CN105367657A - Anti-HER3 antibody, preparing method thereof and applications of the antibody - Google Patents

Anti-HER3 antibody, preparing method thereof and applications of the antibody Download PDF

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CN105367657A
CN105367657A CN 201410401545 CN201410401545A CN105367657A CN 105367657 A CN105367657 A CN 105367657A CN 201410401545 CN201410401545 CN 201410401545 CN 201410401545 A CN201410401545 A CN 201410401545A CN 105367657 A CN105367657 A CN 105367657A
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antibody
seqidno
variable region
chain variable
heavy chain
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CN 201410401545
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Chinese (zh)
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瞿爱东
李翱翔
梁红远
祝婧烨
吴丽娜
黄海武
陆瑾
赵鑫
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上海生物制品研究所有限责任公司
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Abstract

The invention provides an anti-HER3 antibody, a preparing method thereof and applications of the antibody and particularly provides a novel anti-HER3 antibody. The antibody is high in affinity with HER3 molecules and can specifically combine an antigen molecule. In particular, a human-mouse chimeric antibody effectively reduces immunogenicity of a mouse antibody. By combined application of the anti-HER3 antibody and cetuximab, activity of the cetuximab is significantly improved, and the using amount of the cetuximab is reduced.

Description

抗HER3抗体、其制法及其应用 Anti-HER3 antibodies, their preparation and use

技术领域 FIELD

[0001] 本发明属于生物医药领域,具体地说,本发明涉及抗肥R3抗体、其制法及其应用。 [0001] The present invention belongs to the biomedical field, in particular, the present invention relates to anti-fat R3 antibody, their preparation and their applications.

背景技术 Background technique

[0002] 肥R3 巧rbB-3、ERBB3、c-erbB-3、c-erbB3、受体酪氨酸蛋白质激酶erbB-3、沈Q IDNO. :41)是编码表皮生长因子受体巧GFR)酪氨酸激酶家族的一名成员,该家族还包括肥Rl(又称为EGFR)、肥R2、和肥R4等。 [0002] R3 fertilizer Qiao rbB-3, ERBB3, c-erbB3, c-erbB3, a receptor tyrosine protein kinase erbB3, Shen Q IDNO:. 41) encoding the epidermal growth factor receptor is a clever GFR) a member of the tyrosine kinase family, the family also includes fertilizer Rl (also known as EGFR), fat R2, R4 and fertilizer and so on. 肥R3结构包括结合配体的胞外区,α螺旋的跨膜区,具有酪氨酸激酶结构域和富含酪氨酸的C端磯酸化位点胞内区。 R3 fat binding structure comprising an extracellular region, a transmembrane helix region [alpha] ligand, having the tyrosine kinase domain and a C-terminal tyrosine-rich site was acidified Angeles cell region. 其中胞外区又可分为I(L1)、II(S1)、111化2)和IV(S2),结构域I和III是配体结合区,而II和IV结构域是结合配体后发生构象改变暴露的二聚化结构域。 Wherein the extracellular region can be divided into I (L1), II (S1), 111 for 2) and IV (S2), domains I and III is a ligand-binding region, II and IV and the binding domain is a ligand conformational changes dimerization domain exposed. 化regulin(HRG)是肥R3的特异性配体,肥R3的表达和功能受到配体时空表达的影响,HRG通过促进肥R3与肥R家族的其他成员形成异二聚体来激发胞内信号。 Of regulin (HRG) is a specific ligand for fertilizer R3, R3 fat expression and function by the temporal expression of the ligand, intracellular signal of HRG excited by promoting heterodimer formation with other members of manure fertilizer R R3 family . 因此,它能结合此配体,但是不能经由蛋白质磯酸化将信号传送入细胞中。 Thus, it may bind a ligand, but not via the Rocky acidified protein signaling into cells. 然而,它与具有激酶活性的其它肥R家族成员形成异二聚体。 However, it forms heterodimers with other family members have fertilizer R kinase activity. 异二聚化导致受体介导的信号传导途径的激活及其胞内域的转磯酸化。 Heterodimerization cause receptor-mediated signal transduction pathway to activate its rotation Angeles acidified intracellular domain. 肥R家族成员间的二聚体形成扩大HER3 的信号传导潜力,并且是一种不仅用于信号多样化,而且用于信号放大的手段。 R fertilizer heterodimers between family members is formed of HER3 signaling the potential to expand, and is a signal only for diversity, and means for signal amplification. 例如,肥R2/ 肥R3异二聚体在肥R家族成员间经由PI3K和AKT途径诱导最重要的促有丝分裂信号之一, 已经在许多癌症,包括前列腺、膀脫、和乳腺肿瘤中报告了此基因的扩增和/或其蛋白质的过表达。 For example, fat R2 / R3 heterodimer between fat fat R family members via PI3K and AKT pathways induced by one of the most important pro-mitogenic signals, has been in many cancers, including prostate, bladder removal, and breast cancer in this report overexpression of gene amplification and / or protein. 已经表征了编码不同同等型的可变转录剪接变体。 Isoforms have been characterized alternative transcripts encoding different splice variants. 一种同等型缺乏膜间区,并且分泌到细胞外。 Isoforms lack one kind of inter-membrane region, and secreted extracellularly. 此形式作用为调控膜结合形式的活性。 This effect is in the form of membrane-bound form of activity regulation. 还已经报告了别的剪接变体,但是它们尚未彻底表征。 It has also been reported in other splice variants, but they have not been completely characterized.

[0003]W097/35885涉及肥R3抗体。 [0003] W097 / 35885 relates to fat R3 antibody. W02003/013602涉及肥R活性抑制剂,包括肥R抗体。 W02003 / 013602 relates to fertilizer R activity inhibitor, comprising a fertilizer R antibody. W02007/077028和W02008/100624也涉及肥R3抗体。 W02007 / 077028 and W02008 / 100624 also relates to fertilizer R3 antibody. 但是本领域中仍然缺乏一种针对肥R3的灵敏性好特异性强的抗体。 But the art is still the lack of a strong sensitivity and specificity antibodies against fat R3.

发明内容 SUMMARY

[0004] 本发明的目的在于提供一种新型的肥R3特异性抗体或其片段。 [0004] The object of the present invention is to provide a novel fertilizer R3 specific antibody or fragment thereof.

[0005] 本发明的另一目的是提供上述抗体或其片段的制备方法和用途。 [0005] Another object of the present invention is to provide the above-described antibody or fragment thereof preparation and use.

[0006] 本发明的第一方面,提供了一种抗体的重链可变区,所述的重链可变区包括W下Η个互补决定区CDR; [0006] The first aspect of the present invention, there is provided an antibody heavy chain variable region, said heavy chain variable region comprising the complementarity determining regions W Η CDRs of;

[0007] (1)互补决定区CDR1 [0007] (1) complementarity determining regions CDR1

[0008]所述互补决定区CDR1 选自;SEQIDNO. :17、沈QIDNO. :23、沈QIDNO. :29 和沈QIDNO. : 35 ; [0008] The complementarity determining regions CDR1 selected; SEQIDNO:. 17, Shen QIDNO: 23, Shen QIDNO: 29 and Shen QIDNO: 35;...

[0009] 似互补决定区CDR2 [0009] Complementarity determining region CDR2

[0010]所述互补决定区CDR2 选自;SEQIDNO. :18、沈QIDNO. :24、沈QIDNO. :30 和沈QIDNO. : 36 ; [0010] The complementarity determining region CDR2 selected; SEQIDNO:. 18, Shen QIDNO: 24, Shen QIDNO: 30 and Shen QIDNO: 36;...

[0011] 做互补决定区CDR3 [0011] do complementarity determining regions CDR3

[0012] 所述互补决定区CDR3 选自;SEQIDNO. :19、沈QIDNO. :25、沈QIDNO. :31 和沈QIDNO. :37。 [0012] The complementarity determining region CDR3 selected; SEQIDNO:. 19, Shen QIDNO: 25, Shen QIDNO: 31 and Shen QIDNO: 37....

[0013] 在另一优选例中,所述重链可变区的H个互补决定区CDRl、CDR2、CDR3的氨基酸序列分别如沈QIDNO. : 17、SEQIDNO. : 18 和沈QIDNO. : 19 所示。 [0013] In another preferred embodiment, H complementarity determining regions CDRl of the heavy chain variable region, CDR2, CDR3 of the amino acid sequence as Shen QIDNO:. 17, SEQIDNO:. 18 and Shen QIDNO:. 19 Suo shows.

[0014] 在另一优选例中,所述重链可变区的Η个互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如沈QIDNO. : 23、SEQIDNO. : 24 和沈QIDNO. : 25 所示。 [0014] In another preferred embodiment, Η complementarity determining regions CDR1 of the heavy chain variable region, CDR2, CDR3 of the amino acid sequence as Shen QIDNO:. 23, SEQIDNO:. 24 and Shen QIDNO:. 25 Suo shows.

[0015] 在另一优选例中,所述重链可变区的Η个互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如沈QIDNO. : 29、SEQIDNO. : 30 和沈QIDNO. : 31 所示。 [0015] In another preferred embodiment, Η complementarity determining regions CDR1 of the heavy chain variable region, CDR2, CDR3 of the amino acid sequence as Shen QIDNO:. 29, SEQIDNO:. 30 and Shen QIDNO:. 31 Suo shows.

[0016] 在另一优选例中,所述重链可变区的Η个互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如沈QIDNO. : 35、SEQIDNO. : 36 和沈QIDNO. : 37 所示。 [0016] In another preferred embodiment, Η complementarity determining regions CDR1 of the heavy chain variable region, CDR2, CDR3 of the amino acid sequence as Shen QIDNO:. 35, SEQIDNO:. 36 and Shen QIDNO:. 37 Suo shows.

[0017] 在另一优选例中,所述重链可变区具有沈QIDNO.:沈QIDNO. :2、6、10或12所示的氨基酸序列。 [0017] In another preferred embodiment, the heavy chain variable region has a counterbore QIDNO .: Shen QIDNO:. An amino acid sequence of 6, 10 or 12 shown in FIG.

[0018] 本发明的第二方面,提供了一种抗体重链,所述抗体重链具有如权利要求1所述的抗体的重链可变区。 [0018] The second aspect of the invention, there is provided an antibody heavy chain, an antibody heavy chain having a heavy chain variable region of the antibody as claimed in claim 1.

[0019] 在另一优选例中,所述抗体重链具有SEQIDNO. :42、43、44或45所示的氨基酸序列。 [0019] In another preferred embodiment, the antibody heavy chain has SEQIDNO:. An amino acid sequence shown 42,43,44 or 45.

[0020] 本发明的第Η方面,提供了一种抗体的轻链可变区,其特征在于,所述的轻链可变区包括W下Η个互补决定区CDR; [0020] Η first aspect of the invention there is provided a light chain variable region of an antibody, wherein the light chain variable region comprising the W complementarity determining regions CDRs of Η;

[0021] (1)互补决定区CDR1' [0021] (1) complementarity determining regions CDR1 '

[0022] 所述互补决定区CDR1' 选自;SEQIDNO. :20、沈QIDNO. :26、沈QIDNO. :32 和沈QIDNO. : 38 ; [0022] The complementarity determining regions CDR1 'is selected from; SEQIDNO: 20, Shen QIDNO: 26, Shen QIDNO: 32 and Shen QIDNO: 38;....

[0023] (2)互补决定区CDR2' [0023] (2) complementarity determining region CDR2 '

[0024]所述互补决定区CDR2' 选自;SEQIDNO. :21、沈QIDNO. :27、沈QIDNO. :33 和沈QIDNO. : 39 ; [0024] The complementarity determining region CDR2 'is selected from; SEQIDNO: 21, Shen QIDNO: 27, Shen QIDNO: 33 and Shen QIDNO: 39;....

