CN106831995A - Novel bispecific antibodies and application thereof - Google Patents
Novel bispecific antibodies and application thereof Download PDFInfo
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Abstract
This application provides bispecific human IgG1's antibody, it includes two kinds of Fc fragments with identical hinge area, and the amino acid sequence of the hinge area is SEQ ID NO:1 or SEQ ID NO:28 and the 216th 230 bit sequence of natural human IgG1 antibody constant regions is substituted for, antibody constant region amino acid position determines according to EU numbering.Additionally, present invention also provides the purposes of the bispecific antibody.
Description
Technical field
The application relates generally to antibody drug field, specifically, this application provides novel bispecific antibodies and its
Medical science and biological applications.
Background technology
Bispecific antibody (bispecific antibody, BsAb) is a class artificial antibody, its include two it is different
Antigen binding site.Bispecific antibody is widely used in biomedicine field, especially immunotherapy of tumors aspect.
Bispecific antibody can be divided into dual signal blocking-up type and mediated cell functional form from mechanism of action.Generally, it is situated between
Guided cell functional form bispecific antibody refers to the AntiCD3 McAb bispecific antibody that mediate T cell is killed.1985, killed using T cell
The concept of dead tumour cell has just been suggested (Stearz at al.Nature 1985,314:628-631).It has been generally acknowledged that effectively
Activation T cell needs dual signal, and the first signal is from MHC- antigenic compounds on antigen presenting cell and φt cell receptor TCR-
The combination of CD3, secondary signal is the non-antigen produced after T cell interacts with the costimulatory molecules of antigen presenting cell expression
Specific costimulatory signal.Lowered due to the expression of most cancer cell surfaces MHC and even lacked, so that cancer cell escapes immune
Kill.Target CD3 bispecific antibody then can respectively in connection with T cell surface C D3 molecules and cancer cell surface antigens so that
Further cytotoxic T cell (cytotoxic T cell, Tc or CTL) and the distance of cancer cell, guides T cell direct killing cancer
Cell, and it is no longer dependent on the dual activation signal of T cell.Bispecific antibody can be three function antibodies, i.e. one arm target
To the antigen on tumour cell, the CD3 molecules of another arm targeting T-cells, Fc sections combines Fc acceptors.This antibody causes that T is thin
The effector cell of born of the same parents, tumour cell and binding antibody Fc domains formed complex (Muller and Kontermann,
BioDrugs2010;24:89-98).
CD3 molecules have δ, ε, γ, ζ totally 4 subunits, molecular weight be respectively 18.9kDa, 23.1kDa, 20.5kDa,
18.7kDa, respectively including 171,207,182 and 164 amino acid residues.6 polypeptide chains of four kinds of subunit compositions are thin with T
Born of the same parents' acceptor (T cell receptor, TCR) is combined closely, and is formed comprising 8 TCR-CD3 compounds of polypeptide chain, the compound
Conduction t cell activation signal, stabilization TCR structures.The intracellular part of CD3 contains immunity receptor Tyrosine Activating Motifs
(immunoreceptor tyrosine-based activation motif, ITAM), TCR is recognized and is in reference to MHC molecule
The Antigenic Peptide passed so that the tyrosine residue of ITAM in the LCK p561ck phosphorylation CD3 molecules in T cell,
The LCK (such as ZAP-70) containing SH2 (Scr homology 2) domain is raised afterwards.ITAM phosphorylations and
It is one of important biochemical reaction of t cell activation early signal conductive process with reference to ZAP-70.Therefore, CD3 molecules have conduction TCR
The function of the activation signal that identification antigen is produced.
Because early stage is in the deficiency of the aspects such as immunogenicity, structural stability and antibody mass control, double spies are limited
The further development of heterogenetic antibody.In recent years, the improvement of upstream gene engineered antibody and downstream production Technology, overcomes biography
The defect of system bispecific antibody, so as to promote multiclass novel bispecific antibodies to enter clinical development.In order to solve
Two different incomplete antibodies are carried out the problem of correct assembling, the bispecific antibody of various structures has been designed and developed.
One class bispecific antibody is free of Fc areas.The advantage of this kind of structure Antibodies is small molecular weight, can be in prokaryotic
Middle expression, it is not necessary to consider the problem of correct assembling;Have the disadvantage due to no antibody Fc section, it is impossible to mediate corresponding biology work(
Can, and half-life short, thus clinical practice is subject to a definite limitation.Currently reported such bispecific antibody includes
BiTE, DART, TrandAbs, bi-Nanobody etc..BiTE (the bispecific T-cell of German Micromet companies exploitation
Engager) series of products are that AntiCD3 McAb scfv is attached into acquisition by peptide fragment from different antitumor cell surface antigen scfv
, can be in combination with CD3+T cell and tumour cell.This antibody-like overcomes that stability is poor, expression quantity is low, the low life of dissolubility
Product problem, wherein Blinatumomab are successfully listed.
Another kind of bispecific antibody retains antibody Fc domain.This antibody-like forms IgG spline structures, with Fc mediations
Biological function.Currently reported such bispecific antibody includes Triomabs, kih IgG, Cross-mab, ortho
Fab IgG, DVD IgG, IgG scFv, scFv2-Fc etc..These have Half-life in vivo comprising Fc sections of bispecific antibody
The advantages of growing, ADCC and CDC can be mediated.
Good application is preceding by having in oncotherapy for bispecific antibody (for example targetting CD3 and TSA)
Scape.
The content of the invention
In a first aspect, the application provides bispecific human IgG1's antibody, it includes two kinds of Fc pieces with identical hinge area
Section, the amino acid sequence of the hinge area is SEQ ID NO:1 or SEQ ID NO:28 and to substituted for natural human IgG1 antibody permanent
Determine the 216-230 bit sequences in area, antibody constant region amino acid position determines according to EU numbering.
In some embodiments, two kinds of Fc fragments include the mutation for being able to ensure that the different dimerization of heavy chain.In some embodiment party
In case, a Fc fragment includes point mutation S354C and T366W, and another Fc fragment includes point mutation Y349C, T366S, L368A
And Y407V.
In some embodiments, two kinds of single-chain antibodies of the N-terminal difference fusion recognition difference epitope of Fc fragments
(scfv)。
In some embodiments, a kind of N-terminal of Fc fragments merges single-chain antibody (scfv), the N-terminal of another Fc fragments
Fusion Fab fragments, the single-chain antibody epitope different from the identification of Fab fragments.
In some embodiments, two kinds of Fab fragments of the N-terminal difference fusion recognition difference epitope of Fc fragments.
In some embodiments, recognize that the Fab fragments of different epitopes include identical light chain.
