CN1842541A - Compositions and methods for regulating NK cell activity - Google Patents

Compositions and methods for regulating NK cell activity Download PDF

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Publication number
CN1842541A
CN1842541A CN200480021897.0A CN200480021897A CN1842541A CN 1842541 A CN1842541 A CN 1842541A CN 200480021897 A CN200480021897 A CN 200480021897A CN 1842541 A CN1842541 A CN 1842541A
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antibody
cell
kir2dl1
kir
fragment
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CN200480021897.0A
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CN1842541B (en
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S·B·帕德克杰尔
A·莫雷塔
M·D·谢萨
P·安德烈
L·戈捷
P·A·N·R·瓦格特曼
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Novo Nordisk AS
Innate Pharma SA
Universita degli Studi di Genova
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Novo Nordisk AS
Innate Pharma SA
Universita degli Studi di Genova
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Priority to CN201310057089.0A priority Critical patent/CN103588880B/en
Priority claimed from PCT/DK2004/000470 external-priority patent/WO2005003168A2/en
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Abstract

The present invention relates to novel compositions and methods for regulating an immune response in a subject. More particularly, the invention relates to specific antibodies that regulate the activity of NK cells and allow a potentiation of NK cell cytotoxicity in mammalian subjects. The invention also relates to fragments and derivatives of such antibodies, as well as pharmaceutical compositions comprising the same and their uses, particularly in therapy, to increase NK cell activity or cytotoxicity in subjects.

Description

Regulate the composition and the method for NK cytoactive
Invention field
The present invention relates to the two or more inhibition acceptor cross reaction that exists with the NK cell surface and strengthen mammalian subject or biological sample in antibody, antibody fragment and the derivative thereof of NK cell cytotoxicity.The present invention also relates to make the method for this antibody-like, fragment, variant and derivative; The pharmaceutical composition that comprises described material; This quasi-molecule and composition (specifically in treatment) be the purposes in NK cytoactive or the cytotoxicity in increasing subject.
Background
Natural killer (NK) cell is the lymphocyte subgroup that participates in unconventional immunity.Can obtain the NK cell from for example blood sample, cytapheresis (cytapheresis), collection storehouse (collection) etc. by multiple multiple technologies well known in the art.
The feature and the biological characteristics of NK cell comprise: express surface antigen, comprise CD16, CD56 and/or CD57; Cell surface lacks α/β or gamma/delta TCR mixture; By the activation specific lyticase in conjunction with and kill and wound the ability of not expressing the antigenic cell of " self " MHC/HLA; Kill and wound the tumour cell of expression NK activated receptor-part or the ability of other diseased cells; Discharge the ability that stimulates or suppress the cytokine of immunne response; And experience the division of many wheel cellses, produce and have and the ability of the daughter cell of parent cell similar biological.In literary composition of the present invention, " activity " NK cell refers to biologic activity NK cell, more specifically is the NK cell with ability of cracking target cell.For example, " activity " NK cell can kill and wound and express NK activated receptor-part and do not express the antigenic cell of " self " MHC/HLA (the incompatible cell of KIR).
Based on its biological characteristics, multiple treatment and the immunization strategy of regulating the NK cell that depend on proposed in this area.Yet the NK cytoactive is regulated by the complex mechanism that relates to pungency and inhibition signal.Therefore, effectively the cell-mediated treatment of NK may require to stimulate these cells and in and the inhibition signal.
The NK cell is regulated (people such as K_rre, 1986 by major histocompatibility complex (MHC) I class specificity inhibition acceptor is negative; People such as _ hl é n, 1989).MHC I quasi-molecule that exists on these specific receptorss and other cell or the polymorphism determinant of HLA combine, and suppress NK lysis.In the mankind, be called as and kill and wound Ig sample acceptor (killer Ig-likereceptors, some member of receptor family KIRs) discern HLA I class allelotrope.
KIR is the acceptor extended familys that are present on some lymphocyte subgroup (comprising the NK cell).The KIR nomenclature is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS) based on the quantity (KIR2D or KIR3D) and the cytoplasmic tail of extracellular domain.The mankind, in the NK group in being present in independent part, the existence of given KIR or shortage are variable between the NK cell.Also have high-caliber relatively KIR molecule polymorphism in the crowd, some KIR molecule is present in (but being not whole) individuality.When suitable part combined, some KIR gene product caused the stimulation of lymphocyte activity.Certified pungency KIR has and contains electrically charged short cytoplasmic tail of striding the film residue, and described residue combines with the linkers that contains immunostimulating motif (ITAM).Other KIR gene product comes down to inhibition.As if all attested inhibition KIR have long cytoplasmic tail, and interact with the antigenic different subgroups of HLA according to the KIR hypotype.Inhibition KIR part in its kytoplasm shows that one or several recruit the inhibitory motifs of Phosphoric acid esterases.Known inhibition KIR acceptor comprises KIR2DL and KIR3DL subfamily member.KIR acceptor (KIR2D) with two Ig structural domains is identified the epi-position that HLA-C allotype: KIR2DL2 (being appointed as p58.2 originally) or closely-related gene product KIR2DL3 identification are shared by 2 groups of HLA-C allotypes (Cw1,3,7 and 8), and the epi-position that KIR2DL1 (p58.1) identification is shared by mutual (reciprocal) 1 group of HLA-C allotype (Cw2,4,5 and 6).There is Lys residue indication KIR2DL1 identification in 80 in HLA-C allelotrope.There are Asn residue indication KIR2DL2 and KIR2DL3 identification at 80.Importantly, most of HLA-C allelotrope have Asn or Lys at 80.KIR with three Ig structural domains, promptly KIR3DL1 (p70) discerns the epi-position of being shared by HLA-Bw4 allelotrope.At last, homodimer KIR3DL2 (p140) the identification HLA-A3 and the HLA-A11 that have the molecule of three Ig structural domains.
Although inhibition KIR and other I class inhibition acceptor (people such as Moretta, 1997; People such as Valiante, 1997a; Lanier, 1998) may in the NK storehouse of any given individuality (repertoire), there be the cell of expressing single KIR by NK cell coexpression, therefore, corresponding N K cell is only expressed the cell blocking-up of allelic group of specific I class.
Through showing, the NK cell mass of KIR mispairing or clone (promptly expressing the NK cell mass with the inconsistent KIR of host HLA molecule) are the most probable media that sees the transplanting leukemia resisting action of allograft people such as (, 2002) Ruggeri.Again a kind of method that produces this effect in given individuality will be to use the interactional reagent of blocking-up KIR/HLA.
Through showing, KIR2DL1 monoclonal antibody specific blocking-up KIR2DL1 and the allelic interaction of Cw4 (or analogue) people such as (, 1993) Moretta.Also have and describe anti-KIR2DL2/3 monoclonal antibody blocking-up KIR2DL2/3 and the interaction of HLACw3 (or analogue) allelotrope people such as (, 1993) Moretta.Yet this class reagent use in clinical setting two therapeutic mAb that will seek development treat all patients, and no matter whether any given patient expresses 1 class or 2 class HLA-C allelotrope.In addition, before which kind of therapeutic antibodies decision uses, must pre-determine the HLA type that each patient expresses, thereby cause expensive a lot of medical expenses.
People such as Watzl, Tissue Antigens, 56,240 pages (2000) have made the cross-reacting antibody of discerning a plurality of KIR isotypes, but these antibody do not show the enhancing of NK cytoactive.People such as G.M.Spaggiara, Blood, 100,4098-4107 page or leaf (2002) has been finished the experiment of the monoclonal antibody of utilizing many anti-multiple KIR.It is said that one of these antibody (NKVSF1) discerns the common epitope of CD158a (KIR2DL1), CD158b (KIR2DL2) and p50.3 (KIR2DS4).Do not point out NKVSF1 can strengthen the NK cytoactive, also prompting can not use it to be used for the treatment of.Therefore, this area does not still have the feasibility and the effective way of regulating the NK cytoactive at present and can utilize, and still needs to use the specific reagent specificity to intervene HLA allelotrope.
Summary of the invention
The present invention now provides new antibody, composition and method overcoming the existing difficulty in the NK cell activation, and extra favorable characteristics and benefit is provided.One exemplary aspect, the invention provides in nearly all people the single antibody that promotes human NK cell activation.More specifically, the invention provides brand-new specific antibody, described brand-new specific antibody and multiple inhibition KIR group cross reaction and its inhibition signal that neutralizes cause expressing that the NK cell cytotoxicity strengthens in the NK cell of this class inhibition KIR acceptor.Allow antibody of the present invention in most of human experimenters, to be used to effectively increase the NK cytoactive with the ability of multiple KIR gene product cross reaction, and do not pre-determine the burden or the expense of experimenter HLA type.
Aspect first, the invention provides antibody, antibody fragment and arbitrary derivative thereof, at least two kinds of inhibition KIR acceptors cross reaction of wherein said antibody, antibody fragment or derivative and NK cell surface, in and NK cell inhibiting signal and strengthen the NK cytoactive.This antibody more preferably combines with the common determinant of people KIR2DL acceptor.Antibody of the present invention in addition more clearly at least with KIR2DL1, KIR2DL2 and KIR2DL3 receptors bind.For the present invention, term " KIR2DL2/3 " refer in KIR2DL2 and the KIR2DL3 acceptor arbitrary or both.These two acceptors have very high homology, are the allelic forms of the homologous genes of supposition, and are thought interchangeable by this area.Therefore, for the present invention is thought of as single inhibition KIR molecule with KIR2DL2/3, therefore only with KIR2DL2 and KIR2DL3 and not with the antibody of other inhibition KIR acceptor cross reaction not within the scope of the present invention.
Antibodies specific of the present invention suppresses combining of MHC or HLA molecule and at least two kinds of inhibition KIR acceptors and promotes the NK cytoactive.Two kinds of activity inferred in used from here term " in and KIR inhibitory activity ".The ability of antibody of the present invention in literary composition of the present invention " promote NK cytoactive ", " promoting the NK cell cytotoxicity ", " promoting the NK cell ", " strengthening the NK cytoactive ", " strengthening the NK cell cytotoxicity " or " strengthening the NK cell ", refer to this antibody make the NK cell of its surface expression inhibition KIR acceptor can cracking at the cell of the respective ligand of this specific inhibition KIR acceptor (for example specific HLA antigen) of its surface expression.One specific aspect, the invention provides the antibody that specificity suppresses HLA-C molecule and KIR2DL1 and KIR2DL2/3 receptors bind.Another specific aspect, the invention provides the antibody that promotes the NK cytoactive in the body.
Because one of KIR2DL1 or KID2DL2/3 are present at least about among 90% the crowd at least, the preferred antibody of the present invention can promote the NK cytoactive at most of HLA-C allotypes (being respectively 1 group of HLA-C allotype and 2 groups of HLA-C allotypes) relevant cell.Therefore, composition of the present invention can be used for the effectively activation or strengthen the NK cell most of human individuals (general about 90% human individual or more than).Therefore, single antibody compositions according to the present invention can be used for the treatment of most of human experimenters, seldom needs to determine allelic classification or uses mixtures of antibodies.
The present invention proves cross reactivity and the neutrality antibody that can produce anti-inhibition KIR first, and this antibody-like allows effectively to activate the NK cell in the broad range crowd.
Therefore specific purposes of the present invention are positioned antibody, and wherein said antibodies specific is in conjunction with KIR2DL1 and KIR2DL2/3 human receptor, and reverse is by the inhibition of the NK cell cytotoxicity of these KIR mediations.In one embodiment, the monoclonal antibody DF200 competition of this antibody and hybridoma DF200 generation.Alternatively, described antibody with antibody DF200 competition is not antibody DF200 itself.
In another embodiment, antibody and monoclonal antibody NKVSF1 competition, alternatively, wherein the antibody with antibody NKVSF1 competition is not antibody NKVSF1.
In another embodiment, this antibody and antibody 1-7F9 competition.
Described antibody is preferably chimeric antibody, humanized antibody or human antibodies.
When relating to specific monoclonal antibody (for example DF200, NKVSF1,1-7F9, EB6, GL183), term " with ... competition " refer to antibody in conjunction with competing with monoclonal antibody (for example DF200, NKVSF1,1-7F9, EB6, GL183) in measuring, described in conjunction with measuring the KIR molecule that uses reorganization KIR molecule or surface expression.For example, if combine this antibody and DF200 " competition " what reduce DF200 and KIR molecule in conjunction with antibody in measuring.With the antibody of DF200 " competition " can combine with the DF200 competition KIR2DL1 human receptor, KIR2DL2/3 human receptor or KIR2DL1 and KIR2DL2/3 human receptor the two.
In preferred embodiments, the invention provides in conjunction with KIR2DL1 and KIR2DL2/3 human antibodies, reverse and combine the two antibody of KIR2DL1 human receptor, KIR2DL2/3 human receptor or KIR2DL1 and KIR2DL2/3 human receptor by the inhibition of the NK cell cytotoxicity of these KIR mediations and with DF200,1-7F9 or NKVSF1 competition.Described antibody is not NKVSF1 alternatively.That this antibody is chosen as is chimeric, the mankind or humanized antibody.
In another embodiment, the invention provides in conjunction with KIR2DL1 and KIR2DL2/3 human receptor, reverse and combine the KIR2DL1 human receptor by the inhibition of the NK cell cytotoxicity of these KIR mediations and with the EB6 competition, combine the KIR2DL2/3 human receptor with the GL183 competition or combine the KIR2DL1 human receptor with the EB6 competition and compete the antibody that combines the KIR2DL2/3 human receptor with GL183.Described antibody is not NKVSF1 alternatively; This antibody is not DF200 alternatively.That this antibody is chosen as is chimeric, the mankind or humanized antibody.
Aspect favourable, the invention provides antibody with the DF200 competition, described antibody and monoclonal antibody DF200 identification, in conjunction with the epi-position on the basic identical or identical KIR molecule or " epi-position site ", or to wherein epi-position or " epi-position site " have immunologic opsonin.Described KIR molecule is preferably KIR2DL1 human receptor or KIR2DL2/3 human receptor.
Specific purposes of the present invention are positioned antibody, and wherein said antibodies is present in the common determinant on KIR2DL1 and the KIR2DL2/3 human receptor, and reverse the inhibition by the NK cell cytotoxicity of these KIR mediations.These antibody combine upward essentially identical epi-position of KIR with the antibody NKVSF1 that the monoclonal antibody DF200 or the hybridoma NKVSF1 of hybridoma DF200 generation produce more specifically, and wherein said antibody is not NKVSF1.
In preferred embodiments, antibody of the present invention is monoclonal antibody.The most preferred antibody of the present invention is the monoclonal antibody DF200 that is produced by hybridoma DF200.
The hybridoma that produces antibody DF200 is preserved in CNCM preservation center with identifier " DF200 ", number of registration CNCM I-3224 (in registration on June 10th, 2004), CollectionNationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue du Docteur Roux, F-75724 Paris Cedex 15, France.Antibody NKVSF1 can (Cergy Sainte-Christophe France) obtains catalogue reference number MCA2243 from Serotec.Also NKVSF1 is called pan2D mAb at this.
The present invention also provides the functional fragment and the derivative of antibody described here, described functional fragment and derivative have essentially identical antigen-specific and activity (for example can with female antibody cross reaction and the cellular cytoxicity activity that strengthens the NK cell of expression inhibiting KIR acceptor), comprise (but being not limited to) Fab fragment, Fab ' 2 fragments, immunoadhesin, miniature bifunctional antibody (diabodies), CDR and ScFv.In addition, antibody of the present invention can be humanization, people or chimeric.
The present invention also provides antibody derivatives, but described antibody derivatives comprises with toxin, radionuclide test section (for example fluorescent agent) or solid support is puted together or covalently bound antibody of the present invention.
The present invention also provides the pharmaceutical composition that comprises top disclosed antibody, its fragment or its arbitrary derivative.Therefore, the present invention also relates to the purposes of antibody disclosed herein in manufacture method of medicine.In preferred embodiments, described medicine or pharmaceutical composition are used for the treatment of cancer or other proliferative disease, infection or are used for transplanting.
In another embodiment, the invention provides composition, described composition comprises different with two at least human inhibition KIR acceptor gene product bonded antibody, wherein antibody can neutralize and express the inhibition of the NK cell cytotoxicity that is mediated by KIR on the NK cell at least a in described two kinds of different human inhibition KIR acceptors, and wherein antibody is incorporated in the liposome.Said composition randomly comprises additional material, and described additional material is selected from the nucleic acid molecule of carrying gene into gene therapy; Carry the nucleic acid molecule of sense-rna, RNAi or siRNA for suppressor gene in the NK cell; Or be incorporated in the described liposome in addition, the toxin or the medicine of target killing NK cell.
That the present invention also provides is external, exsomatize or body in regulate the method for human NK cytoactive, comprise fragment, its arbitrary derivative of human NK cell and the antibody of the present invention for the treatment of significant quantity, this antibody-like or comprise that at least above-mentioned each pharmaceutical composition contacts.Preferable methods comprises the pharmaceutical composition of the present invention of administering therapeutic significant quantity, and at the cellular cytoxicity activity that in the experimenter who suffers from cancer, infectious diseases or immunological disease, increases human NK cell (most preferably exsomatize or body in).
