6 × histidine-tagged are puted together (antigen E-mmH), and is coupled to carboxylated micro-spheres with 20 μ g/mL concentration amine in MES damping fluid.By mixture incubation at room temperature 2 hours, use 1M pH8.0 Tris to carry out globule inactivation subsequently, then wash with the PBS containing 0.05% (v/v) Tween-20.Afterwards, globule is closed with the PBS (Irvine Scientific, Santa Ana, CA) containing 2% (v/v) BSA (Sigma-Aldrich Corp., St.Louis, MO).In 96 hole screen plates, the supernatant liquor containing antigen E specificity common light chain antibody is diluted by 1: 15 in damping fluid.Preparation is containing having the negative control with the simulation supernatant liquor of the identical nutrient media components for antibody supernatant.The globule that antigen E marks is joined in supernatant liquor, and is incubated overnight at 4 DEG C.Biotinylated part Y albumen is added with the final concentration of 0.06nM, and incubation at room temperature 2 hours.Determine to be bonded to by the R-PE (Moss Inc, Pasadena, MD) being conjugated to Streptavidin the biotinylated part Y of the globule that antigen E-myc-myc-6His marks detection and subsequently based on LUMINEX tMmeasure in the analyser of flow cytometry.The background average fluorescent strength (MFI) of the sample not containing part Y is deducted from all samples.By the MFI of the subtracting background of each sample divided by the negative control value after adjustment, be multiplied by 100, block to calculate per-cent in the value deducting gained from 100.
In similar experiment, detect the ability of the combination of the globule that 98 identical people's common light chain antibody blocking antigen E for antigen E generation and part Y mark.
Briefly, with the concentration of the 20 μ g/mL diluted in MES damping fluid, part Y amine is coupled to carboxylated micro-spheres.By mixture incubation at room temperature 2 hours, use 1MTris pH8.0 to carry out globule inactivation subsequently, wash with the PBS containing 0.05% (v/v) Tween-20 afterwards.Afterwards, globule is closed with the PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w/v) BSA (Sigma-Aldrich Corp., St.Louis, MO).In 96 hole screen plates, the supernatant liquor containing antigen E specificity common light chain antibody dilutes by 1: 15 in damping fluid.Preparation is containing having the negative control with the simulation supernatant liquor of the identical nutrient media components for antibody supernatant.Biotinylated antigen E-mmH is added with the final concentration of 0.42nM, and is incubated overnight at 4 DEG C.Subsequently the globule that part Y marks to be added in antibody/antigen E mixture and incubation at room temperature 2 hours.By the R-PE (Moss Inc, Pasadena, MD) being conjugated to Streptavidin determine the biotinylated antigen E-mmH being bonded to part Y globule detection and subsequently based on LUMINEX tMmeasure in the analyser of flow cytometry.The background average fluorescent strength (MFI) of the sample not containing antigen E is deducted from all samples.By the MFI of the subtracting background of each sample divided by the negative control value after adjustment, be multiplied by 100, block to calculate per-cent deducting income value from 100.
Table 7 and 8 shows at two LUMINEX tMthe per-cent of all 98 the antigen E common light chain antibody tested in mensuration blocks.ND: do not detect under current experiment condition.
Table 7
Table 8
At above-described first LUMINEX tMin experiment, the ability that the block ligand Y testing 80 common light chain antibody containing V κ 1-39J κ 5 through engineering approaches light chain is combined with the globule that antigen E marks.In these 80 common light chain antibody, 68 blocking-up demonstrating > 50%, and 12 blocking-up (blocking-up of the blocking-up of 6 25-50% and 6 < 25%) demonstrating < 50%.18 are contained to the common light chain antibody of V κ 3-20J κ 1 through engineering approaches light chain, for the combination of the globule that part Y and antigen E marks, 12 blocking-up demonstrating > 50%, and 6 blocking-up (blocking-up of the blocking-up of 3 25-50% and 3 < 25%) demonstrating < 50%.