[00巧](3)互补决定区CDR3' [00 Qiao] (3) complementarity determining regions CDR3 '

[0026]所述互补决定区CDR3' 选自;SEQIDNO. :22、沈QIDNO. :28、沈QIDNO. ::34 和沈QIDNO. : 40。 [0026] The complementarity determining regions CDR3 'is selected from; SEQIDNO:. 22, Shen QIDNO: 28, and Shen Shen QIDNO :: 34 QIDNO: 40....

[0027] 在另一优选例中,所述轻链可变区的Η个互补决定区CDR1'、CDR2'、CDR3'的氨基酸序列分别如沈QIDNO. : 20、SEQIDNO. : 21和沈QIDNO. : 22所示。 [0027] In another preferred embodiment, Η complementarity determining region of the light chain variable region CDR1 ', CDR2', CDR3 'amino acid sequences as Shen QIDNO:. 20, SEQIDNO:. 21 and sink QIDNO. : 22.

[0028] 在另一优选例中,所述轻链可变区的Η个互补决定区CDR1'、CDR2'、CDR3'的氨基酸序列分别如沈QIDNO. : 26、SEQIDNO. : 27和沈QIDNO. : 28所示。 [0028] In another preferred embodiment, Η complementarity determining region of the light chain variable region CDR1 ', CDR2', CDR3 'amino acid sequences as Shen QIDNO:. 26, SEQIDNO:. 27 and sink QIDNO. : 28 shown in FIG.

[0029]在另一优选例中,所述轻链可变区的Η个互补决定区CDR1'、CDR2'、CDR3'的氨基酸序列分别如沈QIDNO. : 32、SEQIDNO. : 33和沈QIDNO. : 34所示。 [0029] In another preferred embodiment, Η complementarity determining region of the light chain variable region CDR1 ', CDR2', CDR3 'amino acid sequences as Shen QIDNO:. 32, SEQIDNO:. 33 and sink QIDNO. : 34.

[0030]在另一优选例中,所述轻链可变区的Η个互补决定区CDR1'、CDR2'、CDR3'的氨基酸序列分别如沈QIDNO. : 38、SEQIDNO. : 39和沈QIDNO. : 40所示。 [0030] In another preferred embodiment, Η complementarity determining region of the light chain variable region CDR1 ', CDR2', CDR3 'amino acid sequences as Shen QIDNO:. 38, SEQIDNO:. 39 and sink QIDNO. : 40 below.

[0031] 在另一优选例中,所述轻链可变区具有SEQIDNO. :4、8、12或16所示的氨基酸序列。 [0031] In another preferred embodiment, the light chain variable region having SEQIDNO:. 8, 12, or the amino acid sequence shown in 16.

[0032] 本发明的第四方面,提供了一种抗体的轻链,所述的轻链具有如权利要求3所述的抗体的轻链可变区。 [0032] The fourth aspect of the present invention, there is provided an antibody light chain, said light chain having a light chain variable region of the antibody as claimed in claim 3.

[0033] 在另一优选例中,所述抗体轻链具有SEQIDNO. :46、47、48或49所示的氨基酸序列。 [0033] In another preferred embodiment, the antibody light chain has SEQIDNO:. An amino acid sequence of 47, 48 or 49 shown in FIG.

[0034] 在另一优选例中,所述重链的恒定区和/或所述轻链的恒定区是人源的。 [0034] In another preferred embodiment, the heavy chain constant region and / or constant regions of the light chain of human origin.

[0035] 本发明的第五方面,提供了一种特异性结合肥R3的单克隆抗体,其特征在于,所述单克隆抗体具有W下特性: [0035] The fifth aspect of the present invention, there is provided a monoclonal antibody that specifically binds the fertilizer R3, wherein said monoclonal antibody has the characteristic W:

[0036] (1)与肥R3蛋白亲和力常数Μ> 1X10"; [0036] (1) R3 protein and fat affinity constant Μ> 1X10 ";

[0037] 似结合肥R3胞外区的DI或Dili结构域。 [0037] Incorporation of fertilizer DI Dili or R3 domain extracellular region.

[0038] 在另一优选例中,所述单克隆抗体抑制肥R3与其配体化regulin结合的ICs。 [0038] In another preferred embodiment, the monoclonal antibody inhibits fat R3 regulin its ligand binding of ICs. 值《3nM。 Value "3nM.

[0039] 在另一优选例中,所述单克隆抗体与肥R3的结合位点包括选自下组的一个或多个位点:第125位的丝氨酸、第150位天冬氨酸、151位精氨酸和第467位组氨酸。 [0039] In another preferred embodiment, the monoclonal antibody R3 fat binding site comprises one or more sites selected from the group consisting of: serine at position 125, aspartic acid at position 150, 151 Arg and the 467th histidine.

[0040] 在另一优选例中,所述单克隆抗体具有: [0040] In another preferred embodiment, the monoclonal antibody has:

[0041] (1)如权利要求1所述的重链可变区;和/或 [0041] (1) as heavy chain variable region according to claim 1; and / or

[0042] 似如权利要求3所述的轻链可变区。 [0042] The similar light chain variable region as claimed in claim 3.

[0043] 在另一优选例中,所述单克隆抗体具有: [0043] In another preferred embodiment, the monoclonal antibody has:

[0044] (1)如权利要求2所述的重链;和/或 [0044] (1) a heavy chain as recited in claim 2; and / or

[0045] 似如权利要求4所述的轻链。 [0045] The similar light chain as claimed in claim 4.

[0046] 在另一优选例中,所述单克隆抗体包括:单链抗体、双链抗体、嵌合抗体(如人鼠嵌合抗体)、鼠源抗体、人源化抗体或其组合。 [0046] In another preferred embodiment, the monoclonal antibody comprising: a single chain antibodies, diabodies, chimeric antibodies (e.g., human-mouse chimeric antibody), a murine antibody, a humanized antibody, or a combination thereof.

[0047] 在另一优选例中,所述的单克隆抗体,为IgG(IgGl)型抗体。 [0047] In another preferred embodiment, the monoclonal antibody is IgG (IgGl) antibodies.

[0048] 本发明的第六方面,提供了一种重组蛋白,所述的重组蛋白具有: A sixth aspect [0048] of the present invention, there is provided a recombinant protein, a recombinant protein having:

[0049](i)如权利要求1所述的抗体的重链可变区、如权利要求2所述的抗体重链、如权利要求3所述的抗体的轻链可变区、如权利要求4所述的抗体轻链、或如权利要求5所述的单克隆抗体;W及 [0049] (i) a heavy chain variable region as recited in claim 1 antibody as claimed in claim antibody heavy chain according to claim 2, the light chain variable region of the antibody as claimed in claim 3, 4 the antibody light chain, or a monoclonal antibody as claimed in claim 5; and W is

[0050](ii)任选的协助表达和/或纯化的标签序列。 [0050] (ii) optionally assist the expression and / or purification tag sequence.

[005。 [005. 在另一优选例中,所述的标签序列包括細is标签。 In another preferred embodiment, the tag sequences include fine label is.

[0052] 在另一优选例中,所述的重组蛋白特异性抗肥R3。 [0052] In another preferred embodiment, the recombinant protein fertilizer specific anti-R3.

[0053] 在另一优选例中,所述的重组蛋白选自下组: [0053] In another preferred embodiment, the recombinant protein is selected from the group consisting of:

[0054] (a)具有SEQIDNO. :42-49所示的氨基酸序列的多肤; [0054] (a) having SEQIDNO: 42-49 plurality skin amino acid sequence;.

[00巧]化)将(a)中的氨基酸序列经过一个或多个(如1-20个)氨基酸残基的取代、缺失或添加而形成的、由(a)衍生的且特异性抗肥R3的多肤。 [Qiao 00] of) the (e.g. 1-20) amino acid residues substituted with an amino acid sequence in (a) through one or more deletions or additions formed from (a) and the derived specific anti-fat R3 multi skin.

[0056] 本发明的第走方面,提供了一种免疫偶联物,该免疫偶联物含有: [0056] The first aspect of the present invention take, there is provided an immunoconjugate, the immunoconjugate comprises:

[0057] (a)载体部分,所述载体部分含有如权利要求1所述的抗体的重链可变区、如权利要求2所述的抗体重链、如权利要求3所述的抗体的轻链可变区、如权利要求4所述的抗体轻链、或如权利要求5所述的单克隆抗体或权利要求7所述的重组蛋白;和 Light [0057] (a) a carrier portion, the carrier portion comprising a heavy chain variable region of the antibody as claimed in claim 1, an antibody heavy chain according to claim 2, said antibody as claimed in claim 3, chain variable region of the antibody light chain of claim 4, or claim 5 or the monoclonal antibody as claimed in claim in claim 7, the recombinant protein; and

[0058] 化)选自下组的偶联部分;可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。 [0058] of) the coupling portion is selected from the group; detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0059] 在另一优选例中,所述偶联物选自;英光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体化片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如,DT-必肌黄酶值TD)或联苯基水解酶-样蛋白质度™l))、化疗剂(例如,顺笛)或任何形式的纳米颗粒等。 [0059] In another preferred embodiment, the conjugate is selected; British light or luminescent marker, a radioactive marker, the MRI (magnetic resonance imaging) or CT (computer tomography X-ray) contrast agents, or can be an enzyme producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), an antibody, antibody fragment, antibody scFv fragments, gold nanoparticles / nanorods, viral particle, a liposome, nano-magnetic tablets, prodrug activating enzymes (e.g., DT- diaphorase value will TD) or biphenyl hydrolase - like protein of the ™ l)), chemotherapeutic agents (e.g., cis flute), or any other form of nanoparticles.

[0060]本发明的第八方面,提供了一种多核巧酸,它编码选自下组的蛋白质: [0060] The eighth aspect of the present invention, there is provided a multicore clever acid which encodes a protein selected from the group consisting of:

[0061]如本发明第一方面所述的抗体的重链可变区、如本发明第二方面所述的抗体重链、如本发明第Η方面所述的抗体的轻链可变区、如本发明第四方面所述的抗体轻链、或如本发明第五方面所述的单克隆抗体或本发明第六方面所述的所述的重组蛋白。 [0061] The heavy chain variable region of an antibody of the first aspect of the present invention, as the present invention is an antibody heavy chain according to a second aspect, the light chain variable region of an antibody as the first aspect of the present invention Η, the recombinant protein of the antibody light chain according to a fourth aspect of the invention, or as in the fifth aspect of the invention is a monoclonal antibody or according to a sixth aspect of the present invention.

[0062]在另一优选例中,所述的多核巧酸具有沈QIDNO. :1、3、5、7、9、11、13、15或50-57 所示的DM序列。 [0062] In another preferred embodiment, the acid has a counterbore clever multi-core QIDNO:. DM sequence shown 1,3,5,7,9,11,13,15 or 50-57.

[0063]本发明的第九方面,提供了一种载体,它含有本发明第八方面所述的多核巧酸。 [0063] The ninth aspect of the present invention, there is provided a vector comprising polynuclear eighth aspect of the present invention skillfully acid.

[0064]在另一优选例中,所述的载体包括;细菌质粒、瞻菌体、酵母质粒、植物细胞病毒、 哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。 [0064] In another preferred embodiment, the vectors include; bacterial plasmids, looking cells, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors.

[0065]本发明的第十方面,提供了一种遗传工程化的宿主细胞,它含有本发明第九方面所述的载体或基因组中整合有本发明第八方面所述的多核巧酸。 A tenth aspect of the [0065] present invention, there is provided a genetically engineered host cell, the genome or vector comprising a ninth aspect of the invention incorporating the multinuclear an eighth aspect of the invention clever acid.