On the other hand, the invention provides hybridoma, comprise: (a) from the B cell of mammalian hosts (being generally the non-human mammal host), it is to contain the antigen immune of the epi-position that exists on the inhibition KIR polypeptide, described B cell and (b) immortalized cell line (for example myeloma cell) fusion, wherein said hybridoma produces monoclonal antibody, and this monoclonal antibody is in conjunction with at least two kinds of different human inhibition KIR acceptors, and in can be at least basic with in the NK cell mass of expressing described at least two kinds of different human inhibition KIR acceptors by the inhibition of the NK cell cytotoxicity of KIR mediation.Randomly, this hybridoma does not produce monoclonal antibody NKVSF1.Described antibody preferred combination KIR2DL1 and KIR2DL2/3 acceptor.This antibody preferred combination KIR2DL1 and KIR2DL2/3 go up the common determinant that exists.Described hybridoma preferably produces and is suppressed at 80 HLA-c allelotrope molecules with Lys residue and human KIR2DL1 receptors bind and has the HLA-C allelotrope molecule of Asn residue and the antibody of human KIR2DL2/3 receptors bind at 80.Described hybridoma preferably produces the antibody that the monoclonal antibody DF200 that produces with hybridoma DF200 combines KIR2DL1 or KIR2DL2/3 or KIR2DL1 and the last essentially identical epi-position of KIR2DL2/3.The example of this hybridoma is DF200.
The present invention also provides the method that produces antibody, described antibody and the cross reaction of multiple KIR2DL gene product, and the inhibitory activity of this class KIR that neutralizes, and described method comprises that step is:
(a) to contain the immunogen immune non-human mammal of KIR2DL polypeptide;
(b) prepare antibody from described immune Mammals, the described KIR2DL polypeptide of wherein said antibodies,
(c) antibody of the KIR2DL gene product cross reaction different in the selection (b) with at least two kinds, and
(d) antibody of enhancing NK cell in the selection (c).In one embodiment, described non-human mammal is that the transgenic animal that are designed to the express human antibody storehouse (for example contain the human immunoglobulin gene seat and lack the non-human mammal of native immunoglobulin gene, for example Xenomouse TM(Abgenix-Fremont, CA USA), or contain the non-human mammal at human Ig encoding gene minigene seat (minilocus), for example HuMab-mouse TM(Medarex-Princeton, NJ, USA)).This method randomly comprises the antibody of selection in conjunction with primate (preferred macaque) NK cell or KIR polypeptide in addition.The present invention randomly comprises the method for estimating antibody in addition, and wherein the antibody that produces according to aforesaid method is applied to primate (preferred macaque), preferably monkey is observed the existence or the shortage of antibody toxicity indication.
The inventor also provides the method that produces antibody, shown at least two kinds of different human inhibition KIR acceptor gene products of antibodies, wherein said antibody can in in the inhibition of expressing on the NK cell mass of described at least two kinds of different human inhibition KIR acceptor gene products by the NK cell cytotoxicity of KIR mediation, the method comprising the steps of is:
A) to contain the immunogen immune non-human mammal of inhibition KIR polypeptide;
B) prepare antibody from described animal through immunity, the described KIR polypeptide of wherein said antibodies,
C) antibody of the human inhibition KIR acceptor gene product cross reaction different in the selection (b) with at least two kinds, and
Select in (c) can in on the NK cell mass of expressing described at least two kinds of different human inhibition KIR acceptor gene products by the antibody of the inhibition of the NK cell cytotoxicity of KIR mediation, wherein step (c) and order (d) can be put upside down arbitrarily, and arbitrary steps is optional to repeat 1 or repeatedly.The inhibition KIR polypeptide that is used for immunity is preferably the KIR2DL polypeptide, the antibody that is selected from step (c) at least with KIR2DL1 and KIR2DL2/3 cross reaction.This antibody is preferably discerned and is present at least two kinds of common determinants on the different KIR acceptor gene products; Described KIR most preferably is KIR2DL1 and KIR2DL2/3.This method is selected and primate (preferred macaque) NK cell or KIR polypeptide bonded antibody optional comprising in addition.The present invention randomly comprises the method for estimating antibody in addition, and wherein the antibody that produces according to aforesaid method is applied to primate (preferred macaque), preferably monkey is observed the existence or the shortage of antibody toxicity indication.
Be selected from step c) or d in the aforesaid method) antibody be not NKVSF1 alternatively.The antibody of preparation is preferably monoclonal antibody in the aforesaid method step (b).Being selected from antibody in the aforesaid method step (c) preferably is suppressed at combining of 80 HLA-C allelotrope molecules with Lys residue and human KIR2DL1 acceptor and has the HLA-C allelotrope molecule of Asn residue and combining of human KIR2DL2/3 acceptor at 80.The antibody that is selected from the aforesaid method step (d) preferably causes the Cytotoxic enhancing of NK, the for example Cytotoxic any remarkable enhancing of NK or strengthen at least 5%, 10%, 20%, 30% or higher, for example target NK cytotoxicity strengthens at least about 50% (for example NK cell cytotoxicity strengthen at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% or at least about 95% (for example about 65-100%)).This antibody preferably combines KIR2DL1 and/or KIR2DL2/3 and goes up essentially identical epi-position with monoclonal antibody DF200.Randomly, this method also or in addition contains makes selected monoclonal antibody fragment, makes selected monoclonal antibody derivative (for example by puting together with radionuclide, cytotoxic agent, reporter molecules etc.) or makes the additional step of antibody fragment derivative, and described antibody fragment produces or contains sequence corresponding to this class monoclonal antibody sequence from this class monoclonal antibody.
The present invention provides the method for producing antibody in addition, at least two kinds of different human inhibition KIR acceptor gene products of described antibodies, wherein said antibody can in in the inhibition of expressing on the NK cell mass of described at least two kinds of different human inhibition KIR acceptor gene products by the NK cell cytotoxicity of KIR mediation, the method comprising the steps of is:
(a) from library or storehouse, select the monoclonal antibody or the antibody fragment of different with two kinds at least human inhibition KIR2DL acceptor gene product cross reactions, and
(b) select in (a) can in on the NK cell mass of expressing described at least two kinds of different human inhibition KIR2DL acceptor gene products by the antibody of the inhibition of the NK cell cytotoxicity of KIR mediation.This antibody preferred combination is present in the common determinant on KIR2DL1 and the KIR2DL2/3.The antibody that is selected from step (b) is not NKVSF1 alternatively.Being selected from antibody in the step (b) preferably is suppressed at combining of 80 HLA-c allelotrope molecules with Lys residue and human KIR2DL1 acceptor and has the HLA-C allelotrope molecule of Asn residue and combining of human KIR2DL2/3 acceptor at 80.The antibody that is selected from step (b) preferably causes the Cytotoxic enhancing of NK, the for example Cytotoxic any remarkable enhancing of NK or strengthen at least 5%, 10%, 20%, 30% or higher, for example target NK cytotoxicity strengthens at least about 50% (for example NK cell cytotoxicity strengthen at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% or at least about 95% (for example about 65-100%)).This antibody preferably combines KIR2DL1 and/or KIR2DL2/3 and goes up essentially identical epi-position with monoclonal antibody DF200.This method randomly contains the additional step of making selected monoclonal antibody fragment, the selected monoclonal antibody derivative of making or making the derivative of selected monoclonal antibody fragment.
In addition, the invention provides the method for producing antibody, at least two kinds of different human inhibition KIR acceptor gene products of described antibodies, wherein said antibody can in in expressing the NK cell mass of described at least two kinds of different human inhibition KIR acceptor gene products by the inhibition of the NK cell cytotoxicity of KIR mediation, the method comprising the steps of is:
A) under the condition that allows described monoclonal antibody to produce, cultivate hybridoma of the present invention; And
B) separate described monoclonal antibody from this hybridoma.This method randomly contains the additional step of making this monoclonal antibody fragment, making monoclonal antibody derivative or making the derivative of this class monoclonal antibody fragment.The antibody preferred combination is present in the common determinant on KIR2DL1 and the KIR2DL2/3.
The present invention also provides the method for producing antibody, at least two kinds of different human inhibition KIR acceptor gene products of described antibodies, wherein said antibody can in in expressing the NK cell mass of described at least two kinds of different human inhibition KIR acceptor gene products by the inhibition of the NK cell cytotoxicity of KIR mediation, the method comprising the steps of is:
A) separate the nucleic acid of the described monoclonal antibody of coding from hybridoma of the present invention;
B) randomly modify this nucleic acid, coding is modified or the modification of nucleic acids of the sequence of the antibody of deriving thereby obtain to contain, described antibody contains the aminoacid sequence corresponding to described monoclonal antibody function sequence or similar substantially with it (for example with this class sequence at least about 65%, at least about 75%, at least about 85%, at least about 90%, consistent at least about 95% (for example about 70-99%)), and wherein antibody is selected from the immunoreactivity fragment of humanized antibody, chimeric antibody, single-chain antibody, antibody or contains the reactive segmental fused protein of this para-immunity;
C) described nucleic acid or modification of nucleic acids (or related nucleic acid of coding same acid sequence) are inserted expression vector, wherein when this expression vector was present under optimum conditions in the host cell grown, the antibody or the antibody fragment capable that are encoded accessed expression;
D) with described expression vector transfection host cell, wherein said host cell does not produce immunoglobulin (Ig) protein in addition;
E) causing the host cell of cultivating described transfection under the condition that described antibody or antibody fragment are expressed; And
F) separation is by the antibody or the antibody fragment of the host cell generation of transfection.The antibody preferred combination is present in the common determinant on KIR2DL1 and the KIR2DL2/3.
Be to be understood that the present invention also provides the composition that contains antibody, at least two kinds of different human inhibition KIR acceptor gene products of wherein said antibodies, wherein said antibody can in in the NK cell of expressing one of described two kinds of different human inhibition KIR acceptors at least by the inhibition of the NK cell cytotoxicity of KIR mediation, antibody exists with the amount that can strengthen the patient effectively with detecting or contain NK cell cytotoxicity in the biological sample of NK cell; Also contain pharmaceutically useful carrier or vehicle.Antibody preferably be present in KIR2DL1 and KIR2DL2/3 on common determinant combine.Described composition can randomly contain another kind of therapeutical agent, and this therapeutical agent is selected from for example immunomodulator, hormone agent, chemotherapeutic, anti-angiogenic agent, apoptosis agent, combination and suppresses another antibody, anti-infection agent, target agent or the complementary compound (adjunct compound) of inhibition KIR acceptor.Favourable immunomodulator can be selected from IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β or IFN-γ.The example of described chemotherapeutic comprises alkylating reagent, antimetabolite, cytotoxic antibiotics, Zorubicin, dactinomycin, mitomycin, carminomycin, zhengdingmeisu, Dx, tamoxifen, safe plain, taxotere, vincristine(VCR), vinealeucoblastine(VLB), vinorelbine, Etoposide (VP-16), 5 FU 5 fluorouracil (5FU), cytosine arabinoside, endoxan, plug is for group, methotrexate, camptothecine, dactinomycin, ametycin, cis-platinum (CDDP), aminopterin, combretastatin, other vinca alkaloids and derivative thereof or prodrug.The example of hormone agent comprises Leuprolide, goserelin, triptorelin, buserelin, tamoxifen, toremifene, flutamide, Nilutamide, cyproterone bicalutamide Anastrozole, Exemestane, letrozole, fadrozole medroxy, Verton, megestrol, other LHRH agonist, other estrogen antagonist material, other antiandrogen, other aromatase inhibitor and other progestogen.Described combination and another antibody that suppresses inhibition KIR acceptor are preferably antibody or derivatives thereof or the fragment in conjunction with inhibition KIR acceptor epi-position, wherein said epi-position be different from be present at least two kinds of different human inhibition KIR acceptor gene products on common determinant bonded antibody institute bonded epi-position.
The present invention is provided at the method that can strengthen the NK cytoactive among the patient who needs enhancing NK cytoactive in addition with detecting, is included as the step that the patient uses composition of the present invention.The patient who needs enhancing NK cytoactive can be any suffer from disease or disorderly patient, wherein, this enhancing may promote, enhancing and/or inductive treatment effect (or promote, strengthen and/or induce such effect having this disease or disorder or have in patient's the signal portion of basic similar features at least, can measure by for example clinical trial).Need the patient of this class treatment may suffer from for example cancer, other proliferative disease, infectious diseases or Immunological diseases.This method preferably includes the additional step that uses suitable additional therapeutic agent for the patient, described therapeutical agent is selected from immunomodulator, hormone agent, chemotherapeutic, anti-angiogenic agent, apoptosis agent, combination and suppresses another antibody, anti-infection agent, target agent or the complementary compound of inhibition KIR acceptor, and wherein said additional therapeutic agent is applied to the patient with described antibody with single agent form or with the dosage form of separating.The dosage of antibody (or antibody fragment/derivative) and the dosage integral body of additional therapeutic agent are enough to can induce, promote and/or strengthen with detecting (comprise and strengthen the NK cytoactive) therapeutic response in the patient.During separate administration, (for example about selection of time, medicament quantity etc.) administration of antibodies, fragment or derivative and additional therapeutic agent as required under the condition that the patient is produced detectable combination therapy benefit.
Other content that the present invention comprises be of the present invention can specificity in conjunction with the antibody of non-human primate's (preferred monkey) NK cell and/or monkey KIR acceptor.Also comprise evaluation as toxicity, dosage and/or the activity of the antibody of the present invention of drug candidate or the method for effect.On the one hand, the present invention comprises the method for determining animal or the deleterious antibody dosage of target tissue, comprise antibody of the present invention is applied to the non-human primate's acceptor with NK cell, evaluate this reagent animal or preferred any toxicity or harmful or undesirable action to target tissue.On the other hand, the present invention is the method for identifying animal or the deleterious antibody of target tissue, comprise antibody of the present invention is applied to the non-human primate's acceptor with NK cell, evaluate this reagent animal or preferred any toxicity or harmful or undesirable action to target tissue.On the other hand, the present invention is a method of identifying treatment the infected, disease or the effective antibody of tumour, comprise the infection, disease or the cancer model that antibody of the present invention are applied to the non-human primate, identify the antibody that improves infection, disease or cancer or its symptom.Antibody of the present invention is preferably the human KIR acceptor cross reaction of inhibition of (a) and at least two kinds of human NK cell surfaces and (b) and the antibody of non-human primate's NK cell or the cross reaction of KIR acceptor.
Other content that the present invention comprises is to exist its cell surface to have the method for the NK cell of inhibition KIR in detection of biological sample or the live organism, and described method comprises step:
A) described biological sample or live organism are contacted with antibody of the present invention, but wherein said antibody is puted together with the test section or is covalently bound; And
B) existence of this antibody of detection in biological sample or live organism.
The present invention provides also that purifying has the method for the NK cell of inhibition KIR at its cell surface from sample, comprises step:
A) allowing its cell surface to have under the condition of the NK cell of inhibition KIR and described antibodies, described sample is contacted with antibody of the present invention, wherein antibody and solid support (for example pearl, matrix etc.) are puted together or are covalently bound; And
B) from puting together with solid support or covalently bound antibody elution bonded NK cell.
On the other hand, the invention provides antibody, antibody fragment or its arbitrary derivative, described antibody, antibody fragment or its arbitrary derivative contain variable region of light chain or the one or more variable region of light chain CDR of antibody DF200 or antibody Pan2D, as shown in figure 12.On the other hand, the invention provides antibody, antibody fragment or its arbitrary derivative, described antibody, antibody fragment or its arbitrary derivative contain the whole or basic all highly similar sequences to one or more variable region of light chain CDR of the light chain variable region sequence of DF200 or Pan2D or these antibody one or both of.
On the other hand, the invention provides antibody, antibody fragment or its arbitrary derivative, described antibody, antibody fragment or its arbitrary derivative contain variable region of heavy chain or the one or more variable region of light chain CDR of antibody DF200, as shown in figure 13.On the other hand, the invention provides antibody, antibody fragment or its arbitrary derivative, described antibody, antibody fragment or its arbitrary derivative contain whole to the weight chain variabl area sequence of DF200 or basic all highly similar sequences.
These and other favourable aspect of the present invention and characteristics will further specify in addition at this.
The accompanying drawing summary
Fig. 1 describes monoclonal antibody DF200, and described antibody combines with the common determinant of multiple human KIR2DL acceptor.
Fig. 2 describes monoclonal antibody DF200, and the cytotoxicity by the positive NK cell of KIR2DL1 of KIR2DL mediation in the described antibody and on the Cw4 positive target cell suppresses.
Fig. 3 describes monoclonal antibody DF200, DF200Fab fragment and KIR2DL1 or KIR2DL2/3 specificity conventional antibody, in the described antibody and suppressed by the cytotoxicity of the positive NK cell of KIR2DL1 of KIR2DL mediation on the Cw4 positive target cell and suppressed by the cytotoxicity of the positive NK cell of KIR2DL2/3 of KIR2DL mediation on the Cw3 positive target cell.
Fig. 4 is described in DF200 and EB6 antibody F (ab ') 2 fragments and exists down, the lysis that is caused by the NK clone of HLA Cw4 positive target cell.
Fig. 5 and 6 describes monoclonal antibody DF200, NKVSF1 (pan2D), people's antibody 1-7F9,1-4F1,1-6F5 and 1-6F1 and KIR2DL1 or KIR2DL2/3 specificity conventional antibody, cytotoxicity by the positive NK cell of KIR2DL1 of KIR2DL mediation in the described antibody and on the Cw4 positive target cell suppresses (being the Cw4 transfectional cell among Fig. 5, is the EBV cell among Fig. 6).