In some embodiments, a kind of Fab fragments include natural human IgG1's antibody constant region CH1 fragments, another
Human IgG1 antibody constant region CH1 fragment of the Fab fragments comprising mutation, the human IgG1 antibody constant region CH1 fragments of mutation include point
Any 1,2 or 3 in mutation G137E, N203D and R214T, the wherein amino acid sequence of CH1 fragments is antibody constant
The amino acid sequence that area is 118-215.In some embodiments, the ammonia of natural human IgG1's antibody constant region CH1 fragments
Base acid sequence is SEQ ID NO:2.In some embodiments, the amino acid of human IgG1's antibody constant region CH1 fragments of mutation
Sequence is SEQ ID NO:30.
In some embodiments, point mutation L234F, L235E and P331S are included in two kinds of CH2 fragments of Fc fragments,
Wherein the amino acid sequence of CH2 fragments is the amino acid sequence of antibody constant region 231-340.In some embodiments,
The amino acid sequence of two kinds of CH2 fragments of Fc fragments is SEQ ID NO:31.
In some embodiments, bispecific human IgG1 antibody recognition people immune cell surface antigenic and tumour cell table
Face antigen.
In some embodiments, people's immune cell surface antigenic is CD3E.
In some embodiments, the TSA be selected from HER1, HER2, HER3, EpCAM, CEA, PSMA,
CD19, CD20, CD22, CD38 and BCMA.
Second aspect, this application provides the drug regimen comprising the bispecific human IgG1's antibody described in first aspect
Thing.
In some embodiments, pharmaceutical composition is also comprising pharmaceutically acceptable carrier, excipient, diluent etc..
The third aspect, this application provides the bispecific human IgG1's antibody described in first aspect, or second aspect institute
Purposes of the pharmaceutical composition stated in the medicine for being used for preventing or treat tumour is prepared.
Fourth aspect, this application provides prevention or the method for the treatment of tumour, including gives first to individuality in need
The pharmaceutical composition described in bispecific human IgG1 antibody or second aspect described in aspect.
The 3rd or fourth aspect some embodiments in, targeted swollen of tumour expression bispecific human IgG1's antibody
Knurl surface antigen.
The 3rd or fourth aspect some embodiments in, tumour be selected from breast cancer, stomach cancer, colorectal cancer, prostate
Cancer, cancer of pancreas, leukaemia, Huppert's disease and malignant lymphoma.
Brief description of the drawings
Four kinds of structural representations of different structure bispecific antibody that Fig. 1 a- Fig. 1 d are designed for the application.
Fig. 2 shows and detect various bispecific antibodies in combination with two kinds of antigens of CD3E and HER2 using ELISA method
Result.
Fig. 3 is shown using the various bispecific antibodies of flow cytometry and MDA-MB-453 human breast cancer cell tables
The result that face HER2 is combined.
Fig. 4 shows the result combined with PBMC surface Cs D3 using the various bispecific antibodies of flow cytometry.
Fig. 5 a-5g show lethal effect result of the different bispecific antibodies to tumour cell, and wherein Fig. 5 a show
Mortaility results of the bispecific antibody to HER2 positive cells;Fig. 5 b show that bispecific antibody is killed to HER2 negative cells
Hinder result;Fig. 5 c- Fig. 5 e show influence of the different hinge plot structures of bispecific antibody to killing activity;Fig. 5 f and Fig. 5 g
The influence to killing activity is designed for the different antigen-binding portions of bispecific antibody.
Fig. 6 a-6c show different Mediated by Bi-specific Antibodies HER2 positive target cells, and (MDA-MB-453 human breast carcinomas are thin
Born of the same parents) activation to T cell, wherein Fig. 6 a and Fig. 6 b show different bispecific antibodies activation T cells expression early activation marks
The result of will molecule CD69;Fig. 6 c show that different bispecific antibody inducing T cells produce the result of cell factor IL-2.
Sequence explanation
SEQ ID NO:The amino acid sequence of 1 hinge area included for the Exemplary bispecific antibodies of the application, it is
The hinge area of natural human IgG2's antibody.
SEQ ID NO:2 is the amino acid sequence of natural human IgG1's antibody constant region CH1 fragments.
SEQ ID NO:3 is the amino acid sequence of the HCDR1 of the exemplary anti-human CD3E monoclonal antibodies of the application.
SEQ ID NO:4 is the amino acid sequence of the HCDR2 of the exemplary anti-human CD3E monoclonal antibodies of the application.
SEQ ID NO:5 is the amino acid sequence of the HCDR3 of the exemplary anti-human CD3E monoclonal antibodies of the application.
SEQ ID NO:6 is the exemplary anti-human CD3E monoclonal antibodies and anti-HER 2 monoclonal antibody of the application
The amino acid sequence of LCDR1.
SEQ ID NO:7 is the exemplary anti-human CD3E monoclonal antibodies and anti-HER 2 monoclonal antibody of the application
The amino acid sequence of LCDR2.
SEQ ID NO:8 is the exemplary anti-human CD3E monoclonal antibodies and anti-HER 2 monoclonal antibody of the application
The amino acid sequence of LCDR3.
SEQ ID NO:9 is the amino acid sequence of the HCDR1 of the exemplary anti-HER 2 monoclonal antibody of the application.
SEQ ID NO:10 is the amino acid sequence of the HCDR2 of the exemplary anti-HER 2 monoclonal antibody of the application.
SEQ ID NO:11 is the amino acid sequence of the HCDR3 of the exemplary anti-HER 2 monoclonal antibody of the application.
SEQ ID NO:12 is the amino acid sequence of the weight chain variable district of the exemplary anti-human CD3E monoclonal antibodies of the application
Row.
SEQ ID NO:13 for the application exemplary anti-human CD3E monoclonal antibodies and anti-HER 2 monoclonal antibody it is light
The amino acid sequence of chain variable region.
SEQ ID NO:14 is the amino acid sequence of the weight chain variable district of the exemplary anti-HER 2 monoclonal antibody of the application.
SEQ ID NO:15 is arm (the anti-HER2 containing anti-HER2 scfv of the Exemplary bispecific antibodies of the application
Scfv-Fc fusion proteins) amino acid sequence.
SEQ ID NO:16 is (anti-human for the arm containing anti-human CD3E scfv of the Exemplary bispecific antibodies of the application
CD3E scfv-Fc fusion proteins) amino acid sequence.
SEQ ID NO:17 is the heavy chain moiety of the arm containing anti-HER2 Fab of the Exemplary bispecific antibodies of the application
Amino acid sequence.
SEQ ID NO:18 for the application Exemplary bispecific antibodies containing anti-HER2 Fab or containing anti-human CD3E
The amino acid sequence of the chain moiety of the arm of Fab.
SEQ ID NO:19 is the heavy chain portion of the arm containing anti-human CD3E Fab of the Exemplary bispecific antibodies of the application
The amino acid sequence for dividing.
SEQ ID NO:20 is the heavy chain moiety of the arm containing anti-HER2 Fab of the Exemplary bispecific antibodies of the application
Amino acid sequence.
SEQ ID NO:21 is arm (the anti-HER2 containing anti-HER2 scfv of the Exemplary bispecific antibodies of the application
Scfv-Fc fusion proteins) amino acid sequence.