[0066]本发明的第十一方面,提供了一种制备重组多肤的制备方法,该方法包含: [0066] The eleventh aspect of the present invention, there is provided a process for preparing a recombinant multiple skin preparation, the method comprising:

[0067] (a)在适合表达的条件下,培养本发明第十方面所述的宿主细胞; [0067] (a) under conditions suitable for expression, culturing the host cell of the tenth aspect of the present invention;

[0068] 化)从培养物中分离出重组多肤,所述的重组多肤是本发明第五方面所述的所述的单克隆抗体或本发明第六方面所述的所述的重组蛋白。 [0068] of) a culture isolated from a recombinant multiple skin, the skin is a monoclonal antibody recombinant polynucleotides according to the fifth aspect or the sixth aspect of the present invention, the recombinant protein .

[0069]本发明的第十二方面,提供了一种药物组合物,所述组合物中含有: [0069] The twelfth aspect of the present invention, there is provided a pharmaceutical composition, said composition comprising:

[0070](i)如权利要求1所述的抗体的重链可变区、如权利要求2所述的抗体重链、如权利要求3所述的抗体的轻链可变区、如权利要求4所述的抗体轻链、或如权利要求5所述的单克隆抗体或权利要求7所述的重组蛋白;W及 [0070] (i) a heavy chain variable region as recited in claim 1 antibody as claimed in claim antibody heavy chain according to claim 2, the light chain variable region of the antibody as claimed in claim 3, monoclonal antibody or recombinant protein as claimed in claim 7, the antibody light chain of claim 4, or as claimed in claim 5; and W is

[0071](ii)药学上可接受的载体。 [0071] (ii) a pharmaceutically acceptable carrier.

[0072]在另一优选例中,所述的药物组合物为注射剂型。 [0072] In another preferred embodiment, the pharmaceutical composition is in injectable form.

[0073]在另一优选例中,所述的药物组合物用于制备治疗肿瘤的药物,所述的肿瘤选自下组;非小细胞肺癌、黑色素瘤、头颈肿瘤、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、肾上腺肿瘤、或膀脫肿瘤。 [0073] In another preferred embodiment, the pharmaceutical composition the preparation of a medicament for the treatment of tumors, the tumor is selected from the group; non-small cell lung cancer, melanoma, head and neck cancer, stomach cancer, liver cancer, leukemia, kidney tumor, lung carcinoma, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, adrenal tumors, or of bladder tumors.

[0074]在另一优选例中,所述组合物还包含西妥昔单抗。 [0074] In another preferred embodiment, the composition further comprises cetuximab.

[0075]在另一优选例中,所述组合物中含有本发明第五方面所述的单克隆抗体和西妥昔单抗,其中,所述的单克隆抗体和所述西妥昔单抗的重量比为1-10 :10-1 ;优选地,所述的单克隆抗体和西妥昔的重量比为1-5 :10-1;更优选地,所述的单克隆抗体和西妥昔的重量比为1-5 ;1 [0075] In another preferred embodiment, the composition and the monoclonal antibody cetuximab was contained in the fifth aspect of the present invention, wherein said monoclonal antibody and said cetuximab the weight ratio of 1-10: 10-1; preferably, the monoclonal antibody cetuximab and the weight ratio of 1-5: 10-1; more preferably, the monoclonal antibody cetuximab and Xi is a weight ratio of 1-5; 1

[0076]本发明的第十Η方面,提供了如本发明第一方面所述的抗体的重链可变区、如本发明第二方面所述的抗体重链、如本发明第Η方面所述的抗体的轻链可变区、如本发明第四方面所述的抗体轻链、或如本发明第五方面所述的单克隆抗体或本发明第六方面所述的所述的重组蛋白的用途,用于: [0076] Η a tenth aspect of the invention there is provided a heavy chain variable region of an antibody of the first aspect of the present invention, such as an antibody heavy chain according to a second aspect of the present invention, as a first aspect of the present invention Η light chain variable region of said antibody, recombinant antibody light chain protein of the fourth aspect of the invention or as in the fifth aspect of the invention is a monoclonal antibody or a sixth aspect of the invention is thereof, for:

[0077] (a)细胞的分离、制备、提取、检测;或 [0077] (a) Separation of cells, preparation, extraction, detection; or

[0078] 化)制备用于分离、制备、提取、检测细胞的产品。 [0078] technology) for the preparation of isolation, preparation, extraction, detection of cell products.

[0079]在另一优选例中,所述的细胞为表达肥R3分子的细胞;较佳地为人表达肥R3分子的细胞。 [0079] In another preferred embodiment, the cell is a cell expressing R3 fat molecule; preferably R3 fat cells expressing human molecules.

[0080] 在另一优选例中,所述的分离、制备、提取、检测细胞用的产品包括;介质、磁珠、英光标记抗体、化学标记物标记抗体、放射性同位素标记抗体、胶体金标记抗体、酶标记抗体等。 [0080] In another preferred embodiment, the isolation, preparation, extraction, detection of cells comprising a product; medium, magnetic beads, Eiko labeled antibody, chemical markers labeled antibody, radiolabeled antibody, the colloidal gold-labeled antibody , enzyme-labeled antibody and the like.

[0081] 在另一优选例中,所述的分离、制备、提取、检测细胞用的产品包括;装置、试剂盒等。 [0081] In another preferred embodiment, the isolation, preparation, extraction, detection of cells comprising a product; devices, kits and the like.

[0082] 本发明的第十四方面,提供了一种体外分离人表达肥R3分子的细胞的方法,包括步骤:将本发明第五方面所述的抗体或本发明第六方面所述的重组蛋白(或分离、制备、提取、检测细胞用的产品)与人表达肥R3分子的细胞共赔育或结合,分离(如洗脱或纯化) 出与所述的抗体结合的细胞,从而实现人表达肥R3分子的细胞的分离。 [0082] The fourteenth aspect of the present invention, there is provided a method of in vitro isolated human fat cells expressing R3 molecule, comprising the steps of: a recombinant antibody of the present invention according to a fifth aspect or sixth aspect of the present invention protein (or isolation, preparation, extraction, product detected cells) R3 fat cells expressing human molecules were incubated lost or binding, separation (e.g., elution or purifying) the antibody bound to the cells, enabling human R3 fat cells expressing an isolated molecule.

[0083] 在另一优选例中,所述的分离、制备、提取、检测细胞用的产品包括;含有本发明第五方面所述的单克隆抗体的介质、磁珠、英光标记抗体、化学标记物标记抗体、放射性同位素标记抗体、胶体金标记抗体、酶标记抗体等。 [0083] In another preferred embodiment, the isolation, preparation, extraction, detection of cells comprising a product; medium containing the monoclonal antibody of the fifth aspect of the present invention, magnetic beads, Eiko labeled antibody, a chemical label labeled antibody, radiolabeled antibody, the colloidal gold labeled antibodies, enzyme-labeled antibody and the like.

[0084] 本发明的第十五方面,提供了一种检测样品中是否含有肥R3蛋白的方法,所述方法包括步骤: [0084] The fifteenth aspect of the present invention, there is provided a method of detecting whether the sample contains fat R3 protein, said method comprising the steps of:

[0085] (1)将样品与本发明第五方面所述的单克隆抗体接触; [0085] (1) contacting the sample with a monoclonal antibody of the present invention according to a fifth aspect;

[0086] (2)检测是否形成抗原-抗体复合物,其中形成复合物就表示样品中存在肥R3蛋白。 [0086] (2) detecting the formation of antigen - antibody complex, wherein the complex is formed on the fertilizer R3 represents a protein present in the sample.

[0087] 在另一优选例中,在步骤(1)中,将样品与两种针对肥R3蛋白的抗体接触,并且在步骤(2)中通过化ISA法进行检测,所述两种针对肥R3蛋白的抗体中的至少一种为本发明第五方面所述的抗体。 [0087] In another preferred embodiment, in step (1), the sample with two kinds of antibodies against fat R3 protein contacts, and in step (2) is detected by a method of the ISA, for the two kinds of fertilizer R3 antibody protein of at least one antibody according to the fifth aspect of the present invention.

[0088] 在另一优选例中,所述"抗原-抗体复合物"为"第一抗体-抗原-第二抗体"Η 元复合物,其中,所述第一抗体是本发明第五方面所述的抗体,并且所述第二抗体的结合表位与所述第一抗体的结合表位不同。 [0088] In another preferred embodiment, the "antigen - antibody complex" as the "first antibody - antigen - second antibody" composite element [eta], wherein the first antibody is a fifth aspect of the present invention said antibody binding epitope and the second antibody binding to an epitope different from the first antibody.

[0089] 在另一优选例中,所述第一抗体或所述第二抗体上带有可检测标记。 [0089] In another preferred embodiment, the first antibody or the second labeled with a detectable antibody.

[0090] 在另一优选例中,所述可检测标记为生物素标记、胶体金标记、辣根过氧化物酶标记、放射性核素标记、英光素标记、英光蛋白标记。 [0090] In another preferred embodiment, the detectable label is biotin label, colloidal gold, horseradish peroxidase-labeled, radiolabeled, biotinylated Eiko, British light protein labeling.

[0091] 在另一优选例中,所述样品包括;人或动物组织样品、肿瘤切除样品、培养的人或动物细胞样品。 [0091] In another preferred embodiment, the sample comprises; human or animal tissue samples, tumor resection samples, cultured human or animal cell sample.

[009引在另一优选例中,在步骤似中通过化ISA法进行检测。 [009] In another preferred embodiment, primers, by detection of the ISA method similar step.

[0093] 在另一优选例中,所述方法用于非诊断性的目的。 [0093] In another preferred embodiment, the method for non-diagnostic purposes.

[0094] 本发明的第十六方面,提供了一种治疗与肥R3分子相关的病症的方法,包括步骤:给需要抑制或需要治疗的对象施用本发明第五方面所述的单克隆抗体或本发明第六方面所述的重组蛋白或本发明第十二方面所述的药物组合物。 [0094] The sixteenth aspect of the present invention, there is provided a method of treating disorders associated with fat R3 molecule, comprising the steps of: in need of inhibition or to a subject in need of treatment is administered a monoclonal antibody according to the fifth aspect of the present invention or the the recombinant proteins of the sixth aspect of the present invention or the twelfth aspect of the present invention is a pharmaceutical composition.

[0095] 在另一优选例中,所述的与肥R3分子相关的疾病包括肿瘤、枯抗器官移植免疫排斥反应。 [0095] In another preferred embodiment, the R3 associated with fat molecules diseases include tumor, anti-dry organ allograft rejection.

[0096] 在另一优选例中,所述的对象是哺乳动物(包括人)。 [0096] In another preferred embodiment, the subject is a mammal (including a human).

[0097] 应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可W互相组合,从而构成新的或优选的技术方案。 [0097] It should be understood, within the scope of the present invention, W can be combined with one another between the technical features of the invention and in the following technical features (as described in Example) specifically described, thereby constituting a new or preferred Technical solutions. 限于篇幅,在此不再一一累述。 Due to space limitations, which is not eleven tired.

附图说明 BRIEF DESCRIPTION

[009引图1显示流式检测仪检测的检测结果,其中图1A为抗体927的检测结果,图1B为993的检测结果,图1C为抗体1044的检测结果,图1D为抗体1050的检测结果。 [009 Primer 1 shows a detection result detected by flow cytometry, where FIG. 1A is a detection result of antibody 927, FIG. 1B is a detection result 993, and FIG 1C is a detection result of antibody 1044, FIG. 1D is an antibody detection result 1050 .