At above-described second LUMINEX tMin experiment, test the ability that 80 identical blocking-up antigen E containing the common light chain antibody of V κ 1-39J κ 5 through engineering approaches light chain are combined with the globule that part Y marks.In these 80 common light chain antibody, 36 blocking-up demonstrating > 50%, and 44 blocking-up (blocking-up of the blocking-up of 27 25-50% and 17 < 25%) demonstrating < 50%.18 are contained to the common light chain antibody of V κ 3-20J κ 1 through engineering approaches light chain, for the combination of the globule that antigen E and part Y marks, 1 blocking-up demonstrating > 50%, and 17 blocking-up (blocking-up of the blocking-up of 5 25-50% and 12 < 25%) demonstrating < 50%.
Table 7 and 8 data presentation table 5 and 6 in the rearrangement that describes create antigen E specificity common light chain antibody with the combination of effect block ligand Y in various degree and its homoreceptor antigen E, this and the patient's condition have relative to the overlap of antigen E and the antibody of non-overlapped epitope specificity table 5 and 6 anti-antigen E common light chain antibody consistent.
Embodiment 8
The blocking ability of antigen-specific common light chain antibody is measured by ELISA
In ELISA experiment, to the people's common light chain antibody test produced for antigen E, it blocks the ability of the surface bonding that antigen E and part Y applies.
To be coated in the part Y of the concentration being diluted in 2 μ g/mL in PBS on 96 orifice plates and be incubated overnight, washing 4 times with the PBS containing 0.05% Tween-20 subsequently.Subsequently with the PBS (Irvine Scientific, Santa Ana, CA) containing 0.5% (w/v) BSA (Sigma-Aldrich Corp., St.Louis, MO) room temperature blocking of plates 1 hour.In an independent plate, by the supernatant liquor containing antigen E common light chain antibody in damping fluid by 1: 10 dilution.Use the simulation supernatant liquor with the identical component of antibody as negative control.Antibody E-mmH (above-mentioned) to be added with the final concentration of 0.150nM and incubation at room temperature 1 hour.Subsequently antibody/antigen E-mmH mixture is added in the plate containing part Y, and incubation at room temperature 1 hour.By being conjugated to horseradish peroxidase (the HRP) (Qiagen of anti-Penta-His antibody, Valencia, CA) detection being bonded to the antigen E-mmHg of part Y is measured and by tetramethyl benzidine (TMB) substrate (the BD BiosciencES that neutralized by sulfuric acid of use, San Jose, CA) standard colorimetric reaction develop.Absorbancy is read 0.1 second at OD450.The Background absorbance of the sample not containing antigen E is deducted from all samples.By the MFI of the subtracting background of each sample divided by the negative control value after adjustment, be multiplied by 100, and block to calculate per-cent from 100 values deducting gained.
The per-cent that table 9 and all 98 the antigen E common light chain antibody of 10 display detect in ELISA experiment blocks.ND: do not detect under current experiment condition.
Table 9
Table 10
As described in the embodiment, testing its 80 of blocking the ability of the surface bonding that antigen E and part Y applies containing in the common light chain antibody of V κ 1-39J κ 5 through engineering approaches light chain, 22 blocking-up demonstrating > 50%, and 58 blocking-up (blocking-up of the blocking-up of 20 25-50% and 38 < 25%) demonstrating < 50%.18 are contained to the common light chain antibody of V κ 3-20J κ 1 through engineering approaches light chain, for the combination of antigen E and part Y coated surface, 1 blocking-up demonstrating > 50%, and 17 blocking-up (blocking-up of the blocking-up of 5 25-50% and 12 < 25%) demonstrating < 50%.
These results also with comprise have relative to the overlap of antigen E and the antigen-E specificity common light chain antibody library of the specific antibody of non-overlapping epitopes consistent.