Fig. 7 describes and shows the epitope mapping of competition in conjunction with experimental result, and described result obtains by surface plasma resonance (BIAcore_) analysis with the anti-KIR antibody at KIR2DL1, and wherein the eclipsed circle refers to overlapping in conjunction with KIR2DL1.The result shows that 1-7F9 competes with EB6 and 1-4F1 on KIR 2DL1, but does not compete with NKVSF1 and DF200.Antibody 1-4F1 competes with EB6, DF200, NKVSF1 and 1-7F9 successively.Antibody NKVSF1 competes with DF200,1-4F1 and EB6 on KIR 2DL1, but does not compete with 1-7F9.DF200 competes with NKVSF1,1-4F1 and EB6 on KIR2DL1, but does not compete with 1-7F9.
Fig. 8 describes and shows the epitope mapping of competition in conjunction with experimental result, and described result obtains to analyze by BIAcore_ at the anti-KIR antibody of KIR2DL3, and wherein the eclipsed circle refers to overlapping in conjunction with KIR2DL3.The result shows that 1-4F1 competes with NKVSF1, DF200, g1183 and 1-7F9 on KIR2DL3.1-7F9 competes with DF200, g1183 and 1-4F1 on KIR2DL 3, but does not compete with NKVSF1.NKVSF1 competes with DF200,1-4F1 and GL183 on KIR2DL3, but does not compete with 1-7F9.DF200 competes with NKVSF1,1-4F1 and 1-7F9 on KIR2DL3, but does not compete with GL183.
Fig. 9 describes and shows the epitope mapping of competition in conjunction with experimental result, and described result obtains to analyze by BIAcore_ at the anti-KIR antibody of KIR2DS1, and wherein the eclipsed circle refers to overlapping in conjunction with KIR2DS1.The result shows that antibody 1-4F1 competes with NKVSF1, DF200 and 1-7F9 on KIR2DS1.Antibody 1-7F9 competes with 1-4F1 on KIR2DS1, but does not compete with DF200 and NKVSF1.NKVSF1 competes with DF200 and 1-4F1 on KIR2DS1, but does not compete with 1-7F9.DF200 competes with NKVSF1 and 1-4F1 on KIR2DS1, but does not compete with 1-7F9.
Figure 10 describes proof NKVSF1 (pan2D) mAb and macaque NK cell bonded NKVSF1 (pan2D) mAb titration.With macaque NK cell (total NK16 days) and the Pan2DmAb of different amounts, goat F (ab ') the anti-mouse IgG of 2 fragments (H+L) antibody incubation puted together with PE subsequently.Determine positive percentage with isotype contrast (the mouse IgG1 of purifying).Sample is measured in duplicate.Average fluorescent strength=MFI.
Figure 12 provides the comparison of antibody DF200 and Pan2D mAb variable region of light chain and variable region of light chain cdr amino acid sequence.
Figure 13 provides the variable region of heavy chain of antibody DF200.
Detailed Description Of The Invention
Antibody
The invention provides brand-new antibody and fragment thereof or derivative, the common determinant of described antibody and fragment thereof or derivative and human inhibition KIR acceptor (preferably being present at least two kinds of determinants on the different K IR2DL gene outcome) combination, and cause the enhancing of the NK cell of expressing at least one of those KIR acceptors. The present invention discloses this class cross reaction first and neutralizing antibody can be produced, and this has represented unpredictable consequence, and has opened road to the brand-new effective treatment (particularly in the human experimenter) based on NK. In preferred embodiments, antibody is not monoclonal antibody NKVSF1.
In literary composition of the present invention, " common determinant " refers to by several human inhibition KIR acceptor gene products shared determinant or epi-position. Common determinant is preferably shared by at least two kinds of KIR2DL acceptor group memberships. This determinant is more preferably by KIR2DL1 and KIR2DL2/3 share at least. Except identifying a plurality of KIR2DL gene outcomes, some antibody of the present invention also can be identified the determinant that is present on other inhibition KIR, for example gene outcome of KIR3DL acceptor group. Determinant or epi-position can represent fragments of peptides or the comformational epitope of being shared by described member. In more specific embodiment, the essentially identical epi-position that antibody specific binding of the present invention and monoclonal antibody DF200 identify. This determinant is present on KIR2DL1 and the KIR2DL2/3.
In literary composition of the present invention, the antibody of the common determinant of term " combination " refers to have specificity and/or affinity ground in conjunction with the antibody of described determinant.
Unless stated otherwise or with the obvious contradiction of context, term used herein " antibody " refers to fragment and the derivative of polyclone and monoclonal antibody and described polyclone and monoclonal antibody. According to the type of CH, generally full length antibody is appointed as one of five main classes: IgA, IgD, IgE, IgG and IgM. Wherein several Further Divisions are subclass or isotype, such as IgG1, IgG2, IgG3, IgG4 etc. To be called corresponding to the CH of inhomogeneity immunoglobulin (Ig) respectively " α ", " δ ", " ε ", " γ " and " μ ". Subunit structure and the 3-d modelling of inhomogeneity immunoglobulin (Ig) are widely known by the people. IgG and/or IgM are the preferred class that adopts antibody among the present invention, because they are prevailing antibody and because they produce in laboratory environment the easiliest under the physiological status. Antibody of the present invention is preferably monoclonal antibody. Because one of target of the present invention is the interaction of blocking-up inhibition KIR HLA part corresponding with it in the body and do not consume the NK cell, general preferably hang down the isotype of the Fc acceptor of effector function, for example IgG4 corresponding to mediation.
Can produce antibody of the present invention by multiple technology known in the art. Generally by producing antibody of the present invention with the immunogen immune non-human animal (preferred mouse) of containing inhibition KIR polypeptide (preferred KIR2DL polypeptide, more preferably human KIR2DL polypeptide). Inhibition KIR polypeptide can contain full length sequence or its fragment or the derivative of human inhibition KIR polypeptide, is generally immunogenic fragments, namely contains the polypeptide portion of the epi-position of the cell surface that is exposed to expression inhibiting KIR acceptor. This class fragment generally contains about 7 continuous amino acids of mature polypeptide sequence at least, even more preferably its about at least 10 continuous amino acids. The general basic extracellular domain derived from acceptor of fragment. More preferably human KIR2DL polypeptide, this polypeptide comprise at least one (more preferably two) extracellular Ig domain of total length KIRDL polypeptide, and can simulate at least one and be present in comformational epitope in the KIR2DL acceptor. In another embodiment, this polypeptide contains at least about 8 continuous amino acids of the extracellular Ig domain of KIR2DL1 polypeptide amino acid 1-224 position (amino acid coding is according to the PROW website of describing the KIR gene familyhttp://www.ncbi.nlm.nih.gov/prow/guide/1326018082.htm)。
In the most preferred embodiment, immunogene contains the human KIR2DL polypeptide of wild type of (generally at cell surface) in the adipose membrane. In specific embodiment, immunogene contains complete NK cell, is specially optional complete human NK cell through processing or cracking.
Can with stimulation well known in the art mouse produce antibody any mode (see for example E. Harlow and D.Lane, "Antibodies：A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)) carry out the step with the antigen immune non-human mammal. Immunogene is randomly suspended with adjuvant (for example complete Freund's adjuvant) or be dissolved in the buffer solution. The method of determining immunogenic amount, buffer type and adjuvant amount is well known to those skilled in the art, and does not limit in any form the present invention. For different immunogenes, these parameters may differently still be easy to illustrate.
Similarly, immune position and the frequency that is enough to stimulate antibody producing also known in this area. In typical immunization protocol, rear with antigen intraperitoneal injection non-human animal at first day and an about week. Be about the 20th day antigen memory injection (recall injection) subsequently, randomly contain for example incomplete Freunds adjuvant of adjuvant. Intravenous is carried out the memory injection, and a couple of days repeats continuously. After this be booster shots in the 40th day intravenous or the peritonaeum, generally do not contain adjuvant. This scheme caused the generation of the B cell that produces antigen-specific antibodies after about 40 days. Also can utilize other scheme, as long as they cause the generation of B cell, described B cellular expression is for the antibody of the antigen that is used for immunity.
For the preparation polyclonal antibody, obtain serum from the non-human animal of immunity, by known technical point from the antibody that wherein exists. Can use the above-mentioned any immunogene that is connected to solid support to the serum affinity purifying, thus the antibody of acquisition and inhibition KIR receptor response.
In alternate embodiment, separate and the non-immune non-human mammal lymphocyte of in vitro culture. Then it is exposed to immunogene in cell culture. Then gather in the crops lymphocyte, carry out following fusion steps.
For monoclonal antibody, next step is from the non-human mammal separating Morr. cell of immunity and subsequently those splenocytes and immortalized cells is merged to form the hybridoma that produces antibody. Be known in the art from the non-human mammal separating Morr. cell, produce single cell suspension the suitable buffer solution thereby relate generally to enter from the nylon mesh that the non-human mammal of anesthesia takes out spleen, is cut into fritter, splenocyte is squeezed out splenic capsule and passes the cell filter screen. With cell washing, centrifugal and be resuspended in any erythrocytic buffer solution of cracking. Solution is again centrifugal, and the lymphocyte that will be retained in the precipitation finally is resuspended in the fresh buffer.
In case separate and exist with single cell suspension, lymphocyte can merge with immortalized cell line. Although many other immortalized cell lines for generation of hybridoma known in this field, it is generally mouse myeloma cell line. Preferred Muridae myeloma is to comprise that (but being not limited to) can be from Salk Institute Cell Distribution Center, San Diego, Calif. U.S.A., X63 Ag8653 obtain derived from MOPC-21 and MPC-11 mouse tumor those and can be from American type culture collection (American Type Culture Collection), Rockville, the SP-2 cell that Maryland U.S.A obtains. Use the realization fusions such as polyethylene glycol. Then the hybridoma that produces is cultivated at the selective medium of the material that contains one or more parent myeloma cells' growths that suppress not fusion. For example, if parent myeloma cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium generally will contain hypoxanthine, aminopterin and thymidine (HAT culture medium), and these materials stop the growth of HGPRT deficient cells.
Generally cultivate hybridoma at the macrophage feeder layer. Macrophage is preferably from the littermate for separating of the non-human mammal of splenocyte, generally before the inoculation hybridoma a few days with initiations such as incomplete Freunds adjuvants. At Goding, " Monoclonal Antibodies:Principles and Practice ", the 59-103 page or leaf has been described fusion method in (Academic Press, 1986), and its disclosure is incorporated herein by reference.
Allow cell in the time of selecting the culture medium growth enough to form the clone and produce antibody. Usually between about 7 to about 14 days. Then measure the antibody of hybridoma clone generation and the cross reaction of multiple inhibition KIR acceptor gene product. Although can adopt any determination method that can be adapted to cultivate the hole of hybridoma, this determination method is generally colorimetric ELISA type and measures. Other mensuration comprises immunoprecipitation and radioimmunoassay. Check whether the positive hole of required antibody producing one or more different clones occur. If exist more than a clone, can with again time cloning and cultivation of cell, only be produced by individual cells to guarantee the clone who produces required antibody. The positive hole that generally will have a single obvious clone is time cloning and again measuring again, to guarantee only to detect and produce a kind of monoclonal antibody.
Also can produce antibody by selecting the immunoglobulin (Ig) combinatorial libraries, such as people such as Ward,Nature, disclosed in 341 (1989) 544 pages.
Antibody of the present invention also can in and the NK cell cytotoxicity of KIR mediation suppress; Specifically by the receptor-mediated inhibition of KIR2DL and more specifically at least KIR2DL1 and KIR2DL2/3 inhibition. Therefore these antibody be " neutralization " or " inhibition " antibody, and with regard to this meaning, when itself and the interaction of MHC I quasi-molecule, they maybe can block by KIR receptor-mediated inhibition signal path at least in part with detecting. More importantly, this inhibitory activity relates to a few type inhibition KIR acceptors, preferred several KIR2DL acceptor gene products, and more preferably at least KIR2DL1 and KIR2DL2/3, thus these antibody can be used for multiple experimenter efficiently. The inhibition that can suppress by the NK cell cytotoxicity that many measure or test (for example combination or raji cell assay Raji) evaluation mediate KIR.
In case determined the antibody with the cross reaction of multiple inhibition KIR acceptor, the ability that can test the inhibition effect of those KIR acceptors in its complete NK cell that neutralizes. In specific variant, can reproduce by described antibody that to cause the ability of HLA-C positive target cell cracking to illustrate neutralization by the positive NK clone of KIR2DL active. In another specific embodiment, ability by antibody suppression HLA-C molecule and KIR2DL1 and KIR2DL3 (or KIR2DL2 in close relations) receptors bind preferably is defined as in addition this antibody with the neutralization activity of this antibody and changes :-HLA-C molecule is combined with KIR2DL2/3, and described HLA-C molecule is selected from Cw1, Cw3, Cw7 and Cw8 (or at 80 HLA-C molecules with Asn residue); And
-HLA-C molecule is combined with KIR2DL1, and described HLA-C molecule is selected from the ability of Cw2, Cw4, Cw5 and Cw6 (or at 80 HLA-C molecules with Lys residue).
In another variant, can in based on the CTA of cell, evaluate the inhibitory activity of antibody of the present invention, as disclosed among the embodiment that provides at this.
In another variant, can in measuring, release of cytokines evaluate the inhibitory activity of antibody of the present invention, wherein with NK cell and test antibody and express the allelic target cell of a kind of HLA-C by NK group KIR molecular recognition system and hatch, stimulate the NK cell to produce cell factor (for example producing IFN-γ and/or GM-CSF). In exemplary scenario, cultivate about 4 days after, the IFN-γ that produces from PBMC by dyeing and flow cytometry evaluation in cell surface and the cytoplasm. In brief, can add at last about 4 hours that hatch the Brefeldin A (Sigma Aldrich) that final concentration is about 5 μ g/ml. Then can be at saturatingization (IntraPrepTM Beckman Coulter) front cell and anti-CD3 and anti-CD56mAb are hatched, and with the anti-IFN-γ of PE-or PE-IgG1 (Pharmingen) dyeing. Can use ELISA in supernatant, to measure GM-CSF and IFN-γ (GM-CSF:DuoSet Elisa, R﹠D Systems, Minneapolis, MN that the NK cell by polyclone activation produces; IFN-γ: OptE1A set, Pharmingen).
Antibody of the present invention can be partially or completely in and the NK cell cytotoxicity of KIR mediation suppress. Term used herein " in suppress with the NK cell cytotoxicity of KIR mediation " refers to when the NK cell mass of expressing given KIR contacts with the target cell of expression related MHC I quasi-molecule (KIR that expresses on by the NK cell identifies), compare with not containing the specificity cracking level that antibody obtains, with not because of the specificity cracking that same ratio was obtained of the NK cell of its KIR blocking-up or NK clone increase at least about 20%, preferably at least about 30%, at least about 40%, at least about the ability (by the classical chromium release of cytotoxicity thermometrically) of 5O% or above (for example about 25-100%). For example, the preferred antibody of the present invention can be Induced matching or that HLA is compatible or from somatic target cell group's (when namely lacking described antibody will not by the cell mass of the effective cracking of NK cell) cracking. Therefore, also antibody of the present invention can be defined as promotion NK cell activity in vivo.
In addition, the term inhibition of KIR mediation " in and " refer to the cytotoxicity level that in chromium is measured, obtains with antibody should be with the anti-MHC I of known blocking-up quasi-molecule (for example anti-MHC I of W6/32 antibody-like) obtain Cytotoxic at least about 20%, preferably at least about 30%, at least about 40%, at least about 50% (for example about 25-100%) or more, wherein chromium is measured and is used NK cell clone or the transfectant of expressing one or more inhibition KIR and only express a kind of allelic target cell of HLA that is identified by one of NK cell KIR.
In specific embodiment, this antibody and monoclonal antibody DF200 (being produced by hybridoma DF200) are in conjunction with essentially identical epi-position. This antibody-like is called " DF200 sample antibody " at this. In another preferred embodiment, antibody is monoclonal antibody. The present invention's preferred " DF200 sample antibody " is the antibody except monoclonal antibody NKVSF1. Monoclonal antibody DF200 (being produced by hybridoma DF200) most preferably.
Term and purpose antibody " in conjunction with essentially identical epi-position or determinant " refer to antibody and described purpose antibody " competition ". Term and monoclonal antibody DF200 " in conjunction with essentially identical epi-position or determinant " refer to antibody and DF200 " competition ". Usually, the antibody with purpose monoclonal antibody (for example DF200, NKVSF1,17F9) " in conjunction with basic identical epi-position or determinant " refers to any (being preferably selected from the KIR molecule of KIR2DL1 and KIR2DL2/3) in antibody and a plurality of KIR molecules of described purpose antibody competition. In other embodiments, on purpose antibody is combined the KIR2DL1 molecule antibody of essentially identical epi-position or determinant and purpose antibody " competition " in conjunction with KIR2DL1. The antibody of basic identical epi-position or determinant and purpose antibody " competition " are in conjunction with KIR2DL2/3 on purpose antibody is combined the KIR2DL2/3 molecule.
Term and purpose antibody " in conjunction with essence identical epi-position or determinant " refer to any and all KIR molecules of antibody and this purpose antibody specific binding of described purpose antibody " competition ". Term and monoclonal antibody DF200 " in conjunction with essence identical epi-position or determinant " refer to any and all KIR molecules of antibody and DF200 " competition " DF200 specific binding. For example, with monoclonal antibody DF200 or NKVSF1 in conjunction with the antibody of the identical epi-position of essence or determinant respectively with DF200 or NKVSF1 competition in conjunction with KIR2DL1, KIR2DL2/3, KIR2DS1 and KIR2DS2.