SEQ ID NO:22 is (anti-human for the arm containing anti-human CD3E scfv of the Exemplary bispecific antibodies of the application
CD3E scfv-Fc fusion proteins) amino acid sequence.
SEQ ID NO:23 is the heavy chain moiety of the arm containing anti-HER2 Fab of the Exemplary bispecific antibodies of the application
Amino acid sequence.
SEQ ID NO:24 is the heavy chain portion of the arm containing anti-human CD3E Fab of the Exemplary bispecific antibodies of the application
The amino acid sequence for dividing.
SEQ ID NO:25 is the heavy chain moiety of the arm containing anti-HER2 Fab of the Exemplary bispecific antibodies of the application
Amino acid sequence.
SEQ ID NO:26 is the heavy chain moiety of the arm containing anti-HER2 Fab of the Exemplary bispecific antibodies of the application
Amino acid sequence.
SEQ ID NO:27 is the heavy chain portion of the arm containing anti-human CD3E Fab of the Exemplary bispecific antibodies of the application
The amino acid sequence for dividing.
SEQ ID NO:The amino acid sequence of 28 hinge areas included for the Exemplary bispecific antibodies of the application, it is
The variant of the hinge area of natural human IgG2's antibody.
SEQ ID NO:The amino acid sequence of 29 hinge areas included for the Exemplary bispecific antibodies of the application, it is
The hinge area of natural human IgG1's antibody.
SEQ ID NO:The ammonia of the CH1 fragments in the 30 Fab fragments included for the Exemplary bispecific antibodies of the application
Base acid sequence.
SEQ ID NO:The amino of the CH2 fragments in the 31 Fc fragments included for the Exemplary bispecific antibodies of the application
Acid sequence.
SEQ ID NO:32 is the amino acid sequence of people CD3E extracellular regions (CD3E).
SEQ ID NO:33 is the amino acid sequence of people CD3D extracellular regions (CD3D).
SEQ ID NO:34 is the amino acid sequence of monkey CD3E extracellular regions (mfCD3E).
SEQ ID NO:35 is the amino acid sequence of monkey CD3D extracellular regions (mfCD3D).
SEQ ID NO:36 is the amino acid sequence of mouse CD3E extracellular regions (mCD3E).
SEQ ID NO:37 is the amino acid sequence of mouse CD3D extracellular regions (mCD3D).
SEQ ID NO:38 is the amino acid sequence of HER2 extracellular regions D1D2D3 parts (HER2).
SEQ ID NO:39 is the amino acid sequence of His labels.
SEQ ID NO:40 is the amino acid sequence of the Fc fragments (mFc) of mouse IgG antibody 2a.
SEQ ID NO:41 is the amino acid sequence of the Fc fragment variants (FcK) of human IgG1's antibody.
SEQ ID NO:42 is the amino acid sequence of the Fc fragment variants (FcH) of human IgG1's antibody.
SEQ ID NO:43 is the amino acid sequence of natural human IgG1's heavy chain constant region.
SEQ ID NO:44 is the amino acid sequence of the variant (IgG1Hn) of human IgG1's heavy chain constant region.
SEQ ID NO:45 is the amino acid sequence of the variant (IgG1Kn) of human IgG1's heavy chain constant region.
SEQ ID NO:46 is the amino acid sequence of the variant (IgG1Hn-m3) of human IgG1's heavy chain constant region.
SEQ ID NO:47 is the amino acid sequence of the variant (IgG1kn-m3) of human IgG1's heavy chain constant region.
SEQ ID NO:48 is the amino acid sequence of the variant (IgG1H3n-m3) of human IgG1's heavy chain constant region.
SEQ ID NO:49 is the amino acid sequence of the variant (IgG1kn1-m3) of human IgG1's heavy chain constant region.
SEQ ID NO:50 is the amino acid sequence of the variant (IgG1H3n1-m3) of human IgG1's heavy chain constant region.
SEQ ID NO:51 is the amino acid sequence of human IgG1's antibody κ hypotype constant region of light chain.
Detailed description of the invention
Definition
Except as otherwise noted, term use herein has the implication that those skilled in the art are generally understood.
When antibody structure is described herein, it is related to the description of amino acid position number with reference to the EU of human IgG1's antibody
Numbering is defined, and this is as well known to those skilled in the art and easily inquires.Additionally, herein in connection with EU
Refer to the mutation produced relative to natural antibody sequences when numbering location expressions are mutated.
Terms used herein " Fc fragments ", " Fc domains ", " Fc parts " or similar term refer to constant heavy chain of antibody
The part in area, including hinge area (hinge), constant region CH2 fragments and CH3 fragments.With reference to the EU of human IgG1's antibody
Numbering is defined, and Fc fragments are the amino acid sequences of 216-447 in antibody constant region.
Terms used herein " Fab (fragment antigen binding) fragment ", " Fab parts " or similar term
Refer to produce after complete antibody Papain ferment treatment can be with the antibody fragment of antigen binding, including complete light chain
(VL-CL), weight chain variable district and CH1 fragments (VH-CH1).
Terms used herein " single-chain antibody (scfv, single chain fragment variable) " refers to general profit
The antibody of the single-stranded structure built with technique for gene engineering, comprising weight chain variable district (VH) and more than of light chain variable district (VL)
Peptide chain.One section of connection peptide (linker) of flexibility would generally be designed between weight chain variable district and light chain variable district so that heavy chain can
Becoming area and light chain variable district can be folded into the correct conformation that can combine antigen.
Terms used herein " bispecific antibody " is that have the antibody for combining two kinds of different antigenic capacities, and it can be by two
Individual Fc fragments and two antigen-binding portions for being merged with it respectively are grouped into, and each antigen-binding portion thereof can be Fab fragments
Form or single-chain antibody form.
Terms used herein " bispecific human IgG1 antibody " refers to the bispecific antibody based on human IgG1's antibody, and
In addition to herein described change structure, its essential characteristic and function for possessing human IgG1's antibody.
In some embodiments of this paper, bispecific antibody is described as have two " arm ", for example, in Fig. 1 a-
In four kinds of structures shown in Fig. 1 d, with centre as boundary, bispecific antibody can be divided into two arms.The arm of bispecific antibody
Can be made up of Fc fragments and antigen-binding portion thereof (Fab fragments or single-chain antibody).For what is be made up of Fc fragments and Fab fragments
Arm, its structure contains complete heavy chain and light chain similar to common antibody.
As well known to those skilled in the art, complementary determining region (CDR generally has CDR1, CDR2 and CDR3) is right in variable region
The maximum region of the affinity and specific effect of antibody.The CDR sequence of VH or VL has two kinds of common definition modes, i.e.,
Kabat is defined and Chothia definition.(refer to such as Kabat, " Sequences of Proteins of Immunological
Interest ", National Institutes of Health, Bethesda, Md. (1991);A1-Lazikani et
Al., J.Mol.Biol.273:927-948(1997);And Martin et al., Proc.Natl.Acad.Sci.USA86:
9268-9272(1989)).For give antibody variable region sequences, can according to Kabat definition or Chothia definition come
Determine CDR region sequence in VH and VL sequences.In the embodiment of the application, CDR sequence is defined using Kabat.