Embodiment 9
The BIACORE of antigen-specific common light chain antibody tMavidity measures
Use BIACORE tMt100 equipment (GE Healthcare) measures the equilibrium dissociation constant (K of the antibody supernatant selected by SPR (surface plasma body resonant vibration) d).HBS-EP (10mM HepES, 150mM NaCl, 0.3mM EDTA, 0.05% tensio-active agent P20, pH 7.4) is obtained all data as operation and sample buffer at 25 DEG C.From original supernatant sample by antibody capture on CM5 sensor chip surface, described chip surface uses standard amine-coupling chemistry method to utilize highdensity Anti-Human Fc antibody to carry out derivatize (derivatized) in advance.In the capture step, supernatant liquor is injected through Anti-Human Fc antibody surface with the flow velocity of 3 μ L/min, carries out 3 minutes altogether.After catching step, the running buffer of 100nM concentration or analyte are injected 2 minutes with the flow velocity of 35 μ L/min.Monitoring antigen, from dissociating the antibody of catching, carries out 6 minutes.The antibody of catching is removed by the glycine of of short duration injection 10mM pH1.5.Carry out two sensing figure all with reference to (double reference) by the sensing figure deducted from analyte sensing figure from buffer injection, thus remove and dissociated on the surface and the illusion that produces from catching by antibody.Use BIAcore T100 Evaluation software v2.1 by each antibody adjust in conjunction with data 1: 1 mass transfer combination model.Result is presented in table 11 and 12.
Table 11
Table 12
In table 11 and 12, display is different separately containing the binding affinity of the common light chain antibody of the rearrangement of display in table 5 and 6, is wherein nearly allly all presented at the K received in the scope of rubbing d.What affinity data and the built up section of the variable domains by the rearrangement described in table 5 and 6 produced have high-affinity, through clonal selection and common light chain antibody through somatic mutation consistent.The data of associating previously display, the common light chain antibody described in table 5 and 6 comprises the specific antibody library demonstrated epi-position one or more on antigen E that is various, high-affinity.
Embodiment 10
Pass through LUMINEX tMthe binding specificity of measuring antigen-specific common light chain antibody
Detect the antigen E common light chain antibodies the selected ability to the ECD variant of the ECD of antigen E and antigen E, described variant comprises cynomolgus monkey homologue (Mf antigen E), and they are different from people's albumen on the amino-acid residue of about 10%; Last 10 the amino acid whose deletion mutants of the C-terminal lacking ECD (antigen E-Δ CT) of antigen E; With two with the interactional computed position of part Y on comprise the mutant (antigen E-Ala1 and antigen E-Ala2) that L-Ala replaces.Antigen E protein is produced and it is separately containing C-terminal myc-myc-His label in Chinese hamster ovary celI.
For associativity research, by being coated with anti-myc monoclonal antibody (MAb 9E10, hybridoma cell line CRL-1729 in room temperature and covalency tM; ATCC, Manassas, VA) 1 × 10 6individual microballoon (LUMINEX tM) globule incubation 2 hours comes ECD albumen or the misfolded proteins (as mentioned above) of capture antigen E from the substratum of 1mL.Globule is washed before use subsequently with PBS.Supernatant liquor containing antigen E common light chain antibody is diluted by 1: 4 in damping fluid and is added in 96 hole screen plates.Simulation supernatant liquor not containing antibody is used as negative control.Subsequently the globule containing the antigen E protein of catching to be joined in antibody samples (3000, every hole globule) and be incubated overnight at 4 DEG C.Next day, washing sample globule and the Anti-Human's IgG antibody using R-PE to put together detect the common light chain antibody combined.With based on LUMINEX tMthe analyser of flow cytometry measures globule (for each antibody samples being bonded to each antigen E protein, about 100 globules) fluorescence intensity, record the median fluorescence intensity (MFI) of globule of interactional at least 100 countings of every globule/antibody.The results are shown in table 13 and 14.
Table 13
Table 14
Antigen E common light chain antibody supernatant demonstrates and is combined with the high specific of the globule being connected to antigen E-ECD.For these globules, negative control simulation supernatant liquor when with produce negligible signal (< 10MFI) during antigen E-ECD globule sample combination, and the supernatant liquor containing antigen E common light chain antibody demonstrates strong binding signal, and (for 98 antibody supernatant, average MFI is 2627; The MFI > 500 of 91/98 antibody samples).