Use any in the multiple Immunological Screening determination method of evaluating antibody competition, can easily determine identity, described antibody and the epi-position that monoclonal antibody described here identification is basic or essence is identical of one or more antibody. Many such experiments are by the conventional enforcement in this area and know (see for example U.S. Patent No. 5,660,827 of announcement on August 26th, 1997, this patent in this specific reference as a reference). Should be appreciated that in fact and require never in any form to determine antibody described here in conjunction with epi-position to identify and the antibody of monoclonal antibody described here in conjunction with identical or basic identical epi-position.
For example, when test antibody to be checked available from the animal of separate sources or or even during different I g isotype, can adopt simple competition assay, wherein, to contrast (for example DF200) and test antibody and mix (or in advance absorption), and put on the sample that contains KIR2DL1 and KIR2DL2/3 (each is known to the DF200 combination). The use of analyzing (for example be recited in embodiment part in) based on the scheme of ELISA, radioimmunoassay, Western blotting and BIACORE is suitable for this class and simply competes in the research.
In certain embodiments, before putting on inhibition KIR antigen samples, should be pre-mixed one period to control antibodies (for example DF200) from the test antibody (for example about 1: 10 or about 1: 100) of different amounts. In other embodiments, can during being exposed to the KIR antigen samples, will contrast with the test antibody of different amounts and simply mix. As long as can distinguish combination and free antibody (for example use separate or cleaning technique is eliminated unconjugated antibody) and distinguish DF200 and test antibody (but for example use species specificity or isotype specific secondary antibody or with tags detected specific marker DF200), can determine whether test antibody reduces the combination of DF200 and these two kinds of different K IR2DL antigens, thereby explanation test antibody and DF200 identify essentially identical epi-position. When not having fully irrelevant antibody, the combination of (mark) control antibodies can be used as the high value of contrast. (DF200) antibody and the unlabelled complete homotype antibody (DF200) of mark are hatched (competition will occur at this, and reduce the combination of labelled antibody), can obtain to contrast low value. In measurements determination, the remarkable reduction of the reactivity of labelled antibody in the presence of test antibody shown the test antibody of the identifying basic identical epi-position antibody of mark (DF200) antibody " cross reaction " (namely with). At DF200: test antibody is under any ratio between about 1: 10 and about 1: 100, with in DF200 and KIR2DL1 and the KIR2DL2/3 antigen each in conjunction with reduce at least about 50%, for example at least about 60% or more preferably at least about the test antibody of 70% (for example about 65-100%), be considered to be combined with DF200 the antibody of basic identical epi-position or determinant. This class testing antibody preferably with in DF200 and the KIR2DL antigen each in conjunction with reducing at least about 90% (for example about 95%).
Can be by for example flow cytometry test evaluation competition. In such test, can be at first will have the cell of given KIR with DF200 for example, then hatch with fluorescent dye or biotin labeled test antibody. If about 80%, preferred about 50%, about 40% or lower (for example about 30%) (by fluoremetry) of the combination that obtains with saturation capacity DF200 preincubate time combination that obtained that is antibody not with the DF200 preincubate time then claim this antibody and DF200 to compete. In addition, if with the cell of saturation capacity test antibody preincubate on the combination that obtains with the DF200 of (with fluorescent dye or biotin) mark be not with about 80%, preferred about 50%, about 40% or lower (for example about 30%) of this antibody combination that preincubate is obtained, then claim this antibody and DF200 to compete.
Also can advantageously adopt simple competition assay, in this is measured, test antibody is adsorbed in advance with saturated concentration and be applied on the surface of also having fixed KIR2DL1 and KIR2DL2/3. The preferred BIACORE chip in surface in the simple competition assay (or other is suitable for the medium that surface plasma resonance is analyzed). Then with control antibodies (for example DF200) with to KIR2DL1 and saturated concentration and the Surface Contact of KIR2DL2/3, measure KIR2DL1 and the KIR2DL2/3 surface conjunction of control antibodies. With the combination of described control antibodies when not having test antibody control antibodies and the surface of containing KIR2DL1 and KIR2DL2/3 in conjunction with relatively. In test experiments, in the presence of test antibody, significantly reduced combination this test antibody of indication and the control antibodies on the surface of containing KIR2DL1 and KIR2DL2/3 by control antibodies and identify essentially identical epi-position, so this test antibody and control antibodies " cross reaction ". To contrast in (for example DF200) antibody and KIR2DL1 and the KIR2DL2/3 antigen each in conjunction with reduce at least about 30% or more preferably from about 40% test antibody can be considered to and contrast (for example DF200) in conjunction with the antibody of basic identical epi-position or determinant. This class testing antibody preferably with in control antibodies (for example DF200) and the KIR2DL antigen each in conjunction with reducing at least about 50% (for example at least about 60%, at least about 70% or more). Should be appreciated that the order that to put upside down contrast and test antibody: namely in competition assay, can at first control antibodies be attached to the surface, thereafter with test antibody and Surface Contact. The antibody that KIR2DL1 and KIR2DL2/3 antigen is had high affinity preferably at first is attached to the surface of containing KIR2DL1 and KIR2DL2/3, because the combination of expection SA (supposition antibody is cross reaction) will reduce larger quantity. Reach for example Saunal and Regenmortel at embodiment, (1995) other example that provides this class to measure among the J.Immunol.Methods 183:33-41, wherein Saunal and Regenmortel, the disclosure of (1995) J.Immunol.Methods 183:33-41 is incorporated herein by reference.
Although describe as an example of DF200 example in order to demonstrate, should be appreciated that above-mentioned Immunological Screening measure also can for the identification of with antibody and other antibody of the present invention of NKVSF1,1-7F9, EB6, GL183 competition.
After the immunity and antibody producing of vertebrate or cell, can carry out specific selection step and separate antibody of the present invention. About this point, in specific embodiment, the method that the present invention also relates to produce this antibody-like comprises:
(a) to contain the immunogen immune non-human mammal of inhibition KIR polypeptide;
(b) from described immune animal Dispersal risk, wherein said antibody is in conjunction with described KIR polypeptide,
(c) antibody of selection (b), the inhibition KIR gene outcome cross reaction that described antibody is different from least two kinds, and
(d) select the antibody of (c), described antibody can in on the NK cell mass of expressing described at least two kinds of different human inhibition KIR acceptor gene products by the inhibition of the NK cell cytotoxicity of KIR mediation.
Can resist the antibody realization of two or more different inhibition KIR antigens to the selection of the antibody of the inhibition KIR gene outcome cross reaction different from least two kinds by screening, for example mentioned above.
In a more preferred embodiment, the antibody of preparation is monoclonal antibody in the step (b). Therefore, term " from described immune animal Dispersal risk " comprises from immune animal acquisition B cell and uses those B cells produce to express the hybridoma of antibody as used herein, and directly obtains antibody from immune serum. In another preferred embodiment, the antibody of in step (c), selecting be at least with the antibody of KIR2DL1 and KIR2DL2/3 cross reaction.
In another preferred embodiment, as measured in the standard chromium release assay, with comparing than the cracking or the cytotoxicity that obtain with identical effector cell/target cell with the NK cell of not blocked by its KIR, the antibody of selecting in the step (d) cause by the NK cell to the target cell mediation at least about 10% specificity cracking, preferably at least about 40% specificity cracking, at least about 50% the specificity cracking or more preferably at least about 70% specificity cracking (for example specificity cracking of about 60-100 %), at least a KIR by this antibody recognition of described NK cell display, described target cell is expressed relevant HLA I quasi-molecule. In addition, when the antibody of selecting in the step (d) is used for the chromium test, the cytotoxicity level that obtains with this antibody should be the anti-MHC I of the property blocked class mAb (for example anti-MHC I of W6/32 antibody-like) obtain Cytotoxic at least about 20%, preferably at least about 30% or more, described chromium test is adopted the NK cell clone of expressing one or more inhibition KIR and is only expressed a kind of allelic target cell of HLA that is identified by one of KIR on the NK clone.
Above just the step of described method (c) and order (d) can change. Randomly, the method also or may comprise in addition the additional step of the derivative of making monoclonal antibody fragment or monoclonal antibody or this class fragment, and is for example described in addition at this.
In preferred embodiments, applicable method according to the present invention is mammal for the production of the non-human animal of antibody, such as rodent (such as mouse, rat etc.), ox, pig, horse, rabbit, goat, sheep etc. Non-human mammal also can produce " mankind " antibody, for example Xenomouse through genetic modification or through transformingTM(Abgenix) or HuMAb-MouseTM(Medarex)。
In another variant, the invention provides the method that obtains antibody, comprising:
(a) from library or storehouse, select monoclonal antibody, monoclonal antibody fragment or its arbitrary derivative, the human inhibition KIR2DL acceptor gene product cross reaction that described monoclonal antibody, monoclonal antibody fragment or its arbitrary derivative are different from least two kinds, and
(b) select antibody, fragment or derivative in (a), described antibody, fragment or derivative can in on the NK cell mass of expressing described at least two kinds of different human inhibition KIR2DL acceptor gene products, suppressed by the NK cell cytotoxicity of KIR mediation.
Described storehouse can be any (restructuring) storehouse of antibody or its fragment, is randomly showed by any appropriate configuration (such as bacteriophage, bacterium, synthesising complex etc.). The selection of inhibiting antibody can be according to execution disclosed above, and is further set forth in an embodiment.
According to another embodiment, the invention provides the hybridoma that contains non-human host B cell, wherein said B cell produce be present at least two kinds of different human inhibition KIR acceptor gene products on the antibody of determinant combination, described antibody can in the inhibitory activity of this receptor. The present invention's hybridoma in this respect more preferably is not the hybridoma that produces monoclonal antibody NKVSF1. Can be as mentioned above the non-human mammal splenocyte of immunity and immortalized cell line be merged and produce the present invention's hybridoma in this respect. Can screen the in addition existence of described this class cross reacting antibody such as this paper to merge the hybridoma that produces by this. Hybridoma preferably produces the antibody that identification is present in two kinds of determinants on the different K IR2DL gene outcome at least, and described antibody causes at least enhancing of the NK cell of one of those KIR acceptors of expression. Hybridoma more preferably produces the antibody of being combined essentially identical epi-position or determinant with DF200 and strengthening the NK cytoactive. Hybridoma most preferably is the hybridoma DF200 of manufacture order clonal antibody DF200.
Can in suitable culture medium (for example DMEM or RPMI-1640), cultivate in a large number the hybridoma of verified generation monoclonal antibody of the present invention. In addition, can be with hybridoma as cultivating in the animal ascites tumour body.
After fully cultivating the required monoclonal antibody of generation, will contain growth medium (or ascites liquid) and the cell separation of monoclonal antibody, the monoclonal antibody that purifying wherein exists. Generally realize purifying by gel electrophoresis, dialysis, chromatogram, wherein chromatogram use a-protein or protein G-Sepharose or with solid support for example the anti-mouse Ig that is connected of agarose or Sepharose pearl (all are all at for example " Antibody Purification Handbook ", Amersham Biosciences, publication number No.18-1037-46, illustrate in the AC version that wherein " Antibody Purification Handbook " is incorporated herein by reference). Normal operation hangs down pH buffer solution (pH3.0 or lower glycine or acetate buffer solution) and contains antibody level part from the antibody of a-protein/protein G post elution of bound by direct neutralization. As required these grades part is concentrated, dialysed and concentrates.
According to alternate embodiment, the DNA that separates encoding antibody from hybridoma of the present invention, and it is inserted the suitable expression vector of transfection suitable host, described antibody is in conjunction with being present at least two kinds of determinants on the different human inhibition KIR acceptor gene products. Then the host is used for recombinant production antibody or its variant (for example the active fragment of the humanization form of this monoclonal antibody, this antibody or contain the chimeric antibody of the antigen recognition site of this antibody). The DNA optimized encoding identification that is used for this embodiment is present in the antibody of at least two kinds of determinants on the different K IR2DL gene outcome, and described antibody causes at least enhancing of the NK cell of one of those KIR acceptors of expression. This DNA more preferably encodes and is combined basic identical epi-position with DF200 and strengthens the antibody of NK cytoactive. This DNA monoclonal antibody DF200 that most preferably encodes.
Use conventional scheme to be easy to that DNA with coding monoclonal antibody of the present invention separates and order-checking (for example using can specific binding coding mouse-anti body weight and the oligonucleotide probe of the gene of light chain). In case separate, DNA can be inserted expression vector, this expression vector is then transfected in host cell (for example not producing in addition Escherichia coli (E.coli) cell, ape COS cell, Chinese hamster ovary (CHO) cell or the myeloma cell of immunoglobulin (Ig)), obtains monoclonal antibody synthesizing in recombinant host cell. Antibody coding DNA in bacterium recombinant expressed for known in the art (see such as people such as Skerra,Curr.Opinion in Immunol., 5,256 pages (1993); Pluckthun,Immunol.Revs., 130,151 pages (1992).
Monoclonal antibody fragment and derivative
Can by technology well known in the art produce antibody fragment of the present invention and derivative (unless stated otherwise or with the obvious contradiction of context, term " antibody " used among the application comprises antibody fragment and derivative), preferred DF-200 sample antibody. " immunoreactivity fragment " contains the part of complete antibody, is generally antigen binding site or variable region. The example of antibody fragment comprises Fab, Fab ', Fab '-SH, F (ab ')2With the Fv fragment; Double function miniature antibody; Any antibody fragment, described antibody fragment is the polypeptide (referred to here as " single chain antibody fragments " or " single chain polypeptide ") that primary structure has the uninterrupted sequence of continuous amino acid residue, include but not limited to that (1) scFv (scFv) molecule (2) contains the only single chain polypeptide of a light chain variable domain, or its fragment and (3) of containing three CDR of light chain variable domain, relevant heavy chain part contain the only single chain polypeptide of a weight chain variable domain, or it contains the fragment of three CDR of weight chain variable domain, relevant light chain part; And the multi-specificity antibody that is formed by antibody fragment. For example, can produce according to the antibody that routine techniques separates by protease digestion Fab or F (ab ') 2 fragments. Should be appreciated that and to use known method to modify the immunoreactivity fragment, thereby for example delay the interior pharmacokinetics feature of removing and more being needed of body. Can modify fragment with polyethylene glycol (PEG). Such as people such as Leong,Cytokine16 (3): the people such as 106-119 (2001) and Delgado,Br.J.Cancer73 (2): described PEG among the 175-182 (1996) to being coupled and the locus specificity conjugation methods of Fab ' fragment, the people's such as the people such as Leong and Delgado disclosure is incorporated herein by reference.
Aspect specific, the invention provides the antibody, antibody fragment and the antibody derivatives that contain the DF200 light chain variable region sequence of listing among Figure 12. Another particular aspects the invention provides the antibody, antibody fragment and the antibody derivatives that contain the Pan2D light chain variable region sequence of listing among Figure 12. On the other hand, the invention provides antibody, antibody fragment and the derivative thereof of the one or more variable region of light chain CDR that contain the DF200 that lists among Figure 12. On the other hand, the invention provides antibody, antibody fragment and the derivative thereof of the one or more variable region of light chain CDR that contain the Pan2D that lists among Figure 12. Can the Application standard technology (also can carry out sequence relatively) by in these disclosed amino acid sequences, carrying out suitable replacement, interpolation and/or deletion to produce the functional variant/analog of this class sequence. Therefore, for example, CDR residue conservative between Pan2D and the DF-200 may be suitable modification target, because this class residue may not contributed (although Pan2D and DF-200 competition) about the different characteristic in the competition of other antibody disclosed herein to these antibody, therefore may be to these antibody for its separately not contribution of specificity of defined epitope. On the other hand, the position of (but not another) in residue is present in the sequence of one of these antibody, this position may be suitable for deletion, replaces and/or insert.
Aspect specific, the invention provides the antibody, antibody fragment and the antibody derivatives that contain the DF200 weight chain variabl area sequence of listing among Figure 13. On the other hand, the invention provides antibody, antibody fragment and the derivative thereof of the one or more variable region of heavy chain CDR that contain the DF200 that lists among Figure 13. Can the Application standard technology by in these disclosed amino acid sequences, making suitable replacement, interpolation and/or deletion to produce the functional variant/analog of this class sequence, this will have benefited from sequence relatively. On the other hand, those wherein residue be present in the sequence of one of these antibody but the position that is not present in other sequence may be suitable for deletion, replaces and/or insert.
In addition, can modify produce antibody of the present invention (preferred DF-200 sample antibody) thus the hybridoma DNA fragment of the present invention of encoding. Then modifying DNA is inserted expression vector and be used for conversion or the suitable cell of transfection, then described cell expresses required fragment.
In alternative embodiment, can be before inserting expression vector, by such as and light chain constant domain coded sequence replacement homology non-human sequence heavy with the mankind (such as people such as Morrison,Proc.Natl.Acad.Sci.U.S.A., 81,6851 pages (1984)) or by all or part of NIg polypeptid coding sequence and the covalently bound modification of immunoglobulin coding sequence being produced the hybridoma DNA of antibody of the present invention (preferred DF-200 sample antibody). In this form, preparation has " chimeric " or " hybridization " antibody of original antibody binding specificity. Generally, replace the constant domain of antibody of the present invention with this class NIg polypeptide.