For the variable region sequences for giving antibody, CDR region sequence, example in variable region sequences can be in several ways analyzed
(http such as can be determined using online software Abysis://www.abysis.org/).
Term " specific binding " as used herein, refers to the nonrandom association reaction between two molecules, such as antibody
To the combination of epitope.
In a first aspect, bispecific human IgG1's antibody, it includes two kinds of Fc fragments with identical hinge area, the hinge
The amino acid sequence in area is SEQ ID NO:1 or SEQ ID NO:28 and substituted for the 216- of natural human IgG1 antibody constant regions
230 bit sequences, antibody constant region amino acid position determines according to EU numbering.
SEQ ID NO:1 is the hinge area of natural human IgG2's antibody, SEQ ID NO:28 is natural human IgG2's antibody
Hinge area variant.Present inventor is by research it has been unexpectedly found that drawing in bispecific human IgG1's antibody
Entering the hinge area of human IgG2's antibody can improve some functions and property of bispecific antibody.
When the bispecific antibody for retaining antibody Fc domain is built, can be from following two orientation optimization bispecifics
The structure of antibody:One is the different dimerization of heavy chain, and two is the correct assembling of light chain and heavy chain.In some embodiments, two kinds of Fc pieces
Section includes the mutation for being able to ensure that the different dimerization of heavy chain.KIH technologies (knob-in-hole, KIH) are solve the different dimerization of heavy chain one
Plant strategy.Generally, KIH technologies refer to the amino acid sequence by transforming CH3 areas, are formed with and are mutually paired beneficial to xenogenesis incomplete antibody
Structure, can constitute bispecific antibody while again as much as possible keep normal antibody structure.In some embodiment party
In case, the KIH technologies for being utilized include, a Fc fragment is included point mutation S354C and T366W, and another Fc fragment is included
Point mutation Y349C, T366S, L368A and Y407V.Guidance on KIH technologies, see, for example, " An efficient
Route to human bispecific IgG ", A.Margaret Merchant et al., Nature
, be incorporated by the document herein by reference by Biotechnology, Volume16,1998.
In some embodiments, two kinds of single-chain antibodies of the N-terminal difference fusion recognition difference epitope of Fc fragments
(scfv)。
In some embodiments, a kind of N-terminal of Fc fragments merges single-chain antibody (scfv), the N-terminal of another Fc fragments
Fusion Fab fragments, the single-chain antibody epitope different from the Fab fragments identification.
In some embodiments, two kinds of Fab fragments of the N-terminal difference fusion recognition difference epitope of Fc fragments.By
The structure of this bispecific antibody for being formed, close to natural antibody, is also a kind of preferred embodiment.
In some embodiments, recognize that the Fab fragments of different epitopes include identical light chain.The embodiment has
It is also a kind of preferred embodiment beneficial to the correct assembling of light chain and heavy chain.
In some embodiments, a kind of Fab fragments include natural human IgG1's antibody constant region CH1 fragments, another
Human IgG1 antibody constant region CH1 fragment of the Fab fragments comprising mutation, the human IgG1 antibody constant region CH1 fragments of mutation include point
Any 1,2 or 3 in mutation G137E, N203D and R214T, the wherein amino acid sequence of CH1 fragments is antibody constant
The amino acid sequence that area is 118-215.In some embodiments, the ammonia of natural human IgG1's antibody constant region CH1 fragments
Base acid sequence is SEQ ID NO:2.In some embodiments, the amino acid of human IgG1's antibody constant region CH1 fragments of mutation
Sequence is SEQ ID NO:30.
Without intending to be bound to any theory, above-mentioned mutation is introduced in a kind of Fab fragments can not change antibody constant region knot
On the premise of structure/function and immunogenicity, change the charge characteristic (isoelectric point, pI) of the heavy chain containing the mutation, Jin Eryou
Beneficial to the later-period purification of bispecific antibody.137th, 203 and 214 these three sites are located at the hydrophilic area (domain of CH1 domains
Surface), mutation is carried out to it will not change the conformation of CH1, and involved mutation is that basic amino acid is changed into neutral amino
Sour (such as R214T), or neutral amino acid is changed to acidic amino acid (such as G137E, N203D), such mutation can cause to contain
The isoelectric point (pI) for having this heavy chain of mutation declines, and less than another unmutated isoelectric point of heavy chain, this is conducive to the later stage
Purpose bispecific antibody is had with such as issuable homopolymer of KIH technologies using isoelectric point difference in purge process
The separation of effect.And, above-mentioned mutation is also naturally occurring in the antibody (such as IgG2, IgG4) of other hypotypes, it is contemplated that will not cause bright
Aobvious Immunogenicity.
In some embodiments, point mutation L234F, L235E and P331S are included in two kinds of CH2 fragments of Fc fragments,
Wherein the amino acid sequence of CH2 fragments is the amino acid sequence of antibody constant region 231-340.In some embodiments,
The amino acid sequence of two kinds of CH2 fragments of Fc fragments is SEQ ID NO:31.
Above-mentioned mutation is introduced in CH2 fragments can reduce the antibody-dependent cytotoxicity effect of antibody Fc section mediation
(ADCC), consequently, it is possible to reducing bispecific antibody caused side effect in vivo.Guidance on above-mentioned mutation, for example, can join
See " The binding affinity of human IgG for its high affinity Fc receptor is
determined by multiple amino acids in the CH2domain and is modulated by the
Hinge region ", Stephen M.Canfield et al., J.Exp.Med.Volume 173,1991, by what is quoted
Be incorporated by the document herein by mode.
In some embodiments, bispecific human IgG1 antibody recognition people immune cell surface antigenic and tumour cell table
Face antigen.
In some embodiments, people's immune cell surface antigenic is CD3E.
AntiCD3 McAb E antibody can combine the CD3E subunits in the TCR receptor complexes of T cell surface, using the teaching of the invention it is possible to provide t cell activation
The first signal (being attached to TCR similar to the MHC- peptide complexes on antigen presenting cell), be conducive to the activation of T cell.And
And comprising the bispecific antibody of the antigen-binding portion for CD3E, it is possible to achieve T cell tumour cell periphery enrichment,
Improve killing-efficiency of the T cell to tumour cell.
In some embodiments, the TSA be selected from HER1, HER2, HER3, EpCAM, CEA, PSMA,
CD19, CD20, CD22, CD38 and BCMA.