As the measurement of ability of different epi-position on the ECD of the antigen E common light chain antibody recognition antigen E selected, determine the relative combination of antibody and variant.As above for described by natural antigen E-ECD binding, all four kinds of antigen E variants are captured to anti-myc LUMINEX tMglobule, and measure relative combining ratio (MFI variant/ MFI antigen E-ECD).For 98 common light chain antibody supernatant detected of display in table 12 and 13, the average ratio (MFI of each variant variant/ MFI antigen E-ECD) different, may reflect albumen quantity of the catch (for antigen E-Δ CT, antigen E-Ala1, antigen E-Ala2 and Mf antigen E, average ratio is 0.61,2.9,2.0 and 1.0 respectively) different on globule.For each protein variant, the combination of a subgroup in 98 common light chain antibody detected demonstrates the combination greatly reduced, and shows the susceptibility to the sudden change characterizing given variant.Such as, 19 in common light chain antibody samples with the MFI of < 8% variant/ MFI antigen E-ECDbe bonded to Mf antigen E.Because many in this group comprise high or medium high-affinity antibody (5 K with < 5nM d, 15 K with < 50nM d), therefore lower in this group signal may be come from the susceptibility to sequence (epi-position) difference between natural antigen E-ECD and given variant, instead of comes from lower avidity.
These data show that the common light chain antibody described in table 5 and 6 represents diversified antigen E specificity common light chain antibody group, more than 1 epi-position on its specific recognition antigen E.
Embodiment 11
Light chain reorganization in common light chain antibody
After heavy chain and germline V κ 1-39J κ 5 or germline V κ 3-20J κ 1 through engineering approaches light chain (as described in example 1 above) being matched again, detect the heavy chain of antigen-specific common light chain antibody and the combination of antigen E selected.
Briefly, by the transfection and pass through LUMINEX together with germline V κ 1-39 or germline V κ 3-20 through engineering approaches light chain of 247 heavy chains of antigen E specificity common light chain antibody (V κ 1-39J κ 5 and V κ 3-20J κ 1) tMmeasure (as described in embodiment 7 and embodiment 10) again to screen for the combination with antigen E.Pass through BIACORE tM(as described in example 9 above) combination with antigen E is confirmed.Result is presented in table 15.
As shown in this embodiment, when the germline form with light chain matches, 28 common light chain antibody special to antigen E can be bonded to antigen E.
Table 15
Embodiment 12
Heavy chain gene in common light chain antibody uses and somatic hypermutation frequency
Will mouse (such as US 6,596,541 and US 7,105,348) heavy chain of the antibody produced in and sequence of light chain (> 6000) use and somatic hypermutation frequency with the heavy chain gene section that the heavy chain and sequence of light chain (> 600) that pass through the common light chain antibody adopting many antigen immunes scheme of through engineering approaches light chain mouse (above-described) to obtain carry out collecting to compare antibody chain.
Heavy chain gene uses.The heterodimer (antigen F) of employment cell surface receptor (antigen E), two people's cell surface glycoproteins, human cytokine receptors (antigen G) and people's Tumor Differentiation antigen (antigen H) immunity are containing employment V h, D hand J hconstant gene segment C to the displacement of endogenous letter mouse heavy chain locus and with through engineering approaches germline V κ 1-39J κ 5 people's light chain district or through engineering approaches germline V κ 3-20J κ 1 people's light chain district (as described in example 2 above) to the displacement of endogenous mouse kappa light chain locus mouse, the heavy chain gene section analyzing heavy chain and the sequence of light chain obtained uses and records V hand J hconstant gene segment C.Result is presented in table 16-18.Per-cent in table 16-18 represents rounded value and adds up in some cases and may be not equal to 100%.