Therefore, according to another embodiment, antibody of the present invention (preferred DF-200 sample antibody) is humanized. To contain specific chimeric immunoglobulin (Ig), immunoglobulin chain or its fragment derived from the minmal sequence of rat immune globulin (for example Fv of antibody, Fab, Fab ', F (ab ') according to " humanization " form of antibody of the present invention2, or other antigen zygote sequence). In most cases, humanized antibody is human immunoglobulin (receptor's antibody), in the described human immunoglobulin, residue from receptor's complementary determining region (CDR) is replaced by the residue from original antibody (donor antibody) CDR, keeps simultaneously required specificity, affinity and the ability of original antibody. The Fv framework residue that can replace with corresponding non-human residue in some instances, human immunoglobulin. In addition, humanized antibody can contain in the receptor antibody or the residue that does not have in the CDR that introduces or the frame sequence. Making these modifies further make with extra care and optimizes the antibody performance. Usually, humanized antibody will comprise nearly all at least one (being generally two) variable domains, all or nearly all CDR district be corresponding to the CDR district of original antibody in described variable domains, and all or nearly all FR district are the FR districts of human immunoglobulin concensus sequence. Humanized antibody preferably also will comprise at least a portion constant region for immunoglobulin (Fc) (being generally the human immunoglobulin constant region). Further details is seen the people such as Jones,Nature, 321,522 pages (1986); The people such as Reichmann,Nature, 332,323 pages (1988); And Presta,Curr.Op.Struct.Biol., 2,593 pages (1992).
With antibody humanization's of the present invention method is known in the art.Usually, humanized antibody according to the present invention has one or more from original antibody introducing amino-acid residue wherein.Often these Muridaes or other non-human amino-acid residue are called " introducing " residue, generally take from " introducing " variable domains.Can be substantially according to Winter and colleague's method (people such as Jones, Nature, 321,522 pages (1986); People such as Riechmann, Nature, 332,323 pages (1988); People such as Verhoeyen, Science, 239,1534 pages (1988)) and the execution humanization.Therefore, this class " humanization " antibody is chimeric antibody, is replaced substantially less than complete human variable domains people such as (, U.S. Patent No. 4,816,567) Cabilly by the corresponding sequence of original antibody.In fact, humanized antibody according to the present invention is generally human antibodies, and number of C DR residue and some FR residues of possibility are replaced by the residue in the similar site of original antibody in the described human antibodies.
The selection of human variable domains (light and heavy chain) that is used to make humanized antibody is extremely important for reducing antigenicity.According to so-called " the most suitable " method, to the variable domains sequence of the complete library screening antibody of the present invention of known human variable domains sequence.Accept then with the immediate human sequence of mouse sequence as the human framework (FR) of humanized antibody (people such as Sims, J.Immunol., 151,2296 pages (1993); Chothia and Lesk, J.Mol. Biol., 196,901 pages (1987)).Another method is used the specific framework from the concensus sequence of everyone antibody-like of light or the specific subgroup of heavy chain.Same architecture can be used for several different humanized antibodies (people such as Carter, Proc.Natl.Acad.Sci.U.S.A., 89,4285 pages (1992); People such as Presta, J.Immunol., 51,1993 people such as grade)).
What is more important keeps the antibody humanization to the high affinity of multiple inhibition KIR acceptor and other favourable biological characteristics.In order to realize this target, according to preferable methods, the three-dimensional model analysis female parent sequence by using maternal and humanization sequence and the method for multiple notional humanization product prepare humanized antibody.Three-dimensional immunoglobulin (Ig) model generally can obtain, and is that those skilled in the art are familiar with.Can obtain diagram and show the computer program of the possible three-dimensional conformation structure of selected candidate's immunoglobulin sequences.The inspection of these displayings allows to analyze the possible role of residue in candidate's immunoglobulin (Ig) function, the i.e. residue of analyzing influence candidate immunoglobulin (Ig) and its antigen binding capacity.Like this, can make peace from one and select the calling sequence and combination FR residue, thereby realize required antibody feature, for example the avidity to target antigen increases.Usually, directly also main relating to, influence the antigen combination to the CDR residue.
The another kind of method of making " humanization " monoclonal antibody is to use XenoMouse_, and (Abgenix, Fremont is CA) as immune mouse.XenoMouse is according to Muridae host of the present invention, and its immunoglobulin gene is replaced by the functional human immunoglobulin gene.Therefore, passed through humanization by this mouse or the antibody that produces by the hybridoma that this mouse B cell is made.In U.S. Patent No. 6,162, XenoMouse has been described in 963, wherein U.S. Patent No. 6,162, and 963 are incorporated by reference in this text and examine.Use HuMAb-Mouse TM(Medarex) can finish similar approach.
Also can produce human antibodies, for example use other transgenic animal to be used for immunity people such as (, Nature362 (1993) 255) Jakobovitz, or use the phage display method to select antibody library through design express human antibody storehouse according to multiple other technology.This class technology is that the technician is known, and can begin to carry out from the disclosed monoclonal antibody of the application.
Also can deriving be " chimeric " antibody (immunoglobulin (Ig)) with antibody of the present invention (preferred DF-200 sample antibody), as long as they show required biologic activity, consistent or the homology of corresponding sequence of the part of heavy and/or light chain and original antibody in described " chimeric " antibody, and the remainder of chain with derived from other species or belong to the antibody of other antibody class or subclass and the segmental corresponding sequence of this antibody-like is consistent or homology (people such as Cabilly, as above; People such as Morrison, Proc.Natl.Acad.Sci.U.S.A., 81,6851 pages, (1984)).
Other derivative within the scope of the present invention comprises functionalization antibody, promptly with toxin (for example Ricin, diphtheria toxin, toxalbumin and pseudomonas (Pseudomonas) extracellular toxin); Test section but (for example fluorescence part, radio isotope or preparation); Or solid support (for example agarose pearl etc.) is puted together or covalently bound antibody.These other reagent and antibody are puted together or covalently bound method is well known.
Be conjugated with the target killing that benefits at the NK cell of one of its cell surface display cross reactivity KIR acceptor with toxin.In a single day antibody of the present invention combines with the cell surface of this class cell, it will be by internalization, and toxin is released in the cell, this cell of selective killing.This class purposes is an alternative embodiment of the present invention.
When antibody of the present invention is used for diagnostic purpose, be useful but put together with the test section.The comprising of this classification (but being not limited to) biological sample is determined at its cell surface and has the existence of NK cell of cross reactivity KIR and the existence that in live organism, detects NK cell with cross reactivity KIR.This class is measured and detection method also is an alternative embodiment of the present invention.
As having the instrument of the NK cell of cross reactivity KIR from source (biological example liquid) protein affinity purification at its cell surface, it is useful that antibody of the present invention and solid support are puted together.As the NK cell mass of the purifying that produces, described purification process is that of the present invention another is alternative
Embodiment.
In alternative embodiment, antibody (comprising NKVSF1) can be used for being incorporated into liposome (" immunoliposome ") to the material of animal targeted separately or with another kind, wherein said antibody be present at least two kinds of common determinant combinations on the different human inhibition KIR acceptor gene products, can in on the NK cell of expressing one of two kinds of different human inhibition KIR acceptors of described the present invention at least, suppress by the NK cell cytotoxicity of KIR mediation.Other material of this class is included as the nucleic acid that gene therapy is carried gene or carried sense-rna, RNAi or siRNA for suppressor gene in the NK cell; Or be used for toxin or the medicine that the NK cell-targeting kills and wounds.
Based on its crystalline structure of delivering ( MaenakaDeng the people, (1999), people such as Fan, (2001), people such as Boyington, (2000)), the computer simulation of KIR2DL1,2 and 3 (KIR2DL1-3) extracellular domain has been predicted and has been related to interactional some zone or KIR2DL1,2 and 3 between KIR2DL1 and KIR2DL1-3 cross reactivity mouse monoclonal antibody DF200 and the NKVSF1.Therefore, in one embodiment, the invention provides in and the single-minded bonded antibody of KIR2DK1 by the zone of amino-acid residue (105,106,107,108,109,110,111,127,129,130,131,132,133,134,135,152,153,154,155,156,157,158,159,160,161,162,163,181,192) definition.In another embodiment, the invention provides combine with KIR2DL1 and KIR2DL2/3 and not with by the interactional antibody of amino-acid residue outside the zone of residue (105,106,107,108,109,110,111,127,129,130,131,132,133,134,135,152,153,154,155,156,157,158,159,160,161,162,163,181,192) definition.
In another embodiment, the invention provides combine with KIR2DL1 and not with KIR2DL1 mutant bonded antibody, in the described KIR2DL1 mutant, R131 is Ala.
In another embodiment, the invention provides combine with KIR2DL1 and not with KIR2DL1 mutant bonded antibody, in the described KIR2DL1 mutant, R157 is Ala.
In another embodiment, the invention provides combine with KIR2DL1 and not with KIR2DL1 mutant bonded antibody, in the described KIR2DL1 mutant, R158 is Ala.
In another embodiment, the invention provides and KIR2DL1 residue (131,157,158) bonded antibody.
In another embodiment, the invention provides with KIR2DS3 (R131W) combine, but not with wild-type KIR2DS3 bonded antibody.
In another embodiment, the invention provides and KIR2DL1 and KIR2DL2/3 and KIR2DS4 bonded antibody.
In another embodiment, the invention provides with KIR2DL1 and KIR2DL2/3 combine, but not with KIR2DS4 bonded antibody.
Can determine whether combination in one of epi-position district of above-mentioned definition of antibody with the method for well known to a person skilled in the art.As an example of this class mapping/characterizing method, can use the chemically modified of the amino/carboxyl that exposes in KIR2DL1 or the KIR2DL2/3 protein to determine the epi-position district of anti-KIR antibody by epi-position " footprinting (foot-printing) ".A particular instance of this class footprinting technology is to use HXMS (hydrogen-deuterium exchange that mass spectroscopy detects); hydrogen/deuterium exchange, the combination of acceptor and ligandin matter acyl ammonia proton wherein occur and gain (backexchange); the skeleton amido group that wherein participates in protein bound gains protected avoiding, thereby will keep deuterate.Can identify domain of dependence at this point by stomach en-proteolysis, quick micropore high performance liquid chromatography separation and/or electro-spray ionization mass spectroscopy.See for example Ehring H, Analytical Biochemistry, Vol.267 (2) 252-259 page or leaf (1999) and/or Engen, J.R. and Smith, D.L. (2001) Anal.Chem.73,256A-265A.Another example of suitable epi-position authenticate technology is nucleus magnetic resonance epitope mapping (NMR), in the generally more two-dimentional NMR spectrum free antigen and with the antigenic signal location of hla binding peptide (for example antibody) compound.Generally with 15N selectivity isotopic labeling antigen, thus rarely seen on the NMR spectrum corresponding to antigenic signal, and less than the signal from hla binding peptide.Compare with free antigen spectrum, be derived from that relate to generally will displacement in the mixture spectrum with the interactional amino acid whose antigen signals of hla binding peptide, can identify like this to relate to bonded amino acid.See for example Ernst Schering Res Found Workshop.2004; (44): 149-67; People such as Huang, Journal of Molecular Biology, Vol.281 (1) 61-67 page or leaf (1998); And Saito and Patterson, Methods.1996 Jun; 9 (3): 516-24.
Also can use mass spectrometry method to carry out epitope mapping/sign.See for example Downward, J MassSpectrom.2000 Apr; 35 (4): 493-503 and Kiselar and Downard, AnalChem.1999 May1; 71 (9): 1792-801.
Protease digestion also can be useful at epitope mapping with in identifying.Can pass through protease digestion (for example, use with about 1: 50 ratio of KIR2DL1 or KIR2DL2/3 and use trypsinase) and peptide subsequently and identify mass spectrum (MS) analysis, determine antigenic determinant relevant range/sequence in 37 ℃ and pH7-8 o/n digestion.Sample that subsequently can be by relatively standing tryptic digestion and with antibody incubation, stand for example sample of tryptic digestion (thereby being that binding substances is showed footprint) is then identified the peptide that is avoided the trypsinase cutting by anti-KIR binding substances protection.Also or in addition can be used for similar epi-position characterizing method as other enzymes such as Chymotrypsin, stomach en-s.In addition, enzymic digestion can provide fast method, is used to analyze non-surperficial exposure and also so under most probable and irrelevant the resisting of immunogenicity/antigenicity-KIR polypeptide situation whether has potential antigenic determinant sequence in the KIR2DL1 zone.For example Manca is seen in the discussion of similar techniques, AnnIst Super Sanita.1991; 27 (1): 15-9.
Cross reactivity with macaque
Find that antibody NKVSF1 also combines with macaque NK cell, sees embodiment 7.Therefore the present invention provides antibody and fragment and derivative, the cross reaction of the human KIR acceptor of at least two kinds of inhibitions of wherein said antibody, fragment or derivative and human NK cell surface, and in conjunction with macaque NK cell.In one embodiment, this antibody is not antibody NKVSF1.The present invention also provides test antibody and fragment and derivative thereof toxic method, the cross reaction of the human KIR acceptor of at least two kinds of inhibitions of wherein said antibody, fragment or derivative and human NK cell surface, and wherein method is included in this antibody of test in the macaque.
Composition and using
The present invention also provides the pharmaceutical composition that contains antibody and fragment and derivative with the significant quantity of NK cell cytotoxicity that can strengthen the patient with detecting or contain the biological sample of NK cell in suitable carriers, wherein said antibody, fragment or derivative and the cross reaction of at least two kinds of inhibition KIR of NK cell surface acceptor, its inhibition signal and strengthen those cell activity neutralizes.Said composition contains pharmaceutically acceptable carrier in addition.Also this based composition is called " antibody compositions of the present invention ".In one embodiment, antibody compositions of the present invention contains above disclosed antibody in the antibody embodiment.Antibody NKVSF1 is included in the antibody scope that may come across in the antibody compositions of the present invention.
Term used herein " biological sample " includes, but are not limited to biological liquid (for example serum, lymph liquid, blood), cell sample or tissue sample (for example marrow).
The pharmaceutically acceptable carrier that can be used for these compositions comprises (but being not limited to) ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein (for example human serum albumin), buffer substance is phosphoric acid salt for example, glycine, Sorbic Acid, potassium sorbate, the partial glyceride mixture of saturated vegetable fatty acid, water, salt or ionogen, for example protamine sulfate, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, silica gel, Magnesium Trisilicate, polyvinylpyrrolidone, based on cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, wax, polyethylene-polyoxypropylene-block polymer, polyoxyethylene glycol and lanolin.
In the method that strengthens patient or biological sample NK cytoactive, can adopt composition of the present invention.This method comprises the step that composition is contacted with patient or biological sample.These class methods will be of value to diagnosis and therapeutic purpose.
For using in conjunction with biological sample, depend on properties of samples (liquid or solid), can simply mix with sample or directly apply to sample and the administration of antibodies composition.Can in any suitable device (plate, bag, bottle etc.), biological sample directly be contacted with antibody.For using in conjunction with the patient, said composition must be through formulated for administered in the patient.
Can be oral, parenteral, by suck spraying, part, per rectum, intranasal, through the oral cavity, transvaginal or store storehouse (implanted reservoir) by implantation and use composition of the present invention.That term used herein " parenteral " comprises is subcutaneous, in the intravenously, intramuscular, intraarticular, synovia, in the breastbone, in the sheath, in the liver, intralesional and intracranial injection or infusion techn.Preferred oral, intraperitoneal or intravenously are used said composition.
The sterilization injectable forms of composition of the present invention can be water-based or oily suspensions.Can use suitable dispersion or wetting agent and suspension agent formulated suspension according to technology well known in the art.The agent of sterilization injectable also can be sterilization Injectable solution or the suspension in acceptable nontoxic thinner of parenteral or the solvent, for example solution in the 1,3 butylene glycol.In acceptable carrier and solvent adoptable be water, Ringer solution and etc. open sodium chloride solution.In addition, the conventional disinfectant fixed oil that adopts is as solvent or suspension medium.For this reason, the fixed oil of any gentleness be can adopt, synthetic monoglyceride or triglyceride comprised.Lipid acid (for example oleic acid and glyceride derivative thereof) is useful in the preparation of injection, as natural acceptable oil (for example sweet oil or Viscotrol C, especially its polyoxy ethylization form.These oil solutions or suspension also can contain long-chain alcohol thinner or dispersion agent, for example are usually used in preparing the carboxymethyl cellulose or the similar dispersion agent of the pharmaceutically acceptable dosage form that comprises emulsion and suspension.Other tensio-active agent (for example being usually used in making Tween, Span and other emulsifying agent or the bioavailability reinforce of pharmaceutically acceptable solid, liquid or other dosage form) commonly used also can be used to prepare purpose.
Can be with the Orally administered composition of the present invention of any oral acceptable dosage form, described oral acceptable dosage form comprises (but being not limited to) capsule, tablet, waterborne suspension or solution.Orally using under the situation of tablet, common carrier comprises lactose and W-Gum.Generally also add lubricant, for example Magnesium Stearate.For Orally administered with capsule form, useful thinner comprises lactose and dried corn starch.When requiring waterborne suspension to orally use, with activeconstituents and emulsifying agent and suspension agent combination.If desired, also can add some sweeting agent, seasonings or tinting material.
In addition, can use composition of the present invention with the suppository form of rectal administration.Can be by reagent and suitable nonirritant excipient be mixed with these, described suitable nonirritant excipient is a solid in room temperature, but is liquid in rectal temperature, therefore will melt the release medicine in rectum.This class raw material comprises theobroma oil, beeswax and polyoxyethylene glycol.
Also can topical application composition of the present invention, especially when the treatment target comprises zone that topical application is easy to arrive or organ (comprising eye, skin or lower intestinal tract disease).Be easy to be the suitable topical formulations of each preparation in these zones or the organ.