HER2 genes are located at people's 17q21 chromosomes, and the transmembrane protein of coding molecule amount 185kD, the albumen has tyrosine-kinase
Enzymatic activity, with inactive form presence under normal condition, participates in regulation cell normal differentiation, is generally only expressed in infancy, into
People low expression level in minority tissue.HER2 genes are double copy genes in normal cell, are activated by gene mutation,
Its amplification causes transcriptional upregulation, protein expression increase.HER2 is human epidermal growth factor acceptor (epidermal growth
Factor receptor, EGFR) family second member, belong to I receptor family tyrosine kinases, many normal and
Play important adjustment effect in the growth of abnormal epidermal cell, differentiation and metabolic process, the generation of kinds of tumors, development and disease
State is all related to HER2.The family has 4 kinds of acceptors, is respectively designated as HER1, HER2, HER3 and HER4, and these receive body phase
Interaction forms homologous or heterodimer, especially based on HER2 heterodimers, weight is played during the signal transduction of cell
Act on.HER2 activation suppresses apoptosis of tumor cells, promotes tumor cell proliferation;VEGF/VPF is raised, accelerates tumor vessel life
Into, promoting Nasopharyngeal neoplasms, anti-invasion function (Artufel MV, Valero A C, Llado R R, et are organized in destruction
Al.Clin Transl Oncol, 2005,7. (11):504-511).HER2 protein overexpressions to Cell differentiation inducing activity, propagation and
Conversion and promotion metastases, invasion and attack and adhesion play an important roll (Hynes N E, Stem D F.Biochem
Biophys AcTa, 1994,1198 (2-3):165-184.).Due to the overexpression in 20% breast cancer case, and with not
Good prognosis is related, therefore effects of the HER2 in breast cancer especially attracts attention (Reese et al., Stem Cells 1997;
15:1-8;Andrechek et al., Proc Natl Acad Sci USA 2000;97:3444-3449.Slamon et
Al., Science 1987;235:177-182).
CEA (carcinomebryonic antigen, carcino-embryonic antigen) is a kind of glycoprotein that Colorectal Carcinoma is produced,
The immune response of patient can be caused as antigen.Its Alimentary System for being widely present in endoderm origin, exists in normal
In the digestion tubing of embryo, can also there is micro presence in normal human serum.Carcinomebryonic antigen is a broad spectrum activity tumor-marker
Thing, it can reflect the presence of kinds of tumors, Outcome measure, disease development to colorectal cancer, breast cancer and lung cancer, prison to people
Survey and prognosis estimates it is a preferable tumor markers.
PSMA is the II type membrane-spanning proteins of 110kDa, and the assignment of genes gene mapping is expressed in normal prostatic on the disconnected arm 11q of chromosome
In epithelium and prostate tumor cells, wherein the expression quantity in tumour cell is raised, there is research to think that PSMA take part in prostatitis
The regulation and control of cell migration in gland cancer generating process.The expression of PSMA and the rank of tumour and with the presence or absence of hormone in prostate cancer
Resistance is proportionate, and the strongly expressed of PSMA means recurrence rate higher.Now think that PSMA is one of prostate cancer reference frame.
CD19 is the surface protein for being expressed in bone-marrow-derived lymphocyte and dendritic cells,follicular, belongs to immunoglobulin (Ig) and surpasses
Family member, on No. 16 the short arm of a chromosome (16p11.2), encodes 556 I type transmembrane glycoproteins of amino acid, molecular weight
95KD.CD19 is only expressed in normal and malignant B cell, is expressed hardly in its hetero-organization;Secondly, CD19 is disliked in B cell
Do not lost in property conversion process, refractory/recurrent case is still effective;Furthermore, CD19 is in candidate stem cell and pro-B cells
Do not express, after treatment stops, B cell can be supplemented effectively.The various leukemia treatings of CD19 are thus targetted in recent years
Strategy obtains preferable clinical effectiveness, including the CAR-T of CD3+CD19 bispecific antibodies and targeting CD19 etc..
CD20 antigens are a kind of B cell differentiation antigens, are only expressed in pre B cell and mature B cell surface, it 95% with
On B cell lymthoma in express, and do not expressed in candidate stem cell, plasma cell and other normal structures.Targeting
Multiple monoclonal antibodies (such as Rituxan) of CD20 have clinically been successfully used in the treatment of various lymthomas.
CD38 is the glycoprotein being positioned on film, catalysis cADPR (cADPR, cyclic ADP-
Ribose synthesis and degraded).CD38 is a single transmembrane glycoprotein of 45kDa, and overall structure is divided into the short kytoplasm of N-terminal
Tail, single pass transmembrane domain and C-terminal extracellular region long.In adult, CD38 is single in most of NKs, T cell, B cell
Nucleus/macrophage.Also there is a certain degree of expression on blood platelet and red blood cell.Target the monoclonal antibody of CD38
(daratumumab) treatment for Huppert's disease is had been approved by.
BCMA (B cell maturation antigen, B-cell maturation antigen) is made up of 185 amino acid residues
Type III transmembrane protein.It belongs to TNF receptor family members, with its part B cell activity factor BAFF or proliferation-inducing ligand
APRIL combinations can stimulate B cell hyperplasia.BCMA normal expressions in ripe B cell and thick liquid cell, at Huppert's disease (MM)
In also have extensive expression, be an ideal immunotherapeutic targets of Huppert's disease.
Second aspect, this application provides the drug regimen comprising the bispecific human IgG1's antibody described in first aspect
Thing.
In some embodiments, pharmaceutical composition is also comprising pharmaceutically acceptable carrier, excipient, diluent etc..
In some embodiments, pharmaceutical composition is used to treat tumour, for example, express bispecific human IgG1 antibody institute
For TSA tumour.
In some embodiments, pharmaceutical composition can also include lubricant, such as talcum powder, magnesium stearate and mineral oil;
Wetting agent;Emulsifying agent;Suspending agent;Preservative, such as benzoic acid, sorbic acid and calcium propionate;Sweetener and/or flavor enhancement etc..
In some embodiments, the pharmaceutical composition in the application can be formulated as tablet, pill, pulvis, lozenge, the wine made of broomcorn millet
The forms such as agent, suspension, emulsion, solution, syrup, suppository or capsule.
In some embodiments, it is possible to use any physiologically acceptable administering mode delivers the medicine group of the application
Compound, these administering modes are included but is not limited to:Oral administration, parenteral, nose administration, rectally, intraperitoneal are given
Medicine, intravascular injection, subcutaneous administration, percutaneous dosing, inhalation etc..
In some embodiments, the reagent of purity is pharmaceutically acceptable with optionally needed for can having by mixing
Carrier, excipient etc., the pharmaceutical composition of therapeutical uses is formulated in the form of lyophilized formulations or the aqueous solution to be used to store.
The third aspect, this application provides the bispecific human IgG1's antibody described in first aspect, or second aspect institute
Purposes of the pharmaceutical composition stated in the medicine for being used for preventing or treat tumour is prepared.
Fourth aspect, this application provides prevention or the method for the treatment of tumour, including gives first to individuality in need
The pharmaceutical composition described in bispecific human IgG1 antibody or second aspect described in aspect.
The 3rd or fourth aspect some embodiments in, targeted swollen of tumour expression bispecific human IgG1's antibody
Knurl surface antigen.