Table 16 list from the antibody of mouse (VI), from having homology V κ 1-39 light chain the antibody of mouse (VI-V κ 1-39), from the antibody of V κ 1-39 through engineering approaches light chain mouse (V κ 1-39), from having homology V κ 3-20 light chain the antibody of mouse (VI-V κ 3-20) and the heavy chain families use per-cent from the antibody of V κ 3-20 through engineering approaches light chain mouse (V κ 3-20).Table 17 list from the antibody of mouse (VI), from having homology V κ 1-39 light chain the antibody of mouse (VI-V κ 1-39), from the antibody of V κ 1-39 through engineering approaches light chain mouse (V κ 1-39), from having homology V κ 3-20 light chain the V of the antibody of mouse (VI-V κ 3-20) and the antibody from V κ 3-20 through engineering approaches light chain mouse (V κ 3-20) hand J hgene uses per-cent.Table 18 lists the V of the antibody of the V κ 1-39 through engineering approaches light chain mouse (V κ 1-39 mouse) from each immune group (antigen E, F, G and H) hgene uses per-cent, and the V of antibody of V κ 3-20 through engineering approaches light chain mouse (V κ 3-20 mouse) from the immune group (antigen E and G) selected hgene uses per-cent.
As shown in this embodiment, the feature used for the heavy chain gene of the antigen detected in V κ 1-39J κ 5 through engineering approaches light chain mouse is the V occupied the majority hfamily III subgroup (V h3-7, V h3-9, V h3-11, V h3-13, V h3-20, V h3-23, V h3-30, V h3-33 and V h3-48).Other V hthe feature of the remarkable use of family's subgroup is V h1-18, V h1-69, V h2-5, V h4-59 and V hthe use of 6-1.For the antigen detected in V κ 3-20J κ 1 through engineering approaches light chain mouse, the feature that heavy chain gene uses is the V occupied the majority hfamily III subgroup, V hfamily IV subgroup and V hfamily V subgroup (V h3-11, V h3-30, V h3-33, V h4-39, V h4-59 and V h5-51).Other V hthe feature of the remarkable use of family's subgroup is V h1-18, V h1-69, V h2-70 and V hthe use of 6-1.
Somatic hypermutation frequency.Heavy chain according to every bar heavy chain and/or light chain and light chain gene make for by the heavy chain of mouse and the middle antibody produced of through engineering approaches light chain mouse (above-described) and light chain comparison are to Germline sequences.Calculate the amino acid change of the heavy chain of each sequence and each framework region (FW) of light chain and complementary determining region (CDR).Result is presented in table 19-22.Per-cent in table 21-24 represents rounded value and adds up in some cases and may be not equal to 100%.
Table 19 list from the heavy chain of the antibody of mouse, the number changed from the amino acid (AA) observed in the heavy chain of the antibody of V κ 1-39 through engineering approaches light chain mouse (V κ 1-39 mouse) and each FW and the CDR district from the heavy chain of the antibody of V κ 3-20 through engineering approaches light chain mouse (V κ 3-20 mouse).Table 20 list from the light chain of the antibody of mouse, the number changed from the amino acid (AA) observed in the light chain of the antibody of V κ 1-39 engineered mice (V κ 1-39 mouse) and each FW and the CDR district from the light chain of the antibody of V κ 3-20 engineered mice (V κ 3-20 mouse).Table 21 lists the immune group (antigen E, F and H) for selecting, the number that the amino acid (AA) observed in each FW and CDR district of the heavy chain of the antibody from V κ 1-39 through engineering approaches light chain mouse (V κ 1-39 mouse) changes.Table 22 lists the immune group (antigen E and G) for selecting, the number that the amino acid (AA) observed in each FW and CDR district of the heavy chain of the antibody from V κ 3-20 through engineering approaches light chain mouse (V κ 3-20 mouse) changes.