Can realize the lower intestinal tract topical application by rectal suppository preparation (on seeing) or by suitable enema preparation.Also can use the topical transdermal patch.
For topical application, said composition can be formulated in the suitable ointment, described ointment contains and suspends or be dissolved in activeconstituents in one or more carriers.The carrier that is used for topical application compound of the present invention comprises (but being not limited to) mineral oil, petrosio (liquidpetrolatum), white vaseline, propylene glycol, polyethylene oxide, polyoxypropylene compound, emulsifying wax and water.In addition, said composition can be formulated in the suitable lotion or emulsifiable paste, described lotion or emulsifiable paste contain and suspend or be dissolved in activeconstituents in one or more pharmaceutically acceptable carrier.Suitable carriers comprises (but being not limited to) mineral oil, anhydrosorbitol monostearate, polysorbate 60, cetyl esters wax, hexadecanol, 2-Standamul G, phenylcarbinol and water.
Use for ophthalmology, said composition can be formulated as the grade of regulating pH and open micronization suspension in the disinfection salt solution, or preferably regulate pH etc. open solution in the disinfection salt solution, contain or do not contain this class sanitas such as benzylalkonium chloride.In addition, use, said composition can be formulated in the ointment, for example vaseline for ophthalmology.
Also can use composition of the present invention by nose aerosol or inhalation.Prepare this based composition according to the well-known technology of field of pharmaceutical preparations, and can adopt phenylcarbinol or other suitable sanitas, absorption enhancer, fluorocarbon and/or other the conventional solubilising or the dispersion agent that strengthen bioavailability be prepared as solution in salts solution.
Several monoclonal antibodies are presented in the clinical state effective, for example Rituxan (Rituximab), Herceptin (Trastuzumab) or Xolair (Omalizumab) can use similar application program (being preparation and/or dosage and/or application process) to antibody of the present invention.Can be according to scheme and the dosage of determining to use antibody in the pharmaceutical composition of the present invention about the currently known methods (for example using manufacturers instruction) of these products.For example, can with the antibody that exists in the pharmaceutical composition of the present invention with the concentration deposit of 10mg/mL in 100mg (10mL) or 500mg (50mL) one-trip bottle.The sterilized water that product is formulated in 9.0mg/mL sodium-chlor, 7.35mg/mL Trisodium citrate dihydrate, 0.7mg/mL polysorbate 80 and is used for injecting is used for IV and uses.Regulate pH to 6.5.The suitable exemplary dosage range of antibody can be at about 10mg/m in the pharmaceutical composition of the present invention 2And 500mg/m 2Between.Yet, should be appreciated that these schemes are exemplary, can consider the avidity and the tolerance adjustment preferred plan of specific antibodies in the pharmaceutical composition, the avidity of specific antibodies and tolerance are must determine in the clinical trial in the described pharmaceutical composition.Determine that with considering antibody affinity and pharmacokinetic parameter thereof the antibody in the injection pharmaceutical composition of the present invention soaked into the NK cell 24 hours, 48 hours, 72 hours or the amount and the scheme in a week or one month.
According to another embodiment, antibody compositions of the present invention can contain another kind of therapeutical agent in addition, comprises the reagent of the particular treatment purpose that is generally used for using described antibody.Extra therapeutical agent appears in the composition with the general consumption of this reagent in the monotherapy of treatment specified disease or illness usually.This class therapeutical agent comprises that (but being not limited to) is used for the fragment of the therapeutical agent of cancer therapy, the therapeutical agent that is used for the treatment of infectious diseases, the therapeutical agent that is used for other immunotherapy, cytokine (for example IL-2 or IL-15), other antibody and other antibody.
For example, can utilize the therapeutical agent of many cancer therapy.Antibody compositions of the present invention and method can with any other method combination that is usually used in treating specified disease (being specially other disease or the disorder of tumour, Cancerous disease or patient's performance).As long as know that specific methods of treatment is harmless to the patient in essence, and significantly do not offset the antibody activity in the composition of the present invention, imagination makes up itself and the present invention.
About treatment of solid tumors, can be used in combination pharmaceutical composition of the present invention with classical way (for example operation, radiotherapy, chemotherapy etc.).Therefore the present invention provides combined therapy, wherein, operation or radiotherapy simultaneously, before or after use pharmaceutical composition of the present invention; Or in the immunotoxin or coaguligand of conventional chemical treatment, radiotherapy or anti-angiogenic agent or target, before or after be applied to the patient.
When one or more reagent and the present invention contained the combination of compositions use of antibody in treatment plan, the result who does not require combination was the addition of observed effect when carrying out each treatment separately.Although need addition at least usually, any antitumous effect that increases than a kind of independent treatment will be favourable.Equally, combined therapy there is not particular requirement performance synergy, although this is possible and favourable really.
In order to carry out the combination anticancer therapy, with another kind of carcinostatic agent combination, give animal simple application antibody compositions of the present invention in the mode of the antitumous effect that can effectively cause its combination in animal body.Therefore can cause that its combination in tumor vessel occurs and the significant quantity and the duration of response of compound action in tumor environment provide this reagent.In order to realize this target, can use the different administration approach to be applied to animal simultaneously with the composition of single combination or as two kinds of different compositions antibody compositions of the present invention and carcinostatic agent.
In addition, can be for example before or after the carcinostatic agent treatment, to use antibody compositions of the present invention from several minutes to week and the interval in month scope.People will guarantee the combined effect that the antibody in carcinostatic agent and the antibody compositions of the present invention is favourable to cancer performance.
Most of carcinostatic agents will give before inhibition KIR antibody compositions of the present invention in the angiogenesis inhibitor treatment.Yet, when in antibody compositions of the present invention, using the immunoconjugates of antibody, can simultaneously or use multiple carcinostatic agent subsequently.
In some cases, even may need significant prolongation treatment time-histories, use carcinostatic agent or anticancer therapy respectively and using between the antibody compositions of the present invention interval a couple of days (2,3,4,5,6 or 7), several weeks (1,2,3,4,5,6,7 or 8) or even several months (1,2,3,4,5,6,7 or 8).Be intended to destroy substantially tumour (for example operation or chemotherapy) at anticancer therapy, use antibody compositions of the present invention and be intended to prevent that this will be favourable under the long situation of small transfer or tumor regrowth.
Also anticipation will be used composition or the carcinostatic agent based on inhibition KIR antibody of the present invention more than will utilizing once.Can be in alternative sky or week; Or inhibition KIR antibody compositions treatment cycle of the present invention is alternately used these reagent succeeded by the carcinostatic agent treatment cycle.In any case, realize tumor suppression in order to use combined therapy, application times no matter, all requirements are to carry two kinds of reagent effectively to bring into play the combined amount of antitumor action.
About operation, can make up with the present invention and carry out any surgical intervention.About radiotherapy, consider any mechanism of inducing DNA local damage in cancer cells, for example γ radiation, X ray, UV radiation, microwave even electron emission etc.Consider also that with the radio isotope targeted to cancer cells, this can unite use with targeting antibodies or other target means.
In others, can use immunomodulatory compounds or scheme with antibody compositions combination of the present invention or as its part.The preferred embodiment of immunomodulatory compounds comprises cytokine.In this class combined method, can adopt the various kinds of cell factor.Useful cytokine example comprises IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ in the combination of the present invention's imagination.Use the cytokine that is used for combined therapy of the present invention or composition according to the standard scheme consistent with clinical indication (for example patient's illness) and cytokine relative toxicity.
In certain embodiments, the therapeutic composition that contains cross reaction inhibition KIR antibody of the present invention can with chemotherapy or hormone therapy agent combined administration, perhaps can contain described chemotherapy or hormone therapy agent in addition.Can use multiple hormone therapy and chemotherapeutic in the combination therapy disclosed herein.Be thought of as exemplary chemotherapeutic and comprise (but being not limited to) alkylating reagent, metabolic antagonist, cytotoxic antibody, vinca alkaloids, Zorubicin for example, dactinomycin, mitomycin, carminomycin, zhengdingmeisu, Dx, tamoxifen, safe plain, taxotere, vincristine(VCR), vinealeucoblastine(VLB), vinorelbine, Etoposide (VP-16), 5 FU 5 fluorouracil (5FU), cytosine arabinoside, endoxan, plug is for group, methotrexate, camptothecine, dactinomycin, ametycin, cis-platinum (CDDP), aminopterin, combretastatin and derivative thereof and prodrug.
The hormone agent comprises (but being not limited to) for example lhrh antagonist, for example Leuprolide, goserelin, triptorelin and buserelin; Estrogen antagonist is tamoxifen and toremifene for example; Androgen antagonist is flutamide, Nilutamide, cyproterone and bicalutamide for example; Aromatase inhibitor is Anastrozole, Exemestane, letrozole and fadrozole for example; And progestogen for example medroxy, Verton and megestrol.
As one of ordinary skill in the art will appreciate, the suitable dose of chemotherapeutic will be near the dosage that has adopted in the clinical treatment, wherein chemotherapeutic separately or with other chemotherapeutic agent combined administration.Only as an example, can use for example cis-platinum and other DNA alkylating reagent.Cis-platinum has been widely used in treating cancer, to be used for the effective dose 20mg/m of clinical application 2Per three weeks used 5 days, altogether three courses of treatment.Cis-platinum is oral not to be absorbed, therefore must by in intravenously, subcutaneous, the tumour or peritoneal injection carry.
Other useful chemotherapeutic comprises the compound that disturbs dna replication dna, mitotic division and chromosome segregation and destruction polynucleotide precursor is synthetic and the reagent of fidelity of reproduction.Many exemplary chemotherapeutics that are used for combined therapy are set forth in U.S. Patent No. 6,524, and among 583 the table C, the reagent of described U.S. Patent No. 6,524,583 and the disclosure of explanation are in this specific reference as a reference.Each reagent of listing is exemplary, and is nonrestrictive.The technician is subjected to " Remington ' s Pharmaceutical Sciences " the 15 edition, and the 33rd chapter is specially the 624-652 page or leaf and instructs.According to the illness of being treated, dosage may occur and change.The doctor of administering therapeutic can determine proper dosage for individual subjects.
Cross reaction inhibition KIR antibody compositions of the present invention can use with one or more other angiogenesis inhibitor therapeutic combination, or contains anti-angiogenic agent in addition.The example of this class reagent comprises each neutralizing antibody, sense-rna, siRNA, RNAi, RNA aptamer and ribozyme (U.S. Patent No. 6,524,583, its disclosure is incorporated herein by reference) at VEGF or vegf receptor.Also can adopt the VEGF variant with antagonist properties, described in WO 98/16551, wherein WO 98/16551 is incorporated herein by reference.Other exemplary anti-angiogenic agent useful for combined therapy is set forth in U.S. Patent No. 6,524, and among 583 the table D, the reagent of described U.S. Patent No. 6,524,583 and the disclosure of indication are in this specific reference as a reference.
Inhibition KIR antibody compositions of the present invention also can be advantageously be used in combination with apoptosis-induced method, or contains apoptosis agent.For example, the oncogene of many inhibition apoptosis or apoptosis obtains identifying.Exemplary oncogene in this category comprises (but being not limited to) bcr-ab1, and bc1-2 (is different from bc1-1, cyclin D1; GenBank accession number M14745, X06487; U.S. Patent No. 5,650,491; And 5,539,094; Each is incorporated herein by reference) and the family member, comprise Bc1-x1, Mc1-1, Bak, A1 and A20.In t cell lymphoma, at first found the expression of crossing of bc1-2.Oncogene bc1-2 works by the PROTEIN B ax in combination and the inactivation apoptosis pathway.The function that suppresses bc1-2 stops the inactivation of Bax, and allows apoptosis pathway to carry out.Antisense base sequences, RNAi, siRNA or small molecules chemical compound suppress this class oncogene to consider for example to use, and are used for the present invention to increase apoptosis (U.S. Patent No. 5,650,491; 5,539,094; And 5,583,034; Each is incorporated herein by reference).
Inhibition KIR antibody compositions of the present invention also can comprise the molecule (for example antibody, part or its conjugate) that contains targeting moiety or use with described molecular combinations, and the described molecule of targeting moiety that contains is at target cell (for example target tumour cell) specificity marker (" target agent ").Generally speaking, come-at-able tumour antigen will be preferably discerned in the target agent that is used for these additional aspect of the present invention, and described tumour antigen is preferred or specific expressed in tumor sites.The target agent generally will be expressed in conjunction with surface of tumor cells, surface can composition that reach or surface alignment.The target agent also will preferably be showed high affinity characteristic; And can not bring into play remarkable side effect to the healthy tissues that earns a bare living in vivo, the described healthy tissues that earns a bare living for example is selected from one or more tissues or interior other organ or tissue that earns a bare living of human body in the heart, kidney, brain, liver, marrow, colon, mammary gland, prostate gland, Tiroidina, gall-bladder, lung, suprarenal gland, muscle, nerve fiber, pancreas, the skin.The fact of insignificant or clinical manageable side effect (for example those that run into usually during the chemotherapy) " do not brought into play remarkable side effect " and refer to will only produce when the target agent is used in the body in term used herein.
In oncotherapy, antibody compositions of the present invention can contain ancillary compound in addition or be used in combination with it.As an example, ancillary compound can comprise for example for example benzamide, antihistaminic agent, butyrophenones, cortical steroid, benzene diaza _ class and the Cannabinoids of thiodiphenylamine, replacement of 5-hydroxytryptamine antagonist and therapeutical agent of antiemetic; Double phosphinic acid compounds is Zoledronic acid and pamidronic acid for example; And hemopoieticgrowth factor for example erythropoietin and G-CSF, for example filgrastim, lenograstim and Da Bi ripple booth (darbepoietin).
In another embodiment, two or more antibody (comprising NKVSF1) with different cross reactivities of the present invention can be combined in the single composition, thus the inhibition effect of the inhibition KIR gene product as much as possible that neutralizes.The composition that contains the combination of cross reaction inhibition KIR antibody of the present invention or its fragment or derivative will allow effectiveness widely, may exist because may lack the crowd's of each inhibition KIR gene product of being discerned by single cross reacting antibody low percentage.Similarly, composition of the present invention can contain one or more antibody of discerning single inhibition KIR hypotype in addition.This class combination will provide effectiveness widely once more under the treatment situation.
The present invention also is provided at the method that strengthens the NK cytoactive in the patient's body that need to strengthen the NK cytoactive, comprises to described patient using step according to composition of the present invention.This method is more specifically at strengthen the NK cytoactive in suffering from patient's body of disease, in the described disease, it is useful increasing patient NK cytoactive, this disease relates to, influences or caused by the cell of the cracking susceptible that the NK cell is caused, and perhaps this disease is caused by NK cytoactive deficiency or is feature (for example cancer, another kind of proliferative disease, infectious diseases or immunologic derangement) with it.Method of the present invention more specifically is used for the treatment of multiple cancer and other proliferative disease, comprises (but being not limited to) bladder, mammary gland, colon, kidney, liver, lung, ovary, prostate gland, pancreas, stomach, uterine cervix, Tiroidina and skin (comprising squamous cell cancer) cancer; Lymphatic system hematopoiesis tumour comprises leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, t cell lymphoma, hodgkin's lymphoma, non Hodgkin lymphoma, hair cell lymphoma and Burketts lymphoma; Medullary system hematopoiesis tumour comprises acute and chronic lymphocytic leukemia and promyelocytic leukemia; The tumour in mesenchymal cell source comprises fibrosarcoma and rhabdosarcoma; Other tumour comprises melanoma, spermocytoma, teratoma, neuroblastoma and neurospongioma; Maincenter and peripheral nervous system tumour comprise astrocytoma, neuroblastoma, neurospongioma and schwannoma (schwannomas); The tumour in mesenchymal cell source comprises fibrosarcoma, rhabdosarcoma and osteosarcoma; And other tumour, comprise melanoma, xeroderma pitmentosum, keratoacanthoma, spermocytoma, thyroid follcular carcinoma and teratoma.
The preferred disease that can treat according to the present invention comprises lymphatic system hematopoiesis tumour, and for example T cell and B cell tumour (include, but are not limited to for example T prolymphocyte leukemia (T-PLL) of T cell disease, comprise minicell and gyrus like cell type; The large granular lymphocyte leukemia (LGL) of preferred T cellular type; Sezary syndrome (SS); Adult T-cell leukemia-lymphoma (ATLL); A/d T-NHL liver splenocyte lymphoma; T cell lymphoma behind periphery/thymus gland (polymorphism and immunoblast hypotype); Angioimmunoblastic T cell lymphoma; Blood vessel center (nose) t cell lymphoma; Anaplastic (Ki 1+) large celllymphoma; The intestines t cell lymphoma; The T lymphoblast; And lymphoma/leukemia (T-Lbly/T-ALL).
Also can treat other proliferative disease according to the present invention, comprise hyperplasia for example, fibrosis (especially lung, but comprise the fibrosis of other type, renal fibrosis for example), proliferation of smooth muscle in vasculogenesis, psoriasis, atherosclerosis and the blood vessel, for example narrow or postangioplasty restenosis.Cross reaction inhibition KIR antibody of the present invention can be used for the treatment of or prophylaxis against infection diseases, preferably includes any by virus, bacterium, protozoon, mould or fungus-caused infection.The sexy metachromia biology of this viroid comprises (but being not limited to) hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, I type herpes simplex (HSV-1), herpes simplex types 2 (HSV-2), rinderpest virus, rhinovirus, Europe can be viral, rotavirus, respiratory syncytial virus, papillomavirus (papillomavirus), cytomegalovirus, echinovirus, arboviruses (arbovirus), huntavirus, Coxsackie virus, mumps virus, Measles virus, rubella virus, poliomyelitis virus (polio virus) and I type or 2 type human immunodeficiency virus (HIV-1, HIV-2).