[0099] 图2显示鼠单抗抑制肥R3与配体结合的ICs。 [0099] Figure 2 shows inhibition of murine mAb R3 fertilizer ligand binding ICs. 的检测结果,图2A显示了肥R3抗体927抑制肥R3与配体HRG的结合,图2B显示了肥R3抗体993抑制肥R3与配体HRG的结合,图2C显示了肥R3抗体1044抑制肥R3与配体HRG的结合,图2D显示了肥R3抗体1050 抑制肥R3与配体HRG的结合。 Detection results, FIG. 2A shows fertilizer R3 antibody 927 inhibits binding of fertilizer R3 are ligands of HRG, FIG 2B shows the fat R3 antibody 993 inhibits binding of fertilizer R3 are ligands of HRG, Figure 2C shows the fertilizer R3 antibody 1044 suppressing fat R3 HRG binding of the ligand, Figure 2D shows 1050 fertilizer R3 antibody inhibits binding of the ligand R3 fertilizer of HRG.

[0100] 图3显示人非细胞肺癌A549体内肿瘤模型实验,图3A为肿瘤体积的检测结果,图3B为肿瘤重量的检测结果。 [0100] Figure 3 shows a non-human A549 lung cancer cells in vivo tumor model experiments, FIG. 3A is a result of the detection of the tumor volume, FIG. 3B is a result of the detection of tumor weight.

[0101] 图4显示了肥R3单抗与西妥昔单抗的协同效果,图4A显示了肥R3抗体1044与西妥昔协同抑制A431生长实验、图4B显示了肥R3抗体927与西妥昔协同抑制A431生长实验、图4C显示了肥R3抗体993与西妥昔协同抑制A431生长实验、图4D显示了肥R3抗体1050与西妥昔协同抑制A431生长实验。 [0101] Figure 4 shows the manure and R3 monoclonal antibody cetuximab synergistic effect, FIG. 4A shows 1044 fertilizer R3 antibody cetuximab synergistic inhibition of growth of A431 experiment, Figure 4B shows the fat and R3 antibody cetuximab 927 Xi synergistic inhibition A431 growth experiment, fertilizer R3 4C shows growth experiments to A431 antibody 993 fertilizer R3 antibody cetuximab, 1050 and synergistic inhibitory synergistic inhibition A431 cetuximab growth experiment, shown in FIG. 4D. 图中,西妥昔单独组用量为5μg/ml,协同组西妥昔用量为2. 5μg/ml;各图中横坐标数值单位为μg/ml(抗体用量)。 FIG, cetuximab alone group in an amount of 5μg / ml, the cooperative group in an amount of cetuximab 2. 5μg / ml; FIG abscissa values ​​each unit is μg / ml (the amount of antibody).

具体实施方式 detailed description

[0102] 本发明人通过广泛而深入的研究,获得一类抗肥R3的特异性抗体,实验结果表明,该类抗体能够显著抑制肥R3与其配体的结合。 [0102] The present invention, through extensive and intensive studies to obtain a class of specific antibody fat R3, experimental results show that such antibody is able to significantly inhibit binding to its ligand fertilizer R3. 并且意外的发现使用该抗体与西妥昔单抗联用具有显著的协同作用,能够明显的减少西妥昔的用量。 And accidentally found that the use of the antibody cetuximab combined with a significant synergistic effect, can significantly reduce the amount of cetuximab. 在此基础上完成了本发明。 Based on this completed the present invention.

[0103]肥R3巧rbB-3、ERBB3、c-erbB-3、c-erbB3、受体酪氨酸蛋白质激酶erbB-3、SEQID NO. :41)是编码表皮生长因子受体巧GFR)酪氨酸激酶家族的一名成员,在本发明的一个优选地实施方式中,肥R3的氨基酸序列如下所示: [0103] R3 fertilizer Qiao rbB-3, ERBB3, c-erbB3, c-erbB3, a receptor tyrosine protein kinase erbB3, SEQID NO:. 41) encoding the epidermal growth factor receptor is a clever GFR) casein a member of the tyrosine kinase family, in a preferred embodiment of the present invention, the amino acid sequence of fertilizer R3 are as follows:

[0104]MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTG HNADLS化QWIREVTGYVLVAM肥FSTLPLP化RVVRGTQVYDGKFAIFVMLNYNTNSSHALR化化TQLTEILSGG VYKKNDKLCHMDTIDW畑IV畑畑AEIVV邸NGRSCPPC肥VCKGRCWGPGS邸CQTLTKTICAPQCNG肥FGPNP NQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLT巧LEPNPHTKYQYGGVCVASCPHNFVVDQTS CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIP ALD阳KLNVFRTVRE口GYLNIQSWPPHMHNFSVre化TTIGGRSLYNRGF化LIMKNLNVTSLGFRSLKEISAGRI YISANR化CYH服LNWTKVLRGPT邸RLDIKHNRPR畑CVAEGKVCD化CSSGGCWGPGPGQ化SCRNYSRGGVCVT HCN化NGEPREFA肥AECFSCHPECQPMEGTATCNGSGSDTCAQCAHF畑GP肥VSSCP服VLGAKGPIYKYPDVQN ECRPC肥NCTQGCKG阳LQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGT化YWRGRRIQNKRAMRRYLERGE SIEPLDPSEKANKVLARIFKEWL服LKVLGSGVFGTVHKGVWI阳GESIKIPVCIKVIEDKSGRQSFQAVTDHMLA IGSLDHAHIV化L化CPGS化化VTQYLPLG化LDHVRQHRGALGPQ化LNWGVQIAKGMYYLEE服MVHRNLAARN V化KSPSQVQVADFGVADliPPDDK化LYSEAKTPIKWMALESIHFGKYT册SDVWSYGVTVWELMTFGAEPYAGLR LAEVPDLLEKGERLAQPQICTIDVY [0104] MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTG HNADLS of QWIREVTGYVLVAM fertilizer FSTLPLP of RVVRGTQVYDGKFAIFVMLNYNTNSSHALR technology of TQLTEILSGG VYKKNDKLCHMDTIDW Hata IV Hata Hata AEIVV Di NGRSCPPC fertilizer VCKGRCWGPGS Di CQTLTKTICAPQCNG fertilizer FGPNP NQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLT Qiao LEPNPHTKYQYGGVCVASCPHNFVVDQTS CVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIP ALD male KLNVFRTVRE port GYLNIQSWPPHMHNFSVre of TTIGGRSLYNRGF of LIMKNLNVTSLGFRSLKEISAGRI YISANR of CYH service LNWTKVLRGPT Di RLDIKHNRPR Hata CVAEGKVCD of CSSGGCWGPGPGQ of SCRNYSRGGVCVT HCN NGEPREFA of fertilizer AECFSCHPECQPMEGTATCNGSGSDTCAQCAHF Hata GP fertilizer VSSCP service VLGAKGPIYKYPDVQN ECRPC fertilizer NCTQGCKG male LQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGT of YWRGRRIQNKRAMRRYLERGE SIEPLDPSEKANKVLARIFKEWL service LKVLGSGVFGTVHKGVWI of male GESIKIPVCIKVIEDKSGRQSFQAVTDHMLA IGSLDHAHIV L of CPGS of VTQYLPLG of service of LDHVRQHRGALGPQ of LNWGVQIAKGMYYLEE KSPSQVQVADFGVADliPPDDK MVHRNLAARN V of copies of LYSEAKTPIKWMALESIHFGKYT SDVWSYGVTVWELMTFGAEPYAGLR LAEVPDLLEKGERLAQPQICTIDVY MVMVKCWMIDENIRPTFKELANEFTRMA畑PPRYLVIKRESGPGIAPGPEPH GLTNKKLEEVELE阳LDLDLDLEA邸DNLATTTLGSALSLPVGTLNRPRGSQ化LSPSSGYMPMNQGNLGESC犯SA VSGS沈RCPRPVSLHPMPRGCLA沈SSEGHVTG沈A化犯KVSMCRSRSRSRSPRPRGDSAY服QR服LLTPVTPLS PPGLE邸DVNGYVMPDTHLKGTPSSREGTLSSV化SS化GT邸邸抓邸YEYMNRRRRHSPPHPPRPSSLE化GYEY MDVGSDLSASLGSTgSCPLHPVPIMPTAGTTPD邸YEYMNRg畑GGGPGGDYAAMGACPA沈gGYEEMRAFOGPG册APHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT(SEQIDNO. :41) MVMVKCWMIDENIRPTFKELANEFTRMA Hata PPRYLVIKRESGPGIAPGPEPH GLTNKKLEEVELE Yang LDLDLDLEA Di DNLATTTLGSALSLPVGTLNRPRGSQ of LSPSSGYMPMNQGNLGESC guilty SA VSGS Shen RCPRPVSLHPMPRGCLA Shen SSEGHVTG Shen A of committing KVSMCRSRSRSRSPRPRGDSAY clothes QR clothes LLTPVTPLS PPGLE Di DVNGYVMPDTHLKGTPSSREGTLSSV of the SS of GT Di Di catch Di YEYMNRRRRHSPPHPPRPSSLE of GYEY MDVGSDLSASLGSTgSCPLHPVPIMPTAGTTPD Di YEYMNRg Hata GGGPGGDYAAMGACPA Shen gGYEEMRAFOGPG book APHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT ( SEQIDNO:. 41)

[0105] 如本文所用,术语"重链可变区"与"V/可互换使用。 [0105] As used herein, the term "heavy chain variable region" and "V / are used interchangeably.

[0106] 如本文所用,术语"轻链可变区"与"\ "可互换使用。 [0106] As used herein, the term "light chain variable region" and "\" are used interchangeably.

[0107] 如本文所用,术语"可变区"与。 [0107] As used herein, the term "variable region" and. 互补决定区(complementaritydetermining region,CDR)"可互换使用。 Complementarity determining region (complementaritydetermining region, CDR) "are used interchangeably.

[010引在本发明中,术语"本发明抗体"、"本发明蛋白"、或"本发明多肤"可互换使用,都指特异性结合肥R3的抗体,例如具有重链(如SEQIDNO. :42、43、44、45的氨基酸序列) 和/或轻链(如SEQIDNO. :46、47、48、49的氨基酸序列)的蛋白或多肤。 [010 incorporated in the present invention, the term "antibody of the invention", "present invention protein", or "the present invention is a multi-skin" are used interchangeably and all refer to an antibody that specifically binds fat R3 is, for example, a heavy chain (e.g., SEQIDNO .: 42, 43 amino acid sequence) and / or light chain (e.g., SEQIDNO:. 46, 47 amino acid sequence) of a protein or polypeptide. 它们可含有或不含起始甲硫氨酸。 They may or may not contain the starting methionine.

[0109] 本发明提供了一种抗肥R3的抗体(单克隆抗体)或其片段。 [0109] The present invention provides an antibody of an anti-fat R3 (monoclonal antibody), or fragment thereof.

[0110] 优选地,所述抗体的重链可变区可具有选自下组的互补决定区CDR;SEQID ^.:17、23、29、35所示的〔031,56910^.:18、24、30、36所示的〔01?2,和沈910^.:19、 25、31、37 所示的CDR3。 [0110] Preferably, the heavy chain variable region of the antibody may have a complementarity determining region CDR chosen from the group; shown in [17,23,29,35 SEQID ^ .: 031,56910 ^ .: 18, [01 shown 24,30,36? 2, and CDR3 as 19, 25,31,37 Shen .: 910 ^.

[01U] 更佳地,所述的重链可变区可具有SEQIDNO. :2、6、10、14所示的氨基酸序列。 [01U] More preferably, the heavy chain variable region may have SEQIDNO:. 2,6,10,14 amino acid sequence.