Table 16
V HFamily VI VI-Vκ1-39 Vκ1-39 VI-Vκ3-20 Vκ3-20
1 9.0 14.8 3.3 7.1 4.9
2 2.2 1.8 4.6 0 1.6
3 77.8 69.8 77.3 61.4 29.5
4 8.4 8.3 11.2 27.1 39.3
5 0.9 0 0.7 4.3 23.0
6 1.7 5.3 3.0 0 1.6
Table 17
Table 18
Table 19
Table 20
Table 21
Table 22
Embodiment 13
There is the binding affinity of the bi-specific antibody of universal light chain
Standard recombinant dna technology as known in the art is used to build complete people's bi-specific antibody from the people variable region of heavy chain of the clone of monospecific antigen E common light chain antibody (being described in embodiment 5) selected.Table 23 lists the pairing of the people's heavy chain (HC-1 and HC-2) from the parent's Mono-specific antibodies selected, and people V κ 1-39/J κ 5 light chain that each pairing adopts germline to reset is for each bi-specific antibody of structure.
At BIACORE tM2000 equipment (GE Healthcare) are upper adopts real-time surface plasma resonance biosensor to detect the combination measuring the extracellular domain (ECD) of dual specific or parent's monospecific antigen E antibody and antigen E.Will by using the CM5BIACORE of EDC-NHS chemical utilization anti-c-myc-monoclonal antibody specific (Clone#9E10) derivatize tMsensor surface is used for the ECD (antigen E-mmh) of C-terminal myc-myc-six histidine mark of capture antigen E.The antigen E-mmh of about 190RU is trapped in BIACORE tMon sensor surface, then inject different dual specifics or parent's monospecific antigen E antibody of 300nM and 50nM concentration with the flow velocity of 50 μ l/min.Test in HBST running buffer (0.01M HEPES pH 7.4,0.15M NaCl, 3mM EDTA, 0.05%v/v tensio-active agent P20) at 25 DEG C.The concentration being recorded in 300nM before antibody injection terminates for 3 seconds is bonded to the amount of the antibody on antigen E-mmh surface and charts.
Table 24 and Fig. 8 list the association reaction (BIACORE of each bi-specific antibody (BsAb) and the monospecific parental antibody (PAb-1, PAb-2) observed tMunit; RU).Due to each antibody inject under saturated condition through identical antigen E-mmh surface, therefore association reaction reflect each antibody being bonded to antigen capture surface in conjunction with stoichiometry.
As shown in this embodiment, the about 2 times of association reactions higher than each parent's Mono-specific antibodies (table 24 and Fig. 8) of association reaction of each bi-specific antibody observed, demonstrate the functional structure using the heavy chain of antigen-specific monoclonal antibody and the bi-specific antibody of common light chain, wherein each Fab arm of bi-specific antibody molecule is simultaneously in conjunction with different epi-positions (the antigen E on the extracellular domain of cell surface receptor; See Fig. 7 B, lower-left).
Table 23
Bi-specific antibody Parent HC-1 Parent HC-2
3108 2952 2978
3109 2978 3022
3111 2952 3005
3112 3022 3005
Table 24

Claims (29)

1. prepare a method for Bispecific antigen binding proteins, comprising:
Obtain to derive from and be separated from two kinds of two kinds of same or different mouse different B cell different people's heavy chain variable region gene sequences, described mouse expresses single people's light variable domains and multiple people's heavy-chain variable domains, and
Prepare Bispecific antigen binding proteins, it has and comprises by two kinds of people's heavy chains of the aminoacid sequence of described two kinds of people's heavy chain variable region genes sequence encoding and the people's light chain comprising described people's light variable domains.
2. method according to claim 1, the described single people's light variable domains origin wherein expressed in described same or different mouse comes from the nucleotide sequence coded of single people germline V section through resetting and the single people germline J section through resetting.
3. method according to claim 2, the wherein said single people germline V section through resetting is the people V κ 3-20 constant gene segment C of people V к 1-39 or the rearrangement of resetting.
4. method according to claim 2, the wherein said single people germline J section through resetting is the people J κ 5 reset or people J к 1 constant gene segment C reset.
5. method according to claim 1, wherein said same or different mouse comprises heavy chain gene seat, and described heavy chain gene seat is containing the one or more people V without resetting that may be operably coupled to one or more non-human heavy chain's constant region gene hconstant gene segment C, one or more people D without resetting hconstant gene segment C and one or more people J without resetting hconstant gene segment C.