The 3rd or fourth aspect some embodiments in, tumour be selected from breast cancer, stomach cancer, colorectal cancer, prostate
Cancer, cancer of pancreas, leukaemia, Huppert's disease and malignant lymphoma.
It should be appreciated that it is discussed in detail above only for making those skilled in the art more clearly understand present context,
And be not intended to be any limitation as in any way.Those skilled in the art can carry out various changes and change to the embodiment
Change.
Following examples are merely to illustrate and the purpose of unrestricted the application scope.
Embodiment
The preparation and confirmation of the monoclonal antibody of embodiment 1
As an example of bispecific human IgG1's antibody of displaying the application, it is prepared for targeting people CD3E and HER2
Bispecific human IgG1's antibody.
Used as the basis for preparing bispecific human IgG1's antibody, present inventor is prepared for for people CD3E first
With two kinds of monoclonal antibodies of HER2, the process needed using various different recombinant proteins, including recombined human CD3E's is extracellular
Area (CD3E, SEQ ID NO:32), people CD3D- extracellular regions (CD3D, SEQ ID NO:33), monkey CD3E extracellular regions (mfCD3E,
SEQ ID NO:34), monkey CD3D extracellular regions (mfCD3D, SEQ ID NO:35), mouse CD3E extracellular regions (mCD3E, SEQ ID
NO:36), mouse CD3D extracellular regions (mCD3D, SEQ ID NO:37), restructuring HER2 extracellular regions D1D2D3 parts (HER2, SEQ
ID NO:38).In natural environment, CD3E and CD3D forms heterodimer, therefore in order to prepare the CD3E of native conformation
Antigen, we express CD3E and CD3D simultaneously, and CD3D and CD3E are formed into heterologous using FcK+FcH heterodimerics body method
Dimer, as the antigen in this research.These recombinant proteins have substantial amounts of posttranslational modification (such as:Glycosylation or disulfide bond
Deng), thus would be even more beneficial to keep the 26S Proteasome Structure and Function of recombinant protein using mammalian cell expression system.Additionally, in order to
Convenient purifying, the recombinant protein of non-antibody class with the addition of His-tag (SEQ ID NO in C-terminal:, or mouse IgG antibody 2a 39)
Fc sections of (mFc, SEQ ID NO:40).When prepared by recombinant antibodies, heavy chain constant region can come from human IgG1's antibody (SEQ ID
NO:43), or be the various mutant of IgG1 constant regions, such as:IgG1Hn(SEQ ID NO:44), IgG1Kn (SEQ ID NO:
45), IgG1Hn-m3 (SEQ ID NO:46), IgG1kn-m3 (SEQ ID NO:47), IgG1H3n-m3 (SEQ ID NO:48),
IgG1kn1-m3(SEQ ID NO:49), IgG1H3n1-m3 (SEQ ID NO:50), constant region of light chain is kappa hypotypes (SEQ
ID NO:51).
The amino acid sequence of the various purpose recombinant proteins according to Uniprot databases, designs and synthesizes above-mentioned various heavy
The gene (including His-tag, mFc or Fc encoding gene) of histone.To be synthesized using conventional Protocols in Molecular Biology
Various recombinant protein gene clonings are to suitable carrier for expression of eukaryon (such as pcDNA3.1 of invitrogen companies), Ran Houli
The restructuring egg that will be prepared with liposome (such as 293fectin of invitrogen companies) or other transfection reagents (such as PEI)
White expression plasmid is transfected into HEK293 cells (such as HEK293F of invitrogen companies), under serum free suspension condition of culture
Culture 3-5 days.Then culture supernatant is harvested by modes such as centrifugations.
The recombinant protein of His-tag amalgamation and expressions utilizes metal chelate affinity chromatography post (such as HisTrap FF of GE companies
Deng) a step purifying is carried out to the recombinant protein in culture supernatant.And the recombinant protein and recombinant antibodies of mFc amalgamation and expressions are used
ProteinA/G affinity columns (such as Mabselect SURE of GE companies) carry out a step purifying.Then desalting column is utilized
It is PBS (pH7.0) or other conjunctions that recombinant protein is preserved buffer exchange by (such as Hitrap desaulting of GE companies)
Suitable buffer solution.If necessary, filtration sterilization can be carried out with antagonist sample, then packing is stored in -20 DEG C.
Using recombinant antibodies technology obtain targeting tumour antigen HER2 monoclonal antibody (being appointed as C6G9+L1A7) and
Target the monoclonal antibody (being appointed as H10B7+L1A7) of people CD3E.The weight chain variabl area sequence of C6G9+L1A7 is SEQ ID
NO:14, light-chain variable sequence is SEQ ID NO:13.The weight chain variabl area sequence of H10B7+L1A7 is SEQ ID NO:12,
Light-chain variable sequence is SEQ ID NO:13.C6G9+L1A7 and H10B7+L1A7 have identical light chain.Additionally, C6G9+
The respective antibody functions of L1A7 and H10B7+L1A7 and property are by experimental verification.
The design and preparation of the different structure bispecific antibody of embodiment 2
The two kinds of monoclonal antibodies for being prepared and being verified based on embodiment 1, devise a series of for people CD3E and HER2
Bispecific antibody.
Because each arm of bispecific antibody can include Fab fragments or scfv, therefore according to different combinations
Devise four kinds of bispecific antibodies of structure (referring to Fig. 1 a- Fig. 1 d).
Additionally, to form heterodimer to promote, the human IgG1 based on KIH (Knob-Into-Hole) technology has been used
Fc fragments mutant (FcK, SEQ the ID NO of antibody:41 or FcH, SEQ ID NO:42), i.e. (will be derived from for people CD3E
H10B7+L1A7 antigen binding domain fusion) (will be derived from C6G9+ in the N-terminal of the Fc (FcK) being mutated containing Knob for HER2
L1A7 N-terminal of the antigen binding domain fusion) in the Fc (FcH) being mutated containing Hole.
According to recombinant antibodies technology similar to Example 1, the bispecific antibody (BsAb) being prepared for shown in table 1 and
As the monoclonal antibody (MAb) of control.
The people CD3E+HER2 bispecific antibodies of the different structure of table 1. and the structure of control monoclonal antibody
The affinity analysis of the various bispecific antibodies of embodiment 3
Affinity of antibody measure is carried out with the Biacore X100plus of GE.Amido coupling reagent (Amine coupling
Kit), human antibody capture agent (human antibody capture kit), His capture agents (his capture kit)
And the related reagent such as the 10 × HBS-EP of CM5 chips and pH7.4 and consumptive material are purchased from GE Healthcare companies.
The affinity of different bispecific antibodies is determined using prize law.The monoclonal prepared in embodiment 2 is determined resists
When body or bispecific antibody are to the affinity of CD3E, the antibody coupling of anti-His to CM5 chip surfaces will be marked with His
The CD3E antigens (CD3E-FcK-His/CD3D-FcH) of label capture CM5 chip surfaces as fixing phase, using single-cycle side
Various AntiCD3 McAb E antibody proteins are set a series of concentration gradient and flow through fixed phase surface by method, determine the affine of each antibody protein
Power.