The infectation of bacteria that can treat according to the present invention comprises that (but be not limited to) is by the following infection that causes: staphylococcus; Suis comprises micrococcus scarlatinae (S.pyogenes); Faecalis; Genus bacillus comprises anthrax bacillus (Bacillus anthracis) and lactobacillus; The Li Site bacterium; Diphtheria corynebacterium (Corynebacterium diphtheriae); Gardnerella comprises gardnerella vaginalis (G.vaginalis); Nocardia bacteria; Streptomycete; Thermoactinomyces vulgaris (Thermoactinomyces vulgaris); Treponema; Campylobacter; Pseudomonas comprises Raeruginosa; Legionella; Neisseria comprises Diplococcus gonorrhoeae (N.gonorrhoeae) and Neisseria meningitidis (N.meningitides); Flavobacterium comprises courageous and upright Flavobacterium (F.meningosepticum) of meningeal sepsis and flavobacterium odoratum (F.odoraturn); Brucella; Bordetella comprises Bordetella pertussis (B.pertussis) and bordetella bronchiseptica (B.bronchiseptica); Escherichia comprises intestinal bacteria (E.coli), klebsiella; Enterobacteria, Serratia comprise serratia marcescens (S.marcescens) and liquefied Serratia (S.liquefaciens); Tarda; Bacillus proteus comprises Proteus mirabilis (P.mirabilis) and proteus vulgaris (P.vulgaris); Streptobacillus; Rickettsia comprises R.fickettsfi, and chlamydozoan comprises chlamydia psittaci (C.psittaci) and sand holes chlamydozoan (C.trachomatis); Mycobacterium comprises mycobacterium tuberculosis (M.tuberculosis), Mycobacterium intracellulare (M.intracellulare), M.folluiturn, Mycobacterium leprae (M.laprae), mycobacterium avium (M.avium), Mycobacterium bovis (M.boyis), mycobacterium africanum (M.africanum), mycobacterium kansasii (M.kansasii), Mycobacterium intracellulare (M.intracellulare) and mouse leprosy mycobacterium (M.lepraemurium); And Nocardia bacteria.
Treatable protozoal infections comprises the infection that (but being not limited to) caused by the graceful protozoon of Li Shi (leishmania), kokzidioa and taper worm (trypanosoma) according to the present invention.The national CDC (NCID) of Center for Disease Control (CDC) ( Http:// www.cdc.gov/ncidod/diseases/) complete list of the visible infectious diseases in website, described complete list is incorporated herein by reference.Above-mentioned all diseases all are to use the candidate disease of cross reaction inhibition KIR Antybody therapy of the present invention.
This class is treated the method for multiple infectious diseases can be separately or with other treatment and/or become known for treating this class treatment of diseases agent combination and adopt antibody of the present invention, described treatment reagent comprises antiviral agent, anti-mycotic agent, antibacterial agent, microbiotic, antiparasitic and antiprotozoal.When these methods relate to the additional procedures of using additional therapeutic agent, these reagent can be used with single agent form or with isolating, multi-agent form with antibody of the present invention.When using with isolating dosage form, can be before using antibody of the present invention, simultaneously or use extra reagent subsequently.
Other aspects and advantages of the present invention are open with experimental section below, and it is illustrative that described experimental section should be considered as, and unrestricted the application's scope.
Embodiment 1
The generation of the purifying of PBL and polyclone or clone NK clone.
By Ficoll Hypaque gradient and exhaust the cell that adheres to plastics and obtain PBL from healthy donors.In order to obtain the NK cell of enrichment, PBL and anti-CD3, anti-CD4 and anti-HLA-DRmAb (4 ℃, 30 minutes) are hatched, and are (4 ℃ of goat anti-mouse magnetic beads (Dynal) subsequently, 30 minutes) and immune magnetic selection people such as (, 1999) Pende of using means known in the art.Through the feeder cell of irradiation and 100U/ml interleukin-22 (Proleukin, ChironCorporation) and 1.5ng/ml phytohemagglutinin A (Gibco BRL) go up to cultivate CD3 -, CD4 -, DR -Cell is to obtain polyclone NK cell mass.By limited dilution cloning NK cell, and characterize the NK cell clone about cell surface receptor by flow cytometry.
The mAb that uses is JT3A (IgG2a, anti-CD3), EB6 and GL183 (IgG1, anti-respectively KIR2DL1 and KIR2DL3), XA-141 IgM (with the anti-KIR2DL1 of the specificity identical with EB6), anti-CD4 (HP2.6) and anti-DR (D1.12, IgG2a).Can use and to obtain, to have mutually homospecific mAb by commercial sources (Beckman CoulterInc., Fullerton CA) replace JT3A, HP2.6 and the DR1.12 that is produced by the applicant.EB6 and GL183 can obtain (CA.XA-141 can not obtain by commercial sources for Beckman Coulter Inc., Fullerton, but can according to using EB6 to reproduce cracking in contrast described in people such as (, 1993) Moretta) by commercial sources.
The anti-mouse antibodies of polyclone (the Southern Biotechnology Associates Inc) transfect cell of puting together succeeded by PE or FITC with suitable antibody (4 ℃, 30 minutes).(Becton Dickinson, Mountain View CA) go up by cell fluorescence determination and analysis method analytic sample at the FACSAN instrument.
In this research, use following clone.CP11, CN5 and CN505 are the KIR2DL1 positive colony, by EB6 (the anti-KIR2DL1 of IgG1) or XA-141 (compare have the anti-KIR2DL1 of mutually homospecific IgM with EB6 antibody) dyeing.CN12 and CP502 are the KIR2DL3 positive colony, are dyeed by GL183 antibody (the anti-KIR2DL3 of IgG1).
By standard 4 hours 51Cr discharge to measure evaluation NK clone's dissolved cell activity, described standard 4 hours 51During Cr discharges and measures, fasten test effect NK cell at known its Cw3 or Cw4 positive cell to the sensitivity of NK lysis.Use all target cells, the effector cell with 5000 cells in every hole in the titer plate: the target cell ratio is shown in (4 effector cells of each target cell usually) among the figure.The monoclonal antibody supernatant is carried out molten raji cell assay Raji shown in using or not using _ dilute.Program is with basic identical described in people such as (, 1993) Moretta.
Embodiment 2
The generation of new mAb
According to described in people such as (, 1990) Moretta, with activatory polyclone or mono-clonal NK clone immunity Balb C mouse in 5 age in week to produce mAb.After the different cytogamy, at first to its ability selection mAb with EB6 and the positive NK clone of GL183 and clone's cross reaction.It is reproduced the Cw4 or the Cw3 positive target cell cracked ability that are caused by the EB6 positive or the positive NK clone of GL183 respectively further screen positive monoclonal antibody.
Following execution cell dyeing.The goat F that the goat F that puts together for PE that continues with one group of antibody (1 μ g/ml or 50 μ l supernatants, 4 ℃, 30 minutes) (ab ') anti-mouse IgG of 2 fragments (H+L) or PE put together (ab ') the anti-human IgG of 2 fragments (Fc γ) antibody (Beckman Coulter) transfect cell.Go up execution cell fluorescence determination and analysis at Epics XL.MCL instrument (Beckman Coulter).
One of monoclonal antibody (DF200mAb) and a plurality of KIR family member reactions that comprise KIR2DL1, KIR2DL2/3.KIR2DL1+ and KIR2DL2/3+NK cell are all dyed (Fig. 1) by DF200mAb is bright.
Use the NK clone who expresses one or another kind of (or even two kinds) in these HLA I class specificity inhibition acceptors as effector cell at the allelic target cell of one or more HLA-C of expression.Following execution cytotoxic assay.By standard 4 hours 51Cr discharges the dissolved cell activity of measuring evaluation YTS-KIR2DL1 or YTS-Eco clone.On 721.221 cells of HLA-Cw4 positive or negative EBV clone and HLA-Cw4 transfection, test the effector cell.Use all target cells with 3000 cells in every hole in the titer plate.Effector cell/target cell ratio is shown among the figure.The total length of mono-clonal mouse or human antibodies or F shown in using or not using (ab ') 2 fragments are carried out molten raji cell assay Raji.KIR2DL1 +Even NK clone shows the dissolved cell activity that has also seldom, KIR2DL3 to the target cell of expressing HLA-Cw4 as scheduled +NK is cloned on the Cw3 positive target cell and shows seldom or do not have an activity.Yet in the presence of DF200mAb (being used to cover its KIR2DL acceptor), the NK cell becomes and can not discern its HLA-C part, and Cw3 or Cw4 target cell are shown the intensive dissolved cell activity.
C1R clone (CW4 for example +EBV clone, ATCC n ℃ RL 1993) not by KIR2DL1 +NK clone (CN5/CN505) kills and wounds, but is to use DF200 or conventional anti-KIR2DL1 mAb can effectively reverse this inhibition.On the other hand, expressing K IR2DL2/3 +KIR2DL1 -The NK clone (CN12) of phenotype effectively kills and wounds the C1R cell, and this killing and wounding is not subjected to DF200 mAb to influence (Fig. 2).Be cloned on the Cw3 positive target cell with the positive NK of KIR2DL2 or KIR2DL3 and obtain similar results.
Similarly, Cw4+221EBV clone is not by KIR2DL1 +The NK cell killing of transfection, but be to use DF200, DF200 Fab fragment or conventional anti-KIR2DL1 mAb EB6 or XA141 can effectively reverse this inhibition.Equally, Cw3+221EBV clone is not by KIR2DL2 +The NK cell killing, but be to use DF200 or DF200Fab fragment can reverse this inhibition.At last, the Cw3+221 EBV clone of back is not by KIR2DL3 +The NK cell killing, but be to use DF200 Fab fragment or conventional KIR2DL3 mAb GL183 or Y249 can reverse this inhibition.The results are shown among Fig. 3.
F (ab ') 2 fragments are tested it reproduce Cw4 positive target cell cracked ability.The F of DF200 and EB6 antibody (ab ') 2 fragments can both reverse 221 clones of the NK lysis Cw4 transfection of KIR2DL1 transfection and the inhibition of Cw4+TUBO EBV clone.The results are shown among Fig. 4.
Embodiment 4
The generation of new human mAb
By producing human anti-KIR monoclonal antibody with the transgenic mice of reorganization KIR protein immunity through transforming the express human antibody storehouse.After the different cytogamy, at first to its ability selection mAb with fixed K IR2DL1 and the cross reaction of KIR2DL2 protein.Several monoclonal antibodies (comprising 1-7F9,1-4F1,1-6F5 and 1-6F1) and KIR2DL1 and KIR2DL2/3 reaction.
It is reproduced the Cw4 positive target cell cracked ability that the positive NK transfectant of EB6 by expressing K IR2DL1 causes further screen positive monoclonal antibody.Use the NK cell of expressing HLA I class specificity inhibiting antibody as effector cell (Fig. 5 and 6) at the allelic target cell of one or more HLA-C of expression.Carry out cytotoxic assay as mentioned above.Effector cell/target cell ratio is shown among the figure, uses antibody with 10ug/ml or 30ug/ml.
KIR2DL1 +Even the NK cell shows also seldom dissolved cell activity to the target cell of expressing HLA-Cw4 as scheduled.Yet in the presence of 1-7F9mAb, the NK cell becomes and can not discern its HLA-C part, and the Cw4 target cell is showed the intensive dissolved cell activity.For example, two of test clones (721.221 and CW4 of HLA-Cw4 transfection +EBV clone) not by KIR2DL1 +The NK cell killing, but be to use mAb 1-7F9 or conventional anti-KIR2DL1 mAb EB6 can effectively reverse this inhibition.Antibody DF200 and panKIR (being also referred to as NKVSF1) are compared with 1-7F9.On the other hand, antibody 1-4F1,1-6F5 and 1-6F1 can not reproduce the Cw4 positive target cell lysis that the NK cell causes.
Embodiment 5
The interactional Biacore of DF200 mAb/KIR2DL1 and DF200 mAb/KIR2DL3 analyzes
The production of recombinant protein and purifying
In intestinal bacteria, produce KIR2DL1 and KIR2DL3 recombinant protein.Use PCR to clone the cDNA that 47.11 carriers people such as (, 1993) Biassoni and RSVS (gpt) 183 clone 6 carriers people such as (, 1995) Wagtman amplification coding KIR2DL1 and KIR2DL3 intact cell outer structure territory from pCDM8 respectively, use following primer:
Justice is arranged: 5 '-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3 '
Antisense: 5 '-CGGGATCCCAGGTGTCTGGGGTTACC-3 '
The sequence of itself and encoding human elementization signal is cloned into (people such as Saulquin, 2003) in the pML1 expression vector by reading frame.
In BL21 (DE3) bacterial strain (Invitrogen), carry out protein expression.In the substratum that adds penbritin (100 μ g/ml) in 37 ℃ of microbial culture with transfection to OD 600=0.6, with 1mM IPTG abduction delivering.
(8M urea) reclaims protein from inclusion body under the sex change condition.By reduction urea concentration in six step dialysis (be respectively 4,3,2,1,0.5 and 0M urea), containing L-arginine (400mM, Sigma) and the 20mM Tris of beta-mercaptoethanol (1mM), pH7.8 carries out the folding again of recombinant protein in room temperature in the NaCl 150mM damping fluid.0.5 and 0M urea dialysis step during add reduced form and Sleep-promoting factor B (is respectively 5mM and 0.5mM, Sigma).At last, at 10mM Tris, pH7.5, the NaCl 150mM damping fluid protein of dialysing in a large number.Concentrate and (Pharmacia on Superdex 200 size-exclusion column then; The AKTA system) purification of soluble unfolded protein again.
Going up the execution surface plasma resonance at Biacore instrument (Biacore) measures.In all Biacore experiment, with the HBS damping fluid that adds 0.05% tensio-active agent P20 as running buffer.
Protein is fixed.
With the reorganization KIR2DL1 that produces as mentioned above and KIR2DL3 protein Covalent Immobilization to the carboxyl of sensor chip (Sensor Chip) CM5 (Biacore) dextran layer.With EDC/NHS (N-ethyl-N '-(3-dimethylamino-propyl) carbodiimide hydrochloride and N-hydroxy-succinamide, Biacore) activated sensor chip surface.Injection coupling buffer (10mM acetic acid, pH4.5) protein in.Use 100mM ethanol ammonia pH8 (Biacore) to carry out the passivation of remaining activating group.
Avidity is measured.
For kinetic measurement, with the soluble antibody (1 * 10 of multiple concentration -7To 4 * 10 -10M) put on the fixed sample.Continuous flow velocity with 20 μ l/ minutes is carried out measurement.For each circulation, inject 5 μ l 10mM NaOH pH11 reg sensor chip surfaces.(BIAevaluation 3.1, Biacore) analytical data to use BIAlogueKinetics Evaluation program.On the dextran layer of KIR2DL1 that contains 500 or 540 reflection coefficient units (RU) and difference 1000 or 700RU and KIR2DL3, with the soluble analyte (multiple concentration, 40 μ l) in 20 μ l/ minutes the flow velocitys injection HBS damping fluid.Data represented six independent experiments.The results are shown in the table 1, as follows.
Table 1.DF200mAb analyzes in conjunction with the BIAcore of fixed K IR2DL1 and KIR2DL3.
Protein K D(10 -9M)
KIR2DL1 10.9+/-3.8
KIR2DL3 2.0+/-1.9
K D: dissociation constant.
Embodiment 6
The Biacore competitive binding analysis of mouse and human anti-KIR antibody
According to front explanation (people 1999 such as Gauthier, Saunal and van Regenmortel1995), go up with mouse anti KIR 2D antibody DF200, Pan2D, gl183 and EB6 and the anti-KIR 2D of people antibody 1-4F1,1-6F1,1-6F5 and the analysis of 1-7F9 enforcement epitope mapping at fixed K IR 2DL1 (900RU), KIR 2DL3 (2000RU) and KIR2DS1 (1000RU).
Be injected in the HBS damping fluid with two minutes of the different antibodies of 15 μ g/ml and finish all experiments in 5 μ l/ minutes flow velocity.Divide two steps to every being at war with property of antagonist binding analysis.In first step, first monoclonal antibody (mAb) being expelled on the KIR 2D target protein, is second mAb (not removing first mAb) subsequently, and monitors the RU value (RU2) of second mAb.In second step, at first second mAb is injected directly on the exposed KIR2D protein, and the RU value (RU1) of monitoring mAb.Calculate second mAb causing by first mAb inhibition percentage ratio to KIR 2D protein bound by 100* (1-RU2/RU1).
The results are shown in the table 2,3 and 4, wherein be appointed as the antibody of " first antibody " and list in the vertical column, " second antibody " lists in the horizontal bar.Each antibody combination for test, antibody is directly listed in the table in conjunction with the value (RU) of level chip, wherein second antibody is listed in the top of lattice to the direct combination of KIR2D chip, and second antibody was listed in the bottom of lattice when first antibody existed to the value of KIR2D chips incorporate.What list in each lattice right side is the inhibition per-cent of second antibodies.Table 2 shows the combination on the KIR2DL1 chip, and table 3 shows the combination of antibody to the KIR2DL3 chip, and table 4 shows the combination of antibody to the KIR2DS1 chip.