[0112] 优选地,所述抗体的轻链可变区可具有选自下组的互补决定区CDR;SEQID 備.:20、26、32、38所示的〔01?1',沈910側.:21、27、33、39所示的〔01?2',和沈910側.:22、 28、34、40 所示的CDR3'。 [0112] Preferably, the light chain variable region of the antibody may have a complementarity determining region CDR chosen from the group consisting of; SEQ ID Preparation: [1 01 shown 20,26,32,38 ', 910 sink side.? .:? [01 shown 21,27,33,39 2 ', and the sink side 910:. 22, CDR3 shown 28,34,40'.

[0113] 更佳地,所述的轻链可变区可具有SEQIDNO. :4、8、12、16所示的氨基酸序列。 [0113] More preferably, the light chain variable region may have SEQIDNO:. An amino acid sequence shown in 4-16.

[0114] 在另一优选例中,所述的抗体为抗肥R3人鼠嵌合单克隆抗体,它的重链恒定区和/或轻链恒定区可W是人源化的重链恒定区或轻链恒定区。 [0114] In another preferred embodiment, the antibody is an anti-human fat R3 murine chimeric monoclonal antibodies, which heavy chain constant region and / or light chain constant region may be W is a humanized heavy chain constant region or light chain constant region. 更优选地,所述的人源化的重链恒定区或轻链恒定区为人IgGl、IgG2等的重链恒定区或轻链恒定区。 More preferably, the humanized heavy chain constant region or light chain constant region of a human IgGl, IgG2 heavy chain constant region like or light chain constant region.

[0115] 本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。 [0115] The present invention further provides a fusion with other proteins or antibodies of the present invention the expression product. 具体地,本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物),只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90%同源性,较佳地至少95%同源性。 In particular, the present invention comprises (i.e. immunoconjugate and fusion expression products) any protein or fusion protein conjugate and having a heavy chain and a light chain variable region comprising the expression product, as long as the variable region of an antibody of the present invention the same variable regions of the heavy and light chains, or at least 90% homology, preferably at least 95% homology.

[0116] 一般,抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述,称为可变区域(CDR),将该段间隔成4个框架区域(FR),4个FR的氨基酸序列相对比较保守,不直接参与结合反应。 [0116] In general, the binding properties of the antigen may be located in the heavy and light chain variable regions of the three specific regions will be described, referred to as variable regions (CDRs of), the section frame partitioned into four regions (FR) , FR amino acid sequences of four relatively conserved, not directly involved in binding reaction. 送些CDR形成环状结构,通过其间的FR形成的目折叠在空间结构上相互靠近,重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。 Send some CDR form a cyclic structure, formed by the mesh therebetween FR folding structure close to each other in space, the CDR and CDR of the light chain corresponding to the heavy chain constitutes the antigen-binding site of an antibody. 可W通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。 W may be determined by comparing the amino acid sequences of the same type of antibody is an amino acid which constitutes a FR or CDR regions.

[0117] 本发明抗体的重链和/或轻链的可变区特别令人感兴趣,因为它们中至少部分涉及结合抗原。 The variable regions of the heavy and / or light chain [0117] Antibodies of the present invention is of particular interest, since they are at least partially involved in binding the antigen. 因此,本发明包括郝些具有带CDR的单克隆抗体轻链和重链可变区的分子,只要其CDR与此处鉴定的CDR具有90%W上(较佳地95%W上,最佳地98%W上)的同源性。 Accordingly, the present invention comprises a monoclonal antibody molecule HAO these light chain and heavy chain variable region having CDR's with, as long as it CDR and CDR identified herein having the upper 90% W (preferably 95% W, the best on the 98% W) homology.

[0118] 本发明不仅包括完整的单克隆抗体,还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。 [0118] The present invention includes not only intact monoclonal antibodies, a fusion protein further comprising an antibody fragment or other sequences of immunoreactive antibodies formed. 因此,本发明还包括所述抗体的片段、衍生物和类似物。 Accordingly, the present invention further includes fragments of the antibodies, derivatives and analogs.

[0119] 如本文所用,术语"片段"、"衍生物"和"类似物"是指基本上保持本发明抗体相同的生物学功能或活性的多肤。 [0119] As used herein, the term "fragment", "derivative" and "analog" refers to maintaining substantially the same biological function or activity of the antibody of the invention multiple skin. 本发明的多肤片段、衍生物或类似物可w是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肤,而送样的取代的氨基酸残基可W是也可W不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的多肤,或(iii)成熟多肤与另一个化合物(比如延长多肤半衰期的化合物,例如聚己二醇)融合所形成的多肤,或(iv)附加的氨基酸序列融合到此多肤序列而形成的多肤(如前导序列或分泌序列或用来纯化此多肤的序列或蛋白原序列,或与細is标签形成的融合蛋白)。 Multi skin fragments of the invention, derivatives or analogs may be w is (i) with one or more conservative or non-conservative amino acid residue (preferably a conserved amino acid residue) is substituted with multiple skin, substituted and send samples the amino acid residues may also be W is not W or (ii) a plurality skin substituent group, or (iii) more mature skin and other compounds in one or more amino acid residues encoded by the genetic code ( prolong half-life, such as multi-compound skin, e.g. skin plurality polyhexamethylene glycol) formed by fusion, or (iv) an additional amino acid sequence of this fusion sequence plurality skin formed by multiple skin (such as a leader or secretory sequence or to this purified proprotein sequence or multiple sequences skin, or is formed with a fine-tag fusion protein). 根据本文的教导,送些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。 According to the teachings herein, send some fragments, derivatives and analogs belonging to the person skilled in the art well-known range.

[0120] 本发明抗体指具有人肥R3结合活性的、包括上述CDR区的多肤。 [0120] The present invention refers to a human antibody binding activity R3 fat, skin comprising the above-described multi-CDR regions. 该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肤的变异形式。 The term also includes antibodies having the same function of the present invention, in the form of multiple variants of the skin including the CDR regions. 送些变异形式包括(但并不限于);一个或多个(通常为1-50个,较佳地1-30个,更佳地1-20个,最佳地1-10个) 氨基酸的缺失、插入和/或取代,W及在C末端和/或N末端添加一个或数个(通常为20 个W内,较佳地为10个W内,更佳地为5个W内)氨基酸。 Send some variant forms including (but not limited to); one or more (generally 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids deletion, insertion and / or substitution, W, and add one or several at the C-terminus and / or N-terminus (generally within 20 W, preferably within 10 W, more preferably within five W) amino acid . 例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能。 For example, in the art, when substituted with amino acids similar or like properties, typically it does not change the function of the protein. 又比如,在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。 Further, the addition of one or several amino acids at the C-terminus and / or N-terminus typically will not change the function of the protein. 该术语还包括本发明抗体的活性片段和活性衍生物。 The term also includes the active fragments of the antibodies and active derivatives of the present invention.

[0121] 该多肤的变异形式包括;同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、W 及利用抗本发明抗体的抗血清获得的多肤或蛋白。 [0121] The plurality of variant forms of skin comprising; homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA encodes an antibody of the present invention under high or low stringency conditions of DNA hybridization of the encoded protein, W, and the use of multiple antibodies against skin or proteins of the present invention, antisera obtained.

[0122] 本发明还提供了其他多肤,如包含人抗体或其片段的融合蛋白。 [0122] The present invention further provides a plurality of other skin, such as a human antibody or fragment thereof comprises a fusion protein. 除了几乎全长的多肤外,本发明还包括了本发明抗体的片段。 In addition to almost the entire length of the multi-skin, the present invention also includes fragments of the antibodies of the present invention. 通常,该片段具有本发明抗体的至少约50个连续氨基酸,较佳地至少约50个连续氨基酸,更佳地至少约80个连续氨基酸,最佳地至少约100个连续氨基酸。 Typically, the antibody fragment of the present invention having at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 contiguous amino acids.

[0123] 在本发明中,"本发明抗体的保守性变异体"指与本发明抗体的氨基酸序列相比, 有至多10个,较佳地至多8个,更佳地至多5个,最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肤。 [0123] In the present invention, "conservative variants of the antibodies of the present invention" refers to the amino acid sequence of the antibody as compared with the present invention, having up to 10, preferably up to 8, more preferably up to 5, the best up to 3 amino acids are replaced with amino acids similar or similar properties to form a plurality skin. 送些保守性变异多肤最好根据表A进行氨基酸替换而产生。 Send some conservative variations skin preferably carried out multiple amino acid substitutions according to Table A produced.

[0124] 表A [0124] Table A

[0125] [0125]

Figure CN105367657AD00121

Figure CN105367657AD00131

[0126] [0126]

[0127] 本发明还提供了编码上述抗体或其片段或其融合蛋白的多核巧酸分子。 [0127] The present invention further provides an antibody or fragment thereof encoding the above fusion protein polynuclear clever acid molecule. 本发还提供了编码上述抗体或其片段或其融合蛋白的多核巧酸分子。 This provides reimbursement antibodies or fragments thereof encoding the above fusion protein polynuclear clever acid molecule. 本发明的多核巧酸可W是DNA形式或RNA形式。 W polynuclear Qiao acid form of the present invention is a DNA or RNA form. DNA形式包括cDNA、基因组DNA或人工合成的DNA。 The form of DNA including cDNA, genomic DNA or synthetic DNA. DNA可W是单链的或是双链的。 W is a DNA may be double-stranded or single-stranded. DNA可W是编码链或非编码链。 DNA may be the coding strand or non-coding W strand. 编码成熟多肤的编码区序列可W与SEQID NO. :50-57所示的编码区序列相同或者是简并的变异体。 Encoding the mature coding sequence of a plurality of skin may be W and SEQID NO:. The same as the coding sequence shown in FIG 50-57 or degenerate variant thereof. 如本文所用,"简并的变异体"在本发明中是指编码具有与本发明的多肤相同的氨基酸序列,但与SEQIDNO. :50-57所示的编码区序列有差别的核酸序列。 As used herein, "degenerate variant" is meant in the present invention, and encoding a plurality of skin of the present invention is the same amino acid sequence, but SEQIDNO:. 50-57 coding region of nucleic acid sequence shown in sequence differences.

[012引编码本发明的成熟多肤的多核巧酸包括;只编码成熟多肤的编码序列;成熟多肤的编码序列和各种附加编码序列;成熟多肤的编码序列(和任选的附加编码序列)W及非编码序列。 [012 primers of the invention encodes more mature skin polynuclear acids include clever; encoding only the mature coding sequence of multiple skin; more mature skin and various additional coding sequences coding sequence; the coding sequence for the mature plurality skin (and optionally additional coding sequence) W and non-coding sequences.

[0129] 术语"编码多肤的多核巧酸"可W是包括编码此多肤的多核巧酸,也可W是还包括附加编码和/或非编码序列的多核巧酸。 [0129] The term "polynuclear Coded skin clever acid" W may include multiple skin encoding such polynuclear clever acid, W may further include a multicore additional coding and / or non-coding sequence clever acid.