In affinity of the monoclonal antibody or bispecific antibody prepared in determining embodiment 2 to HER2 antigens,
By the antibody coupling of anti-Fc to CM5 chip surfaces, various Anti-HER 2 albumen are diluted to suitable concn, CM5 is captured respectively
The HER2-His of restructuring is set a series of concentration gradient and flowed through admittedly by chip surface as fixing phase, using single-cycle method
Determine phase surface, determine the affinity of different antibodies albumen.
The description of embodiment 1 is shown in the preparation of people's CD3E and the HER2 antigen used in the present embodiment.
As shown in the result of table 2 and table 3, various bispecific antibodies can effectively combine CD3E and two kinds of HER2 respectively
Antigen.
Table 2. compares the affinity constant of monoclonal antibody and bispecific antibody combination CD3E
Antibody | Ka | Kd | KD |
MAb1 | 1.615E+7 | 4.871E-3 | 3.016E-10 |
BsAb1 | 3.161E+6 | 2.275E-2 | 7.189E-9 |
BsAb2 | 8.260E+5 | 1.118E-2 | 1.353E-8 |
BsAb3 | 4.346E+5 | 4.122E-2 | 9.486E-8 |
BsAb4 | 3.221E+6 | 1.198E-2 | 3.718E-9 |
BsAb5 | 1.276E+6 | 1.228E-2 | 9.622E-9 |
BsAb6 | 2.2E+6 | 8.701E-3 | 3.954E-9 |
BsAb7 | 6.573E+5 | 8.677E-3 | 1.320E-8 |
BsAb8 | 3.111E+6 | 1.843E-2 | 5.924E-9 |
Table 3. compares the affinity constant of monoclonal antibody and bispecific antibody combination HER2
Antibody | Ka | Kd | KD |
MAb2 | 1.194E+5 | 6.676E+-4 | 5.313E-9 |
BsAb1 | 9.659E+4 | 6.846E+-4 | 7.088E-9 |
BsAb2 | 7.427E+4 | 9.622E+-4 | 1.296E-8 |
BsAb3 | 1.32E+5 | 9.474E+-4 | 7.175E-9 |
BsAb4 | 8.231E+4 | 7.118E+-4 | 8.648E-9 |
BsAb5 | 6.666E+-4 | 7.172E+-4 | 1.076E-8 |
BsAb6 | 8.252E+4 | 6.984E+-4 | 8.463E-9 |
BsAb7 | 9.533E+4 | 6.473E+-4 | 6.79E-9 |
BsAb8 | 5.236E+4 | 5.071E-4 | 9.686E-9 |
The various bispecific antibodies of embodiment 4 recognize the ability identification of two kinds of antigens of CD3E and HER2 simultaneously
Using the bispecific antibody prepared in conventional ELISA method detection embodiment 2 to two kinds of antigens of CD3E and HER2
While combine, ELISA flows are as follows:CD3E-FcK/CD3D-FcH antigen coat elisa plates, 4 DEG C of refrigerator overnights are used first;
Then closed 1 hour with 37 DEG C of the confining liquid containing 1%BSA;37 DEG C of bispecific antibody is added to be incubated after washing 1 hour;Wash
Add HER2-His antigens after washing, 37 DEG C are incubated 1 hour;The anti-His IgG of HRP- are added after washing, 37 DEG C are incubated 1 hour;After washing
Adding HRP substrate solutions carries out chromogenic assay.Result is as shown in Figure 2:The various bispecific antibodies for building can be recognized simultaneously
Two kinds of antigens of CD3E and HER2.
The ability identification of the CD3E and HER2 of the various bispecific antibodies of embodiment 5 identification cell surface
By the bispecific antibody and MDA-MB-453 human breast cancer cells that are prepared in flow cytometry embodiment 2
The binding ability of the people CD3 expressed on the people HER2 of upper expression or human PBMC.MDA-MB-453 cells are purchased from China Medical Science
Institute of Basic Medical Sciences of institute preclinical medicine cell centre, and be incubated in RPMI1640 culture mediums.PBMC utilizes Ficoll density
Gradient centrifugation is separated from volunteer's whole blood, and is incubated in RPMI1640 culture mediums.
The blood (each 50mL) of Healthy Volunteers is gathered, wherein the blood for being gathered is by inventor and its colleague's conduct aspiration
Person provides, all equal signed Informed Consent Forms of volunteer.The inclusive criteria of volunteer is:
1. the age is more than 18 one full year of life;
2. without HIV, HBV infection;
3. blood routine detection is normal;
4. non-pregnant woman or women breast-feeding their children.
The cell of culture is collected, and cell viability is assessed using Trypan Blue.Then living cells is being contained 0.1%
Adjusted in the PBS of BSA to 3 × 106Individual cell/ml, to the cell suspending liquid for adding 90 μ l in the orifice plate of round bottom 96 per hole.
To the bispecific antibody that 10 μ l are added in celliferous hole or corresponding two kinds of monoclonal antibodies (MAb1 and MAb2) to obtain
The final concentration of 17.8nM (MDA-MB-453 bindings) or 53.3nM (PBMC bindings).In incubation 30 minutes at 4 DEG C
Afterwards, by cell be centrifuged (5 minutes, 350g), using 150 μ l/ holes containing BSA PBS dye solutions wash, resuspension and with 100 μ
Fluorescent dye conjugated goat anti-human igg antibody in l/ holes is in being incubated extra 30 minutes at 4 DEG C.Then by adding 150 μ l/ holes
PBS dye solutions and under 350g centrifugation carry out washed cell within 5 minutes.Second is carried out using 150 μ l/ holes PBS dye solutions
Individual washing step.Sample is resuspended in 100 μ l/ holes PBS dye solutions, flow cytometer (BD is used
Biosciences) obtain and analyze the sample.Result is as shown in Figures 3 and 4:The bispecific prepared in embodiment 2 resists
Body can respectively recognize the people CD3 expressed on the people HER2 and PBMC expressed on MDA-MB-453 human breast cancer cells.