Evaluation murine antibody DF200, NKVSF1 and EB6 and people's antibody 1-4F1,1-7F9 and 1-6F1 are to the competitiveness combination of fixed K IR2DL1, KIR2DL2/3 and KIR2DS1.Epitope mapping (Fig. 7) from anti-KIR antibodies KIR2DL1 experiment shows (a) antibody 1-7F9 and EB6 and 1-4F1 competition, but does not compete with NKVSF1 and DF200; (b) antibody 1-4F1 competes with EB6, DF200, NKVSF1 and 1-7F9 successively; (c) NKVSF1 and DF200,1-4F1 and EB6 competition, but do not compete with 1-7F9; And (d) DF200 and NKVSF1,1-4F1 and EB6 competition, but do not compete with 1-7F9.Epitope mapping (Fig. 8) from anti-KIR antibodies KIR2DL3 experiment shows (a) 1-4F1 and NKVSF1, DF200, gl183 and 1-7F9 competition; (b) 1-7F9 and DF200, gl183 and 1-4F1 competition, but do not compete with NKVSF1; (c) NKVSF1 and DF200,1-4F1 and GL183 competition, but do not compete with 1-7F9; And (d) DF200 and NKVSF1,1-4F1 and 1-7F9 competition, but do not compete with GL183.Epitope mapping (Fig. 9) from anti-KIR antibodies KIR2DS1 experiment shows (a) 1-4F1 and NKVSF1, DF200 and 1-7F9 competition; (b) 1-7F9 and 1-4F1 competition is not still competed with DF200 and NKVSF1; (c) NKVSF1 and DF200 and 1-4F1 competition, but do not compete with 1-7F9; And (d) DF200 and NKVSF1 and 1-4F1 competition, but do not compete with 1-7F9.
Embodiment 7
Test the ability of anti-KIR antibody NKVSF1 with the anti-KIR mAb of macaque NK cell titration in conjunction with macaque NK cell.Antibody and monkey NK
The combination of cell is shown among Figure 10.
The generation of the purifying of monkey PBMC and total polyclone NK cell.
From Trisodium Citrate CPT pipe (Becton Dickinson) preparation macaque PBMC.Use negative consumption method (negative depletion) to carry out NK cell purification (macaque NK cell enrichment test kit, Stem Cell Technology).(Invitrogen Gibco) goes up cultivation NK cell to obtain polyclone NK cell mass at artificial rearing cell, 300U/ml interleukin-22 (Proleukin, Chiron Corporation) and 1ng/ml phytohemagglutinin A through radiotreatment.
With macaque NK cell titration Pan2D mAb.
With macaque NK cell (total NK16 days) and the Pan2D mAb of different amounts and the goat F that puts together succeeded by PE (ab ') the anti-mouse IgG of 2 fragments (H+L) antibody incubation.Determine positive percentage with isotype contrast (the mouse IgG1 of purifying).The duplicate sample of handling.Average fluorescent strength=MFI.
Table 2:KIR2DL1 epitope mapping
← the two Ab →
The one Ab (following) DF200 Pan2D EB6 1-4F1 1-7F9 1-6F1 1-6F5
DF200 80％ 90％ 490 92％ 40 480 27％ 350 540 15％ 460 400 15％ 340
Pan2D 90％ 90％ 900 95％ 50 860 2％ 840 750 12％ 660 600 13％ 520
EB6 60％ 40％ 460 57％ 200 370 48％ 190 490 65％ 170 260 23％ 200 nd
1-4F1
1-7F9 600 10％ 545 545 2％ 534 460 60％ 180 360 95％ 16 330 9％ 300 nd
1-6F1 350 11％ 310 475 7％ 440 260 18％ 320 360 23％ 275 490 10 ％ 440 nd
1-6F5 350 17％ 290 475 7％ 440 nd 360 17％ 300 nd 290 40％ 170
Table 3:KIR2DL3 epitope mapping
← the two Ab →
The one Ab (following) DF200 Pan2D gl183 1-4F1 1-7F9 1-6F1 1-6F5
DF200 75％ 20％ 1270 75％ 320 520 62％ 200 550 16％ 460 440 4％ 420
Pan2D 95％ 85％ 2250 68％ 730 880 15％ 750 840 8％ 770 560 18％ 460
gl183 8％ 40％ 1300 75％ 330 670 76％ 160 530 18％ 430 nd
1-4F1 1140 82％ 210 2400 63％ 890 1240 73％ 330 1050 87％ 140
1-7F9 770 42％ 450 870 5％ 830 800 75％ 200 1000 63％ 270
1-6F1 790 4％ 760 990 0％ 1090 620 8％ 570
1-6F5 800 5％ 760 990 4％ 950 nd
Table 4:KIR2DS1 epitope mapping
← the two Ab →
The one Ab (following) DF200 Pan2D 1-4F1 1-7F9
DF200 70％ 660 87％ 80 975 15％ 825
Pan2D 100％ 650 100％ -8 920 45％ * 500
1-7F9 900 17％ 1090 1350 11％ 1200 660 96％ 23
Embodiment 8
DF200 and pan2D are in conjunction with the epitope mapping of KIR2DL1
Based on its crystalline structure of delivering ( MaenakaDeng the people, (1999); People such as Fan, (2001); People such as Boyington, (2000)), the computer simulation predicted amino acid R131 of KIR2DL1,2 and 3 (KIR2DL1-3) extracellular domain 1Relate to the interaction between KIR2DL1 and KIR2DL1-3 cross reactivity mouse monoclonal antibody (mAb) DF200 and the pan2D.The preparation fused protein to be verifying this prediction, and described fused protein is by the wild-type that merges with human Fc (hFc) or point mutation (R131W for example 2) the intact cell outer structure territory (amino acid H1-H224) of KIR2DL1 forms.In (Winter and Long (2000)), material and the method that is used to produce and estimate multiple KIR2DL1-hFc fused protein described.In brief, (Quickchange II, the cDNA support C L42-Ig of the production wild-type KIR2DL1-hFc that Promega) has delivered people such as (, (1995)) Wagtmann produce the cDNA carrier of encoded K IR2DL1 (R131W)-hFc in PCR-based mutagenesis.Substantially in the COS7 cell, produce KIR2DL1-hFc and KIR2DL1 (R131W)-hFc according to described people such as (, (1995)) Wagtmann, and it is separated from tissue culture medium (TCM).Correctly folding in order to test it, KIR2DL1-hFc and KIR2DL1 (R131W)-hFc are hatched with the LCL721.221 cell of expressing HLA-Cw3 (no KIR2DL1 part) or HLA-Cw4 (KIR2DL1 part), by the interaction between research cell surface proteins interactional standard technique facs analysis KIR-Fc fused protein and the cell.In Figure 11 A hurdle, provided the example of independent experiment.As what predict, there is not the KIR2DL1-hFc fused protein to combine with the LCL721.221 cell of expressing HLA-Cw3 from document.On the contrary, KIR2DL1-hFc and KIR2DL1 (R131W)-hFc combines with the LCL721.221 cell of expressing HLA-Cw4, thereby confirms that it is correctly folding.
Standard technique ELISA research KIR2DL1 (the R131W)-hFc of use research protein interaction and KIR2DL1-hFc combine with KIR-specificity mAb (DF200, pan2D, EB6 and GL183's).In brief, KIR2DL1 (R131W)-hFc is connected with 96 orifice plates with KIR2DL1-hFc, after this adds KIR specificity mAb with multiple concentration (0-1 μ g/ml is in PBS) by the mountain goat anti-human antibody.Use is to the mouse antibodies spy
1Single-letter amino acid coding
2Amino acid/11 in KIR2DL1 31 (from the N-terminal numbers) replaces the secondary antibody that the different peroxidase of W is coupled with R and transforms tmb substrate, manifests interaction between KIR2DL1-hFc variant and the mAb by spectrophotometry (450nm).In Figure 11 B hurdle, provided the example of independent experiment.Although KIR2DL2-3 specificity mAb GL183 can not be in conjunction with any KIR2DL1-hFc fused protein, KIR2DL1 specificity mAb EB6, DF200 and pan2D in dose-dependent mode in conjunction with the KIR2DL1-hFc variant.Independent point mutation (R131W) influences the combination of DF200 and pan2D, compare with wild-type at the maximum concentration (1 μ g/ml) of mAb and to reduce by 10% combination approximately, confirm that R131 is the part of DF200 and the binding site of pan2D in the extracellular domain 2 of KIR2DL1.
Reference
Moretta，A.，Bottino，C.，Pende，D.，Tripodi，G.，Tambussi，G.，Viale，O.，Orengo，A.，Barbaresi，M.，Merli，A.，Ciccone，E.，and et al.(1990).Identification of four subsets of humanCD3-CD16+ natural killer(NK)cells by the expression ofclonallydistributed functional surface molecules：correlationbetween subset assignment of NK clones and ability to mediatespecific alloantigen recognition.J Exp Med 172，1589-1598.
Moretta，A.，Vitale，M.，Bottino，C.，Orengo，A.M.，Morelli，L.，Augugliaro，R.，Barbaresi，M.，Ciccone，E.，andMoretta，L.(1993).P58 molecules as putative receptors formajor histocompatibility complex(MHC)class I molecules inhuman natural killer(NK)cells.Anti-p58 antibodiesreconstitute lysis of MHC class I-protected cells in NK clonesdisplaying differents pecificities.J Exp Med 178，597-604.Pende，D.，Parolini，S.，Pessino，A.，Sivori，S.，Augugliaro，R.，Morelli，L.，Marcenaro，E.，Accame，L.，Malaspina，A.，Biassoni，R.，et al.(1999).Identification andmolecular characterization of NKp30，a novel triggeringreceptor involved in natural cytotoxicity mediated by humannatural killer cells.J Exp Med 190，1505-1516.
Ruggeri，L.，Capanni，M.，Urbani，E.，Perruccio，K.，Shlomchik，W.D.，Tosti，A.，Posati，S.，Rogaia，D.，Frassoni，F.，Aversa，F.，et al.(2002).Effectiveness of donor naturalkiller cell alloreactivity in mismatched hematopoietictransplants.Science 295，2097-2100.
Wagtmann N，Biassoni R，Cantoni C，Verdiani S，Malnati MS，Vitale M，Bottino C，Moretta L，Moretta A，Long EO.Molecularclones of the p58 NK cell receptor revealimmunoglobulin-related molecules with diversity in both theextra-and intracellular domains.Immunity.1995May；2(5)：439-49.
Biassoni R，Verdiani S，Cambiaggi A，Romeo PH，Ferrini S，Moretta L.Human CD3-CD16+natural killer cells express thehGATA-3T cell transcription factor and an unrearranged 2.3-kbTcR delta transcript.Eur J Immunol.1993 May；23(5)：1083-7.
Saulquin X，Gastinel LN，Vivier E.Crystal structure of thehuman na tural killer cell activating receptor KIR2DS2(CD158j)J Exp Med.2003 Apr 7；197(7)：933-8.
Gauthier，L.，Lemmers，B.，Guelpa-Fonlupt，V.，Fougereau，M.，and Schiff，C.
μ-SLC physico-chemical interactions of the human preB cellreceptor：implications for VH repertoire selection and cellsignaling at the preB cell stage.Journal of Immunology，162.，41-50.(1999).
Saunal，H.and Van Regenmortel，M.H.V.，Mapping of viralconformation epitopes using biosensor measurements.Journal ofImmunology，183：33-41(1995).
Boyington JC； Motyka SA； Schuck P； Brooks AG； Sun PD.Nature，Vol.405(6786)pp.537-543(2000)
Fan QR； Long EO； Wiley DC.Nature immunology，Vol.2(5)pp.452-460(2001)
Maenaka K； Juji T； Stuart DI； Jones EY.Structure withFolding and design，Vol.7(4)pp.391-398(1999)
Wagtmann N； Rajagopalan S； Winter CC； Peruzzi M； Long EO.Immunity，Vol.3(6)pp.801-809(1995)
Winter CC；Long EO.Natural Killer Cells Protocols(editedby Campbell KS and Colonna M).Human Press.pp.219-238(2000)
All are incorporated herein by reference at this reference of enumerating (comprising publication, patent application and patent), are spelt out degree being incorporated herein by reference separately as each reference, and display in full with it at this.
All titles and subtitle only are used for conveniently should not being interpreted as limiting by any way the present invention at this.
Unless point out in addition or other and the obvious contradiction of context that at this present invention comprises any combination of mentioned component in its all possible variant.
Unless point out in addition or in addition and the obvious contradiction of context at this, as describe term used in the literary composition of the present invention " a ", " an " and " the " and similar referring to and to be interpreted as encompasses singular and plural number.
Unless point out in addition at this, only be intended in specification sheets, quote each independent value in the scope of the value of this narration as the simple method of mentioning each the independent value that belongs to this scope separately, just it is at this by statement separately equally.Unless stated otherwise, all (for example represent corresponding approximation in this exact value that provides, the exemplary values accurately all about specificity factor or measurement that provides can be thought of as corresponding approximate measure value also is provided, represent with " approximately " in the time of suitably).
Unless point out in addition or other and the obvious contradiction of context at this, can carry out all methods described here with any suitable order.
Unless otherwise noted, use and only be intended to set forth the present invention better, and scope of the present invention is not caused restriction at these any and all embodiment that provide or exemplary term (for example " for example ").Unless same explicit state should be not that enforcement is essential to the invention for any composition of indication with the terminological interpretation in this specification sheets.
Only quoting and introduce of this patent document, do not reflect this class patent document validity, patentability and/or anyways enforceable to for the purpose of the facility.
Unless stated otherwise or with the obvious contradiction of context, in any aspect of the present invention or the embodiment for composition use that term for example " comprises ", the description of " having ", " comprising " or " containing " be intended to as the present invention " by " special component " forms ", special component " is formed " or the similar aspect or the embodiment of " comprising substantially " special component (for example provide support " substantially by ", unless otherwise indicated or with the obvious contradiction of context, the composition that is described as comprising special component at this should be interpreted as and also describe the composition that is grouped into by this one-tenth).
The present invention governing law allow at utmost be included in this proposition aspect and all modifications and the Equivalent of stating in claims.

Claims (19)

1. produce the method for antibody, the inhibition activity of described antibody and the cross reaction of multiple KIR2DL gene product and this class KIR that neutralizes, described method comprises step:
(a) to contain the immunogen immune non-human mammal of KIR2DL polypeptide;
(b) Mammals from described immunity prepares antibody, and wherein said antibody combines with described KIR2DL polypeptide,
(c) antibody in the selection (b), the KIR2DL gene product cross reaction that described antibody is different with at least two kinds, and
(d) antibody in the selection (c), described antibody strengthens the NK cell
(e) select antibody, described antibody combines with primate NK cell or KIR polypeptide.
2. according to the process of claim 1 wherein that the primate in the step (e) is a macaque.
3. assess the toxic method of antibody, wherein the antibody that will produce according to the method for claim 1 or 2 is applied to primate.
4. according to the method for claim 3, wherein primate is a macaque.
5. antibody, antibody fragment or antibody derivatives, described antibody, antibody fragment or antibody derivatives contain the light chain variable region sequence of the DF-200 that lists among Figure 12.
6. antibody, antibody fragment or antibody derivatives, described antibody, antibody fragment or antibody derivatives contain the light chain variable region sequence of the Pan2D that lists among Figure 12.
7. antibody, antibody fragment or antibody derivatives, described antibody, antibody fragment or antibody derivatives contain one or more variable region of light chain CDR of the DF-200 that lists among Figure 12.
8. antibody, antibody fragment or antibody derivatives, described antibody, antibody fragment or antibody derivatives contain one or more variable region of light chain CDR of the Pan2D that lists among Figure 12.
9. antibody, antibody fragment or antibody derivatives, described antibody, antibody fragment or antibody derivatives contain the weight chain variabl area sequence of the DF-200 that lists among Figure 13.
10. antibody, antibody fragment or antibody derivatives, described antibody, antibody fragment or antibody derivatives contain one or more variable region of heavy chain CDR of the DF-200 that lists among Figure 13.
11. antibody, described antibody combines with KIR2DK1 is single-minded in the zone by amino-acid residue (105,106,107,108,109,110,111,127,129,130,131,132,133,134,135,152,153,154,155,156,157,158,159,160,161,162,163,181,192) definition.
12. combining with KIR2DL1 and KIR 2DL2/3, antibody, described antibody with by the amino-acid residue outside the zone of residue (105,106,107,108,109,110,111,127,129,130,131,132,133,134,135,152,153,154,155,156,157,158,159,160,161,162,163,181,192) definition do not interact.
13. antibody, described antibody combine with KIR2DL1 and do not combine with the KIR2DL1 mutant, R131 is Ala in the described KIR2DL1 mutant.
14. antibody, described antibody combine with KIR2DL1 and do not combine with the KIR2DL1 mutant, R157 is Ala in the described KIR2DL1 mutant.
15. antibody, described antibody combine with KIR2DL1 and do not combine with the KIR2DL1 mutant, R158 is Ala in the described KIR2DL1 mutant.
16. antibody, described antibody combines with KIR2DL1 residue (131,157,158).
17. antibody, described antibody combine with KIR2DS3 (R131W), but do not combine with wild-type KIR2DS3.
18. antibody, described antibody and KIR2DL1 and KIR2DL2/3 and KIR2DS4 combine.
19. antibody, described antibody combines with KIR2DL1 and KIR2DL2/3, but does not combine with KIR2DS4.
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WO2010091542A1 (en) * 2009-02-16 2010-08-19 重庆西南医院 Anti-tumor, anti-bacterial and/or anti-viral applications of ctla4ig and derivatives thereof.
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