[0130] 本发明还涉及与上述的序列杂交且两个序列之间具有至少50%,较佳地至少70%,更佳地至少80%相同性的多核巧酸。 [0130] The present invention further relates to and hybridizes to a sequence between the two sequences having at least 50%, preferably at least 70%, more preferably at least 80% identical multicore clever acid. 本发明特别涉及在严格条件下与本发明所述多核巧酸可杂交的多核巧酸。 The present invention particularly relates to the present invention under stringent conditions to the multi-core multicore Qiao Qiao acid hybridizable acid. 在本发明中,"严格条件"是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0. 2XSSC,0. 1%SDS,6(TC;或(2)杂交时加有变性剂,如50% (v/v) 甲醜胺,0. 1%小牛血清/0. 1%Ficoll,42°C等;或(3)仅在两条序列之间的相同性至少在90%W上,更好是95%W上时才发生杂交。并且,可杂交的多核巧酸编码的多肤与SEQID NO. : 10和/或SEQIDNO. : 15所示的成熟多肤有相同的生物学功能和活性。 In the present invention, "stringent conditions" refers to: (1) hybridization and wash under lower ionic strength and higher temperature, such as 0. 2XSSC, 0 1% SDS, 6 (TC; or (2). plus denaturant, such as 50% (v / v) methyl amine ugly, 01% calf serum / 0 1% Ficoll, 42 ° C hybridization and the like;., or (3) only between two sequences identity on at least 90% W, preferably hybridization occurs when the 95% W and can hybridize polynuclear acids encoded multichannel coincidence skin and SEQID NO:.. 10 and / or SEQIDNO:. mature 15 shown multi-skin have the same biological function and activity.

[0131] 本发明的抗体的核巧酸全长序列或其片段通常可W用PCR扩增法、重组法或人工合成的方法获得。 [0131] The antibodies of the invention nuclear acid Qiao full-length sequences or fragments thereof may generally W by PCR amplification, recombination or synthetic methods available. 一种可行的方法是用人工合成的方法来合成有关序列,尤其是片段长度较短时。 One possible method is a method for synthesis of synthetic sequence, especially when fragments of shorter length. 通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。 Typically, by first synthesizing a plurality of small fragments, and then the obtained long fragment sequence. 此外,还可将重链的编码序列和表达标签(如細is)融合在一起,形成融合蛋白。 Moreover, the coding sequence and may label the expression of the heavy chain (such as fine is) together to form a fusion protein.

[0132] 一旦获得了有关的序列,就可W用重组法来大批量地获得有关序列。 [0132] Once the relevant sequence is obtained, W can be obtained in large quantities by recombinant methods related sequences. 送通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。 Feeding usually cloned into a vector and then into cells, and then isolated from the host cell after the sequences related to proliferation by conventional methods. 本发明所涉及的生物分子(核酸、蛋白等)包括W分离的形式存在的生物分子。 The present invention relates to a biological molecule (nucleic acid, protein, etc.) comprises a biomolecule isolated form of W present.

[0133]目前,已经可W完全通过化学合成来得到编码本发明蛋白(或其片段,或其衍生物)的DNA序列。 [0133] It has been completely W can be obtained encoding the protein of the invention (or fragment or derivative thereof) DNA sequences by chemical synthesis. 然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。 The DNA sequence may then be incorporated in the art known in various existing DNA molecules (or, vector) and cells. 此外,还可通过化学合成将突变引入本发明蛋白序列中。 In addition, mutations may be incorporated by chemical synthesis of the protein sequences of the present invention.

[0134] 本发明还涉及包含上述的适当DNA序列W及适当启动子或者控制序列的载体。 [0134] The present invention further relates to DNA sequences containing the appropriate W and appropriate promoters or control vector sequence. 送些载体可W用于转化适当的宿主细胞,W使其能够表达蛋白质。 These vectors may send W for transformation of a suitable host cell, W so as to express the protein.

[0135] 宿主细胞可W是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。 [0135] W is a host cell may be a prokaryotic cell, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. 代表性例子有:大肠杆菌,链霉菌属;鼠伤寒沙口氏菌的细菌细胞;真菌细胞如酵母;果觸S2或Sf9的昆虫细胞;CH0、C0S7、293细胞的动物细胞等。 Representative examples are: E.coli, Streptomyces; Shakou typhimurium bacterial cells of Salmonella; fungal cells such as yeast; S2 touch or if Sf9 insect cells; CH0, C0S7,293 cells and animal cells.

[0136] 用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 [0136] using conventional techniques well known to those skilled in the host cell transformed with a recombinant DNA. 当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaClz法处理, 所用的步骤在本领域众所周知。 When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaClz method steps used are well known in the art. 另一种方法是使用MgClz。 Another method is to use MgClz. 如果需要,转化也可用电穿孔的方法进行。 If desired, the conversion process may be performed using electroporation. 当宿主是真核生物,可选用如下的DNA转染方法:磯酸巧共沉淀法,常规机械方法如显微注射、电穿孔,脂质体包装等。 When the host is a eukaryote, optional DNA transfection methods as follows: a coprecipitation method Rocky acid Qiao, conventional mechanical methods such as microinjection, electroporation, liposomal packaging.

[0137] 获得的转化子可W用常规方法培养,表达本发明的基因所编码的多肤。 Transformants [0137] W can be obtained by conventional culturing methods, the expression of the gene of the present invention, the multi-skin encoded. 根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。 Depending on the host cell used, the culture medium used may be selected from various conventional culture media. 在适于宿主细胞生长的条件下进行培养。 Cultured under conditions suitable for growing the host cells. 当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导) 诱导选择的启动子,将细胞再培养一段时间。 When the host cells are grown to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells cultured for a period of time.

[013引在上面的方法中的重组多肤可在细胞内、或在细胞膜上表达、或分泌到细胞外。 [013 cited in the above process may be a recombinant multiple skin, expressed on the cell membrane or within the cell, or secreted outside the cell. 如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 If desired, utilizing the physical, chemical and other characteristics of the isolated and purified by various separation methods of recombinant protein. 送些方法是本领域技术人员所熟知的。 These methods are sent to the person skilled in the art. 送些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离必、渗透破菌、超处理、超离必、分子筛层析(凝胶过滤)、 吸附层析、离子交换层析、高效液相层析0PLC)和其它各种液相层析技术及送些方法的结厶口0 Examples of such methods include, but are sent are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), from the must, bacteria osmosis, ultra-treatment, will transcend, molecular sieve chromatography (gel filtration) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography 0PLC) and various other liquid chromatography techniques and send these methods port junction Si 0

[0139] 本发明的抗体可W单独使用,也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何W上送些物质的组合结合或偶联。 [0139] Antibodies of the invention W may be used alone or with a detectable marker (for diagnostic purposes), therapeutic agents, some combination of feed material bound or conjugated on PK (protein kinase) or any modified part W.

[0140] 用于诊断目的的可检测标记物包括但不限于;英光或发光标记物、放射性标记物、 MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。 [0140] for diagnostic purposes detectable labels include, but are not limited to; British light or luminescent marker, a radioactive marker, the MRI (magnetic resonance imaging) or CT (computer tomography X-ray) contrast agents, or capable of generating a detectable enzyme product.

[0141] 可与本发明抗体结合或偶联的治疗剂包括但不限于;1.放射性核素化oppe等, 2005,癌转移评论(Cancermetastasisreviews) 24, 539) ;2.生物毒(Qiau化ary等, 1989,自然(化Uire) 339, 394 ;化el等,2002,癌症免疫学和免疫治疗(CancerImmunology andImmunotherapy)51,565) ;3.细胞因子如IL-2 等(Gillies等,1992,美国国家科学院院刊(PNA巧89,1428 ;Card等,2004,癌症免疫学和免疫治疗(CancerImmunologyand Immunotherapy)53,345;Halin等,2003,癌症研究(CancerResea;rch)63,3202);4.金纳米颗粒/ 纳米棒(Xapotko等,2005,癌症通信(Cancerletters)239,36;Huang等,2006,美国化学学会杂志(JournaloftheAmericanQiemicalSociety) 128,2115) ;5.病毒颗粒任eng等,2004,基因治疗(Genetherapy) 11,1234) ;6.脂质体(Mamot等,2005,癌症研究(Cancerresearch)65,11631) ;7.纳米磁粒;8.前药激活酶(例如,DT-必肌黄酶值TD)或联苯基水解酶-样蛋白质度™d) ;1〇.化 [0141] therapeutic agent may be combined with or conjugated antibodies of the invention include, but are not limited to,;. 1 of radionuclides oppe et al, 2005, Cancer metastasis reviews (Cancermetastasisreviews) 24, 539);. 2 entomotoxicity (Qiau of ary et al., 1989, Nature (of Uire) 339, 394; of el al., 2002, cancer Immunology and immunotherapy (CancerImmunology andImmunotherapy) 51,565);. 3 cytokines such as IL-2, etc. (Gillies et al., 1992, U.S. national Academy of Sciences (PNA clever 89,1428; Card et al., 2004, cancer Immunology and immunotherapy (CancerImmunologyand immunotherapy) 53,345; Halin et al, 2003, cancer Research (CancerResea; rch) 63,3202);. 4 gold nanoparticles / nanorods (Xapotko et al., 2005, cancer communications (Cancerletters) 239,36; Huang et al., 2006, Journal of the American chemical Society (JournaloftheAmericanQiemicalSociety) 128,2115);. 5 viral particles either eng et al, 2004, gene therapy (Genetherapy) 11,1234); 6 liposomes (Mamot et al., 2005, cancer Research (Cancerresearch) 65,11631);.. 7 nano-magnetic particle; 8 prodrug activating enzymes (e.g., DT- diaphorase value will TD) or biphenyl hydrolase - like protein degree ™ d);. 1〇 of 剂(例如,顺笛)或任何形式的纳米颗粒等。 Agents (e.g., cis flute), or any other form of nanoparticles.

[0142] 本发明还提供了一种组合物。 [0142] The present invention further provides a composition. 在优选例中,所述的组合物是药物组合物,它含有上述的抗体或其活性片段或其融合蛋白,W及药学上可接受的载体。 In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment or fusion protein pharmaceutically, W, and a pharmaceutically acceptable carrier. 通常,可将送些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中,其中抑通常约为5-8,较佳地抑约为6-8,尽管pH值可随被配制物质的性质W及待治疗的病症而有所变化。 Typically, these substances may be formulated to send a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the suppressor is usually about 5-8, preferably about 6-8 suppressed, although the pH may vary W formulated material nature and condition to be treated vary. 配制好的药物组合物可W通过常规途径进行给药,其中包括(但并不限于);瘤内、腹膜内、静脉内、或局部给药。 Formulated pharmaceutical composition may be administered by conventional routes W, including (but not limited to); intratumoral, intraperitoneal, intravenous, or topical administration.

[0143] 本发明的药物组合物可直接用于结合人肥R3分子,因而可用于预防和治疗肿瘤。 [0143] The pharmaceutical compositions of the present invention can be directly used for human binding molecules fertilizer R3, which can be used for prevention and treatment of tumors. 此外,还可同时使用其他治疗剂。 Further, optionally in conjunction with other therapeutic agents.

[0144]本发明的药物组合物含有安全有效量(如0. 001-99wt%,较佳地0. 01-90wt%, 更佳地0.l-80wt%)的本发明上述的单克隆抗体(或其偶联物)W及药学上可接受的载体或赋形剂。 The above-described monoclonal antibody of the present invention [0144] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0. 001-99wt%, preferably 0. 01-90wt%, more preferably 0.l-80wt%) of (or conjugate) W, and a pharmaceutically acceptable carrier or excipient. 送类载体包括(但并不限于);盐水、缓冲液、葡萄糖、水、甘油、己醇、及其组合。 Sending class vectors include (but are not limited to); saline, buffered saline, dextrose, water, glycerol, hexanol, and combinations thereof. 药物制剂应与给药方式相匹配。 The pharmaceutical formulation should match the mode of administration. 本发明的药物组合物可W被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 The pharmaceutical compositions of the invention W may be made in the form of injection, for example, prepared with physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. 药物组合物如针剂、溶液宜在无菌条件下制造。 Pharmaceutical compositions such as injections, the solution should be manufactured under sterile conditions. 活性成分的给药量是治疗有效量,例如每天约1微克/千克体重-约5毫克/千克体重。 The dose of the active ingredient is a therapeutically effective amount, e.g. from about 1 microgram per day / kg body weight - about 5 mg / kg body weight. 此外,本发明的多肤还可与其他治疗剂一起使用。 Further, the multi skin may also be used with the present invention with other therapeutic agents.