6. method according to claim 5, wherein said heavy chain gene seat comprises 80 the people V without rearrangement that may be operably coupled to one or more murine heavy chain constant region gene hconstant gene segment C, 27 people D without rearrangement hconstant gene segment C and 6 people J without rearrangement hconstant gene segment C.
7. method according to claim 5, wherein one or more people V hconstant gene segment C comprises people V h1-2, V h1-8, V h1-24, V h1-69, V h2-5, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-23, V h3-30, V h3-33, V h3-48, V h3-53, V h4-31, V h4-39, V h4-59, V h5-51, V h6-1 or its combination.
8. method according to claim 1, wherein said same or different mouse does not comprise the endogenous light chain variable gene segment can resetting the gene forming coding light variable domains.
9. method according to claim 1, each of wherein said two kinds of people's heavy chain variable region gene sequences comprises and derives from identical or different V hthe sequence of section, and wherein said V hsection is selected from V h1-2, V h1-8, V h1-24, V h1-69, V h2-5, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-23, V h3-30, V h3-33, V h3-48, V h3-53, V h4-31, V h4-39, V h4-59, V h5-51 and V h6-1.
10. method according to claim 1, wherein said two kinds of people's heavy chains combine:
Two kinds of different antigens, or
Two kinds of different epi-positions of same antigen.
11. 1 kinds of methods preparing Bispecific antigen binding proteins, comprising:
First mouse is exposed to the first object antigen comprising the first epi-position, described first mouse is expressed origin and comes from the single immunoglobulin light chain variable structural domain of the nucleic acid sequence encoding of the single people's variable region gene sequence through resetting and multiple people's heavy-chain variable domains;
Second mouse is exposed to the second object antigen comprising the second epi-position, described second mouse is expressed origin and comes from the single immunoglobulin light chain variable structural domain of the nucleic acid sequence encoding of the single people's variable region gene sequence through resetting and multiple people's heavy-chain variable domains;
Described first and second mouse are allowed to produce immunne response for object antigen separately;
The first heavy-chain variable domains of the first epi-position of the first object antigen described in the combination identifying described first mouse;
Second people's heavy-chain variable domains of the second epi-position of the second object antigen described in the combination identifying described second mouse;
Preparation encoded packets contains second complete people's heavy chain gene of the second heavy chain of described second people's heavy-chain variable domains containing first complete people's heavy chain gene of the first heavy chain of described the first heavy-chain variable domains and encoded packets;
Described first and second heavy chain genes are introduced the cell containing the single complete people's light chain gene comprising the single people's variable region gene sequence through resetting;
First and second complete people's heavy chains of being encoded by complete people's heavy chain gene at described cells and complete people's light chain of being encoded by described single complete people's light chain gene, to form Bispecific antigen binding proteins; With
Described Bispecific antigen binding proteins is separated from described cell.
12. methods according to claim 11, wherein said single the people V κ 1-39 or the V κ 3-20 constant gene segment C that comprise rearrangement through resetting people's variable region gene sequence.
13. methods according to claim 12, wherein said single the people J к 1 or J к 5 constant gene segment C that also comprise rearrangement through resetting people's variable region gene sequence.
14. methods according to claim 11, wherein:
Described first antigen and described second antigen are different; Or
Described first antigen and described second antigen are identical, and described first epi-position and described second epi-position are different.
15. methods according to claim 11, first epi-position of wherein said complete people's light chain first antigen described in specific binding when matching with described first complete people's heavy chain, and the second epi-position of wherein said complete people's light chain second antigen described in specific binding when matching with described second complete people's heavy chain.
16. methods according to claim 11, wherein said first or described second complete people's heavy chain has and reduces its amino acid modified to the avidity of albumin A, but described first different with described second complete people's heavy chain time there is described modification.
17. methods according to claim 16, wherein said modification is selected from 95R (EUR435R), 96F (EUR436F) and combination thereof.
18. methods according to claim 11, wherein said single be Germline sequences through resetting people's variable region gene sequence.