Killing of the various Mediated by Bi-specific Antibodies T cells of embodiment 6 to HER2 positive tumor cells
Collect MDA-MB-453 human breast cancer cells (HER2 positive cells) or MDA-MB-468 human breast cancer cells (HER2
Negative cells, purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), count, and cell viability is assessed using trypan blue.Adjustment
Cell density is 2 × 105/ ml, 96 porocyte culture plates are inoculated in per the μ l of hole 100, are subsequently adding initial concentration for 3.3nM, and 10
The CD3+HER2 bispecific antibodies prepared in the embodiment 2 of times gradient dilution.Compare for convenience, by different CD3+HER2
Bispecific antibody or control monoclonal antibody (MAb1 and MAb2) are adjusted to identical molar concentration.Added in most backward hole
Human PBMC's cell (effector) is obtaining 5:1 final E:T ratios.Independent target cell (MDA-MB-453 people's mammary gland is set simultaneously
Cancer cell or MDA-MB-468 human breast cancer cells) control, independent PBMC cells (effector cell) control, independent culture medium do sky
White control.After being incubated 20 hours, supernatant is taken, with reference to CytoToxNon-radioactive cell toxicity detection kit specification
(CytoToxNon-Radioactive Cytotoxicity Assay, promega, G1780) detect and the double spies of analysis
Killing rate of the T cell of heterogenetic antibody mediation to target cell MDA-MB-453 human breast cancer cells.Testing result such as Fig. 5 a- Fig. 5 g
With shown in table 4:The application build various bispecific antibodies (BsAb) can effectively mediate T cell to HER2 positive target cells
Killing, it is impossible to killing of the mediate T cell to HER2 negative targets, the form or hinge of antigen-binding portion in bispecific antibody
There is some difference for the activity of the bispecific antibody that the different choice of the sequence of sequence is brought.When using SEQ ID NO:1 or
Person SEQ ID NO:During hinge area shown in 28, bispecific antibody can mediate T cell stronger killing is realized to tumour cell
Hinder ability.
The EC50 that the different Mediated by Bi-specific Antibodies T cells of table 4. are killed to target cell MDA-MB-453
The t cell activation of the various Mediated by Bi-specific Antibodies of embodiment 7
The MDA-MB-453 human breast cancer cells of expression HER2 are collected, is counted and is assessed cell viability using trypan blue.Adjust
The cell density of whole MDA-MB-453 human breast cancer cells is 2 × 105/ ml, 96 porocyte culture plates are inoculated in per the μ l of hole 100,
It is subsequently adding various CD3+HER2 bispecific antibodies prepared by the embodiment 2 being serially diluted.Compare for convenience, will be different
CD3+HER2 bispecific antibodies or control monoclonal antibody (MAb1 and MAb2) are adjusted to identical molar concentration.It is most backward
Human PBMC's (effector) is added in hole to obtain 5:1 final E:T ratios.Independent target cell (MDA-MB-453 people is set simultaneously
Breast cancer cell) control.After being incubated 20 hours, by (5 minutes, 350g) sedimentation cell being centrifuged and being delayed using 150 μ l/ hole PBS
Fliud flushing is washed twice.The antibody (eBioscience, 11-0037-42,12-0699-42) of anti-human CD3 and people CD69 is added, in 4
Lucifuge is incubated 30 minutes at DEG C.Then using 150 μ l/ holes PBSs washed cells twice, it is resuspended to 100 μ l/ hole PBS
In buffer solution, and cell sample is analyzed using flow cytometer (BD Biosciences C6) and compares different samples
After treatment in CD3 positive cell groups activation marker thing CD69 differential expression.Or after being incubated 20 hours, take and asked in culture,
Using IL2 levels in elisa assay supernatant (human IL-2's ELISA reagents, DAKEWE, DKW12-1020-096).Result such as Fig. 6 a-
Shown in Fig. 6 c:Various bispecific antibodies that the application builds can realize specificity of the HER2 positive target cells to T cell
Activation.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. bispecific human IgG1 antibody, it includes two kinds of Fc fragments with identical hinge area, the amino acid of the hinge area
Sequence is SEQ ID NO:1 or SEQ ID NO:28 and the 216-230 bit sequences of natural human IgG1 antibody constant regions are substituted for,
Antibody constant region amino acid position determines according to EU numbering.
2. bispecific human IgG1 antibody as claimed in claim 1, wherein described two Fc fragments are comprising being able to ensure that heavy chain
The mutation of different dimerization;Preferably, a Fc fragment includes point mutation S354C and T366W, and another Fc fragment includes point mutation
Y349C, T366S, L368A and Y407V.
3. bispecific human IgG1 antibody as claimed in claim 1 or 2, wherein the N-terminal of described two Fc fragments is merged respectively
Recognize the single-chain antibody (scfv) of different epitopes.
4. bispecific human IgG1 antibody as claimed in claim 1 or 2, the N-terminal fusion single-chain antibody of one of which Fc fragments
(scfv), the N-terminal fusion Fab fragments of another Fc fragments, the single-chain antibody antigen table different from the Fab fragments identification
Position.
5. bispecific human IgG1 antibody as claimed in claim 1 or 2, wherein the N-terminal of described two Fc fragments is merged respectively
Recognize the Fab fragments of different epitopes;
Preferably, recognize that the Fab fragments of different epitopes include identical light chain.
6. bispecific human IgG1 antibody as claimed in claim 5, one of which Fab fragments include natural human IgG1's antibody
Constant region CH1 fragments, human IgG1 antibody constant region CH1 fragment of another Fab fragments comprising mutation, the human IgG1 of the mutation
Antibody constant region CH1 fragments include any 1, wherein 2 or 3, CH1 fragments in point mutation G137E, N203D and R214T
Amino acid sequence for antibody constant region 118-215 amino acid sequence;
Preferably, the amino acid sequence of natural human IgG1's antibody constant region CH1 fragments is SEQ ID NO:2, and/or institute
The amino acid sequence for stating human IgG1's antibody constant region CH1 fragments of mutation is SEQ ID NO:30.
7. the bispecific human IgG1's antibody as any one of claim 1-6, wherein the CH2 pieces of described two Fc fragments
Duan Zhongjun includes point mutation L234F, L235E and P331S, and the wherein amino acid sequence of CH2 fragments is antibody constant region 231-
The amino acid sequence of 340;
Preferably, the amino acid sequence of the CH2 fragments of described two Fc fragments is SEQ ID NO:31.
8. the bispecific human IgG1's antibody as any one of claim 1-7, its identification people's immune cell surface antigenic
And TCSA;
Preferably, people's immune cell surface antigenic is CD3E.
9. pharmaceutical composition, it includes the bispecific human IgG1's antibody any one of claim 1-8.
10. the drug regimen described in the bispecific human IgG1 antibody or claim 9 any one of claim 1-8
Purposes of the thing in the medicine for being used for preventing or treat tumour is prepared.
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WO2018176992A1 (en) * | 2017-03-31 | 2018-10-04 | 北京百特美博生物科技有限公司 | New type of bispecific antibody and use thereof |
CN110172100A (en) * | 2019-05-06 | 2019-08-27 | 北京智仁美博生物科技有限公司 | Anti-human CD3E antibody and application thereof |
CN113406320A (en) * | 2021-08-18 | 2021-09-17 | 南京立顶医疗科技有限公司 | Directional coupling method based on recombinant gene engineering antibody and microsphere and application |
CN113501878A (en) * | 2021-02-03 | 2021-10-15 | 北京智仁美博生物科技有限公司 | Antibodies against human TSLP and uses thereof |
EP3988573A4 (en) * | 2019-06-19 | 2023-08-09 | Beijing Wisdomab Biotechnology Co., Ltd | Anti-cd3e/bcma bispecific antibody and use thereof |
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