[0145]使用药物组合物时,是将安全有效量的免疫偶联物施用于哺乳动物,其中该安全有效量通常至少约10微克/千克体重,而且在大多数情况下不超过约8毫克/千克体重, 较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。 [0145] When using the pharmaceutical composition, is a safe and effective amount of the immunoconjugate is administered to mammals, wherein the safe and effective amount typically at least about 10 micrograms / kg body weight, and in most cases not more than about 8 mg / kg body weight, preferably the dose is about 10 micrograms / kg body weight - about 1 mg / kg body weight. 当然,具体剂量还应考虑给药途径、病人健康状况等因素,送些都是熟练医师技能范围之内的。 Of course, the specific dose factors should be considered the route of administration, patient health status, to send more physicians are skilled within the skill range.

[0146] 本发明的主要优点在于: [0146] The main advantage of the present invention is that:

[0147] (1)提供了一类新型的抗肥R3抗体,所述抗体与肥R3分子具有很强的亲和力,可特异性结合抗原分子,尤其是人鼠嵌合抗体有效降低了鼠抗的免疫原性。 [0147] (1) provides a class of novel anti-fat R3 antibody, the antibody molecule with R3 fertilizer having strong affinity molecule can specifically bind an antigen, particularly human chimeric antibody effectively reduces the murine antibody immunogenicity.

[014引(2)本发明的抗肥R3抗体具有显著的抑制肿瘤生长的活性。 [014 Primer (2) an anti-fat R3 antibody of the invention has significant activity in inhibition of tumor growth.

[0149] (3)本发明的肥R3抗体与西妥昔联用,能够显著增强西妥昔单抗的活性,并且能够降低西妥昔单抗的用量。 [0149] (3) R3 fertilizer antibody of the invention in combination with cetuximab, can significantly enhance the activity of cetuximab, and can reduce the amount of cetuximab.

[0150] 下面结合具体实施例,进一步阐述本发明。 [0150] The following embodiments with reference to specific embodiments, further illustrate the present invention. 应理解,送些实施例仅用于说明本发明而不用于限制本发明的范围。 It should be understood, send some embodiments of the present invention are illustrative only and are not intended to limit the scope of the present invention. 下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆;实验室手册(NewYork:ColdSpringHarborL油oratory Press, 1989)中所述的条件,或按照制造厂商所建议的条件。 Experimental methods without specific conditions in the examples below, generally in accordance with conventional conditions as described by Sambrook et al, Molecular Cloning; A Laboratory Manual (NewYork: ColdSpringHarborL oil oratory Press, 1989) in the conditions recommended by the manufacturer, or conditions of. 除非另外说明,否则百分比和份数按重量计算。 Unless otherwise indicated, percentages and parts are by weight.

[0151]本发明实施例中所用的实验材料如无特殊说明均可从市售渠道获得,其中,Ba化/c小鼠购自上海斯莱克公司;CHO/化化细胞购自美国ATCC,ATCC的货号ATCC巧C化-9096TM;Sp2/0-Agl4小鼠骨髓瘤细胞购自美国ATCC,ATCC货号ATC;0管.C化-1581™。 [0151] Experimental Examples Materials used in the embodiment of the present invention, if no special instructions are available from commercial sources, wherein, Ba of / c mice were purchased from Shanghai SLAC Company; CHO / of cells purchased from ATCC, ATCC Qiao ATCC item number C of -9096TM; Sp2 / 0-Agl4 mouse myeloma cells were purchased from ATCC, ATCC NO ATC; 0 .C tube of -1581 ™. 阳15引连施例1、肥R3鼠源单克降杭体的制各 Example 15 male primer 1 attached, R3 murine monoclonal fertilizer made hang down each body

[0153] 一、肥R3单克隆抗体杂交瘤细胞株的制备 [0153] a, R3 fertilizer preparation of monoclonal antibodies of hybridoma cell lines

[0154] 1、免疫原 [0154] 1, immunogen

[01巧]免疫原为肥R3胞外区第584位的组氨酸突变成苯丙氨酸胞外区全长(肥R3-ECD-册84F),通过CH0/化化细胞(购自ATCC)稳转表达,纯化细胞培养上清得到。 [01 Qiao] R3 fertilizer immunogen is the extracellular domain of the histidine at position 584 is mutated to phenylalanine extracellular region of the full-length (fertilizer volumes R3-ECD- 84F), by CH0 / of cells (available from ATCC) stably transfected expression and purification of cell culture supernatant obtained.

[0156]2、免疫Ba化/c小鼠 [0156] 2, immunization of Ba / c mice

[0157]Ba化/c小鼠购自上海斯莱克公司,所有免疫用Ba化/c小鼠均为3周龄、雌性、标准化无病、健康的纯种小鼠,符合美国FDA标准。 [0157] of Ba / c mice were purchased from Shanghai SLAC Company, immunization with all of Ba / c mice were 3 weeks old, female, standardized disease, pure healthy mice, consistent with FDA standards.

[0158] 3、Ba化/c小鼠免疫方法 [0158] 3, Ba of / c mice were immunized method

[0159] 第一次免疫;将50μg(250μ1)抗原与250μ1MF59佐剂混匀,配置成500μ1溶液,注射于Ba化/c小鼠皮下多点及足掌。 [0159] The first immunization; and 50μg (250μ1) mixing antigen and adjuvant 250μ1MF59 configured 500μ1 solution of injected Ba / c mice subcutaneously, and the multi-paw. 第二次免疫:距第一次免疫间隔Η周后,同法同剂量进行第二次免疫。 Second immunization: After the interval from the first immunization Η weeks, second immunization with the same dose method. 第Η、四次免疫;距第二次免疫间隔二周后,同法同剂量进行第Η、四次免疫。 Of [eta], fourth immunization; from the second immunization two weeks after an interval, [eta] with the same dose for the first method, a four immunization. 四次免疫后小鼠尾静脉采血,采用常规酶联免疫吸附实验巧LISA法)测定血清抗体滴度,待血清滴度〉1〇5W上时(为3. 3X106),准备做细胞融合。 Four mice immunized tail vein blood using a conventional ELISA method clever LISA) serum antibody titer, serum titer to be> 1〇5W the upper (as 3. 3X106), prepared for cell fusion. 细胞融合前Η天进行加强免疫;小鼠尾静脉注射抗原20μg(100μ1)。 Η days before cell fusion boosted; tail vein of mice injected antigen 20μg (100μ1).

[0160] 4、细胞融合: [0160] 4, cell fusion:

[016。 [016. Sp2/0-Agl4小鼠骨髓瘤细胞购自美国ATCC。 Sp2 / 0-Agl4 mouse myeloma cells were purchased from ATCC.

[0162] (1)融合前一天换液,使Sp2/0-Agl4骨髓瘤细胞保持良好生长状态。 [0162] one day before medium change (1) integration of the Sp2 / 0-Agl4 myeloma cells were maintained good growth state.

[0163] (2)脾细胞:取免疫小鼠,放血,断颈粹死,于75%酒精浸泡3-4min。 [0163] (2) spleen cells: the immunized mice, bled and died pure cervical, soaked in 75% alcohol 3-4min. 无菌条件下取出小鼠脾脏,放入15ml离必管中,加入少许无血清RPM1640培养液,用移液管轻轻吹打, 碼碎,直至没有组织结块、细胞均匀为止。 Mouse spleen removed under sterile conditions, into the tube will be from 15ml, add a little RPM1640 serum-free medium, pipetted gently pipetting, code broken until no lumps tissue, until the cells uniformly. 然后,用无血清RPM1640培养液洗涂小鼠脾脏细胞Η次,计数备用。 Then, serum-free culture RPM1640 spleen cells were washed twice Η coating mice, counted standby.

[0164] (3)取对数生长期的Sp2/0-Agl4骨髓瘤细胞,无血清RPM1640培养液洗涂Η次, 计数备用。 [0164] (3) in the logarithmic phase of Sp2 / 0-Agl4 myeloma cells, serum-free culture medium RPM1640 wash coating Η times, the standby counting.

[016引(4)将小鼠脾脏细胞和Sp2/0-Agl4骨髓瘤细胞W10 ;1的比例混合,150化pm离必7min。 [016 primer (4) The mouse spleen cells and Sp2 / 0-Agl4 myeloma cells W10 of; 1 ratio, will be from 150 pm of 7min. 洗去上清液,准备融合。 The supernatant was washed away, ready integration.

[0166] (5) -分钟内缓缓加入1ml阳G(1450),轻摇90sec;再在2. 5min中内加入5ml无血清RPM1640培养液,最后再加入5ml无血清培液终止反应,静置5min后,128化pm离必8min,弃去上清,加入常规RPM1640培养液(含有10%胎牛血清),制备成细胞悬液。 [0166] (5) - within minutes was slowly added 1ml male G (1450), rocked 90sec; then add 5ml of serum-free medium in RPM1640 medium 2. 5min, then add 5ml of serum-free culture solution to terminate the reaction, static rear 5min, 128 pm of from will 8min, supernatant was discarded, addition of conventional RPM1640 medium (containing 10% fetal bovine serum) to prepare a cell suspension.

[0167] (6)将上述细胞悬液W每孔2X104个细胞的密度种入96孔板,每孔200μ1,置于37°C、5%α)2细胞培养箱中赔育。 [0167] (6) The above cell suspension at a density of 2X104 cells per well W into 96-well plates, each well 200μ1, placed in 37 ° C, 5% α) 2 cell culture incubator incubated lost. 培养24h后,更换含有HAT(25X)的常规RPM1640 培养液,于37°C、5% (¾细胞培养箱中继续赔育。培养14d后用ELISA法检测各个克隆细胞的上清液筛选肥R3抗体的阳性克隆。 After incubation 24h, replacing conventional RPM1640 medium containing HAT (25X) is, at 37 ° C, 5% (¾ lost incubation continued cell incubator. After incubation 14d detect each clonal cell supernatants were screened by ELISA fertilizer R3 antibody positive clones.

[016引5、细胞筛选与亚克隆: [016 lead 5, the cell selection and subcloning:

[0169] 首先使用含有HAT的常规RPM1640培养液进行培养筛选,培养7d后,改用HT的常规RPM1640培养液培,进行再次培养筛选。 [0169] First, using conventional RPM1640 medium containing HAT were screened culture, after culture 7d, conventional RPM1640 culture broth instead of HT, cultured screened again. 培养14d后,用ELISA方法W各个克隆细胞的上清液筛选肥R3抗体的阳性克隆。 After incubation 14d, fat R3 antibody screening positive clones supernatant of each clone by ELISA W cells. 采用有限稀释法,将细胞悬液稀释至60个/ml,于96 孔板中每孔加100μ1 (约6个细胞/孔)。 Using limiting dilution, cell suspensions were diluted to 60 / ml, in 96 well plates each well 100μ1 (about 106 cells / well). 接种2排,剩余细胞悬液用培养液作倍比稀释, 再接种2排。 Inoculated 2 rows, the remaining cell suspension was used as the dilution culture, then inoculated with 2 rows. 重复一次。 repeat. 置37°C、5%C02细胞培养箱中赔育。 Set 37 ° C, 5% C02 incubator cells incubated lost. 每隔2~3天,更换1/2培养液。 Every 2 to 3 days, changing the medium 1/2. 培养约lOd后,选择单个克隆生长的阳性孔进行第二次筛选和亚克隆。 After incubation for about lOd, selecting positive wells individual clones were grown and subcloned second screening. 连续Η次亚克隆后,经化ISA法检测抗体阳性率为100%时确定为稳定表达目的抗体的杂交瘤细胞株, 保种建库。 Η consecutive subcloning, the antibody positive rate was determined at 100% hybridoma cell lines stably expressing the antibody was detected by the ISA technology, conservation storage.