19. methods according to claim 11, wherein said first and described second mouse in described multiple people's heavy-chain variable domains by containing the one or more people V without resetting that may be operably coupled to one or more non-human heavy chain's constant region gene hconstant gene segment C, one or more people D without resetting hconstant gene segment C and one or more people J without resetting hthe heavy chain gene seat coding of constant gene segment C.
20. methods according to claim 19, wherein said first or described second mouse in described heavy chain gene seat comprise 80 of may be operably coupled to one or more murine heavy chain constant region gene without the people V reset hconstant gene segment C, 27 people D without rearrangement hconstant gene segment C and 6 people J without rearrangement hconstant gene segment C.
21. methods according to claim 19, wherein said one or more people V hconstant gene segment C comprises people V h1-2, V h1-8, V h1-24, V h1-69, V h2-5, V h3-7, V h3-9, V h3-11, V h3-13, V h3-15, V h3-20, V h3-23, V h3-30, V h3-33, V h3-48, V h3-53, V h4-31, V h4-39, V h4-59, V h5-51, V h6-1 or its combination.
22. selections are used for the method for two kinds of human immunoglobulin heavy chain's variable domains of Bispecific antigen binding proteins, comprising:
With the first object antigen immune first mouse to obtain the first immunoglobulin heavy chain variable structural domain in conjunction with the first antigen, wherein said first mouse expresses the single human normal immunoglobulin light variable domains and multiple human immunoglobulin heavy chain's variable domains that derive from the single people's variable region gene sequence through resetting; With
With the second object antigen immune second mouse to obtain second human immunoglobulin heavy chain's variable domains in conjunction with the second antigen, wherein said second mouse expresses the single human normal immunoglobulin light variable domains and multiple human immunoglobulin heavy chain's variable domains that derive from the single people's variable region gene sequence through resetting.
23. methods according to claim 22, the wherein said single people's variable region gene sequence through resetting comprises people V к 1-39 or the V к 3-20 constant gene segment C of rearrangement.
24. methods according to claim 23, the wherein said single people's variable region gene sequence through resetting also comprises people J к 1 or J к 5 constant gene segment C of rearrangement.
25. methods according to claim 22, wherein said first and described second mouse containing the endogenous light chain variable gene segment forming light chain immunoglobulin can be reset.
26. methods according to claim 22, wherein said first and described second antigen be:
Different; Or
Identical, and described first and described second heavy-chain variable domains in conjunction with different epi-positions.
27. 1 kinds of host cells, it comprises:
A () coding is in conjunction with the first nucleotide sequence of the first heavy chain immunoglobulin of the first antigen, wherein said the first heavy chain comprises the sequence of the first heavy-chain variable domains and nucleotide sequence coded by available from the first mouse with described first antigen immune of the sequence of described the first heavy-chain variable domains;
B () coding is in conjunction with second nucleotide sequence of the second human immunoglobulin heavy chain of the second antigen, wherein said second people's heavy chain comprises the sequence of second people's heavy-chain variable domains and nucleotide sequence coded by available from the second mouse with described second antigen immune of the sequence of described second people's heavy-chain variable domains
Wherein said first and described second mouse express by the single human normal immunoglobulin light variable domains of the single human normal immunoglobulin chain variable region gene sequence encoding through resetting and multiple human immunoglobulin heavy chain's variable domains separately; With
C () comprises the 3rd nucleotide sequence of the described single human normal immunoglobulin chain variable region gene sequence through resetting, wherein said 3rd nucleic acid encoding human normal immunoglobulin light chain;
Wherein said first, second, and third nucleotide sequence is expressed with the antigen-binding proteins produced in conjunction with described first and described second antigen.
28. host cells according to claim 27, the wherein said single human normal immunoglobulin chain variable region gene sequence through resetting comprises people V к 1-39 or the V к 3-20 constant gene segment C of rearrangement.
29. host cells according to claim 28, the wherein said single human normal immunoglobulin chain variable region gene sequence through resetting also comprises people J к 1 or J к 5 constant gene segment C of rearrangement.
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