[0170] 6、鼠源单抗的制备与纯化 [0170] 6. Preparation and purification of murine monoclonal antibody

[017。 [017. 提前一周注射液体石蜡,0. 5ml/只腹腔注射6周龄W上的Ba化/c小鼠,每种杂交瘤细胞注射Ba化/c小鼠3只。 One week ahead of the injection liquid paraffin, 0. 5ml / Ba of only intraperitoneal / c mice were injected on weeks 6 W, Ba injection of hybridoma cells each / c mice 3. 7天后腹腔注射1~2X106个杂交瘤细胞,隔7~10天取腹水。 7 days after the intraperitoneal injection of 1 ~ 2X106 hybridoma, taken every 7 to 10 days ascites. 抗体纯化过程参照GEproteinG蛋白柱进行: Referring GEproteinG protein antibody purification columns:

[0172] (1)腹水1200化pm离必15min去杂质与上层降脂焼,平衡缓冲液(pH7. 0PB巧5倍稀释,用0. 45um膜过滤。 [0172] (1) 1200 of ascites pm 15min impurities from the upper layer will lowering firing, the equilibration buffer (pH7. 0PB opportunely diluted 5-fold with a 0. 45um membrane filtration.

[0173] (2)proteinG柱用d地20平衡柱子5~10柱体积后用平衡缓冲液平衡柱子10个柱体积; [0173] (2) after the 20 d proteinG column Equilibrate the column with 5 to 10 column volumes of equilibration buffer the column is equilibrated with 10 column volumes;

[0174] (3)按照1mlproteinG胶结合5mg蛋白的载量上样(腹水抗体含量通常为;1~ 5mg/ml) [0174] (3) according to 1mlproteinG 5mg loading of glue-like binding protein (usually antibody content ascites; 1 ~ 5mg / ml)

[0Π5] (4)用平衡缓冲液液平衡柱子10柱体积; [0Π5] (4) with equilibration buffer, the column was balanced by 10 column volumes;

[0176] (5)用洗脱液洗脱(0.1MpH2. 7甘氨酸-HC1) 10个柱体积,分管收集,洗脱蛋白迅速按每1ml加入150ul1MTris-ClpH9. 0的比例进行中和。 [0176] (5) with eluent (0.1MpH2. 7 Glycine -HC1) 10 column volumes, charge collection, protein was eluted quickly added per 1ml 150ul1MTris-ClpH9. 0 ratio of neutralization.

[0177] 化)SDS-PAGE分析洗脱抗体的量和纯度。 [0177] of) SDS-PAGE analysis of quantity and purity of the antibody was eluted. . 刪连施例2、鼠单杭亲巧力测定 Example 2 even deleted, Hang affinity murine single measurement Qiao

[0179]采用proteonTMXPRSGProteinInteractionArraySystem法(可参考文献BronnerV,DenkbergG,PeledΜ,etal.Therapeuticantibodies:Discoveryand developmentusingtheProteOnXPR36biosensorinteractionarraysystem.Anal Biochem2010 ;406 :147-156)测定肥R3单克隆抗体的亲和力。 [0179] The method proteonTMXPRSGProteinInteractionArraySystem (Reference may BronnerV, DenkbergG, PeledΜ, etal.Therapeuticantibodies: Discoveryand developmentusingtheProteOnXPR36biosensorinteractionarraysystem.Anal Biochem2010; 406: 147-156) Determination of the affinity of the monoclonal antibody R3 fertilizer.

[0180] [0180]

Figure CN105367657AD00171

[0181]连施例3、杭化r3单杭对细胸表面化r3外子的识别 [0181] Example 3 even, Hang Hang superficial identification of single r3 r3 of the outer sub-small chest

[0182] 采用流式细胞术(FAC巧进行检测,间接免疫英光染色法。 [0182] Flow cytometry (FAC coincidence detection, Eiko indirect immunofluorescence staining.

[0183] (1)计数;取对数生长期的S邸R-3细胞,调整单细胞悬液浓度为2X106/ml。 [0183] (1) count; logarithmic growth phase S Di R-3 cells, a single cell suspension adjusted to a concentration of 2X106 / ml.

[0184] (2)洗涂;取1ml单细胞悬液加入1.5ml离必管中,100化pmX3min。 [0184] (2) wash-coated; 1ml of a single cell suspension will be added 1.5ml from tube 100 of pmX3min. 弃上清,用2% PBA(PBS加2%的胎牛血清)洗涂并重悬细胞,100化pmX3min,弃上清。 The supernatant was discarded, with 2% PBA (PBS plus 2% fetal calf serum) and resuspended washcoated cells, 100 of pmX3min, the supernatant was discarded.

[0185] (3) -抗赔育:用2 %PBA稀释抗化r2单抗至10μg/ml,加入200μ1,轻轻吹打混匀细胞,4°C冰浴30min。 [0185] (3) - Anti incubated lost: 2% PBA was diluted with inhibitors r2 mAb to 10μg / ml, was added 200μ1, cells were mixed gently pipetting, 4 ° C ice bath was 30min. 同时做空白对照和鼠IgG单抗同型对照。 While doing the blank control and mouse IgG isotype control mAb. 100化pmX3min,弃上清。 100 of pmX3min, the supernatant was discarded.

[0186] (4)英光二抗赔育:用预冷的2%PBA洗涂一次,加入PBA适当稀释的FITC标记羊抗小鼠IgG多抗(lug/106细胞)200μ1。 [0186] (4) a secondary antibody Eiko incubated lose: washcoated once with pre-cooled 2% PBA, PBA was added appropriately diluted FITC-labeled goat anti-mouse IgG polyclonal antibody (lug / 106 cells) 200μ1. 轻轻吹打混匀细胞,4°C避光冰浴30min。 Cells were mixed gently pipetting, 4 ° C in the dark ice bath was 30min.

[0187] (5)用预冷的2%PBA洗涂2次。 [0187] (5) with a pre-cooled 2% PBA washcoat twice. 将细胞重悬于200μ1PBS中,轻轻混匀,置流式管中,避光,用流式检测仪检测。 The cells were resuspended 200μ1PBS gently mixing at flow tube, protected from light, detected by flow cytometry.

[018引检测结果如图1所示,图1A为抗体927的检测结果,图1B为993的检测结果,图1C为抗体1044的检测结果,图1D为抗体1050的检测结果,从图中可W看出相对于阴性对照,抗体927、993、1044、1050都有一定程度的英光偏移,并且1044的英光偏移最大。 [018 lead detection results shown in FIG. 1, FIG. 1A is a result of the detection antibody 927, FIG. 1B is a detection result 993, and FIG 1C is a result of the detection of antibody 1044, an antibody detection result of FIG. 1D 1050, from FIG. W seen with respect to the negative control, we have some degree of antibody 927,993,1044,1050 British light offset and a maximum offset of 1044 British light.

[0189] 图中所示:鼠源抗体927,、993、1044、1050流式细胞术结果。 [0189] Shown in: murine antibody 927,, 993,1044,1050 flow cytometry results. 黑色表示对照组,绿色线表示抗体组,Ml表示对照组英光偏移量,M2表示抗体实验组英光偏移量。 Represents a group of black, green line indicates antibody group, represents a group of Ml Eiko offset, M2 represents an antibody light experiment England offset. 阳190]连施例4、肥R3鼠源杭体结合肥R3胸外区结构城确定 Male 190] connected Example 4, murine R3 Hang fat fat-binding region R3 chest structure City OK

[0191] 为了确定抗体的表位是在HER3胞外区的具体结构域,本发明人分别构建了分段表达肥R3胞外区结构域的真核表达载体。 [0191] To determine the epitope of an antibody specific domain in the extracellular region of HER3, the present inventors constructed the eukaryotic expression vector segment fertilizer R3 intracellular domain extracellular region expression. 根据肥R3胞外区的四个结构域,把胞外区分成DI、DII、DIII、DIV、DI+II、DII+III、DIII+IV、DI+II+IILDII+III+IV九段,分别构建与hGH融合表达的PCDNA3. 1 (+/-)真核表达载体。 The four domains of the extracellular region R3 fertilizer, the extracellular region is divided into DI, DII, DIII, DIV, DI + II, DII + III, DIII + IV, DI + II + IILDII + III + IV 9p were constructed hGH expression of the fusion PCDNA3. 1 (+/-) eukaryotic expression vector. 分别将送九个表达载体进行瞬时转染表达,W鼠抗hGH单抗包板,检测结构域表达水平;W我们筛选的鼠单抗包化ISA板,检测鼠单抗与肥R3胞外区结构域的结合。 Respectively, will be sent to nine expression vectors Transient Transfection, W mouse anti-hGH monoclonal antibody coated plates, detection of the expression level domain; W is We screened Antibody packetized ISA board, fertilizer and R3 Antibody detection extracellular region binding domain.

[0192] [0192]

Figure CN105367657AD00181

阳19引连施例5、肥R3鼠源单杭表份的确定 19 male lead connected Example 5, fertilizer R3 murine single parts table determined Hang

[0194] 通过点突变技术,将肥R3胞外区全长的表达质粒的几个氨基酸突变,突变位点分别为第I结构域的第125位丝氨酸突变成亮氨酸(S12化),第150和151位天冬氨酸和精氨酸突变成鄉氨酸和脯氨酸值150V-R151P),第162位精氨酸突变成甘氨酸巧162G)第III结构域的第467位组氨酸突变酪氨酸(H467Y),第471和472位精氨酸和精氨酸突变成亮氨酸和甘氨酸(R471kR472G)。 [0194] technique by point mutations, several plasmids expressing the amino acid extracellular region of the full-length R3 fat mutation, mutation sites are serine at position 125 of the I domain was mutated to leucine (S12 based), 150 and 151 mutated to aspartic acid and arginine and proline Township value 150V-R151P), 162 Pro197 glycine Qiao 162G) 467 bit domain III tyrosine histidine mutations (H467Y), 471 and arginine 472 and arginine mutated to leucine and glycine (R471kR472G). 完成突变之后,将突变过的表达质粒通过瞬时转染的方式表达, 之后用ELISA的方法检测肥R3单抗与具有突变的肥R3胞外区结合情况。 After completion method of mutation, over-expression plasmids mutations by transient transfection of the expression way, after the detection by ELISA and monoclonal antibody fat fat R3 R3 extracellular region having a mutation binding conditions.

[0195] [0195]

Figure CN105367657AD00191

[0196] 从上表中可已看出肥R3的第125位的丝氨酸、第150位天冬氨酸和151位精氨酸与抗体927结合相关;肥R3的第467位组氨酸与抗体1044结合相关;肥R3的第125位的丝氨酸、第150位天冬氨酸和151位精氨酸与抗体1050结合相关。 [0196] can be seen that the fertilizer has the R3 position 125 from the table serine, aspartic acid at position 150 and arginine 151 associated with the antibody binding 927; 467 bit group and R3 manure histidine antibody 1044 with related; R3 fertilizer of serine 125, aspartic acid at position 150 and arginine 151 associated with the antibody 1050 binding. 阳197] 连施例6、ELISA檢郷I杭体抑制肥R3与配体Heregulin的结合 Male 197] 6 even applied, subject Hongo I binding ELISA inhibition fat R3 Hang ligand Heregulin Example

[019引W10μg/ml浓度Trx-HRG融合蛋白包被ELISA板,10%牛奶封闭。 [019 primer W10μg / ml concentration of Trx-HRG fusion protein ELISA plates were coated, blocked 10% milk.

[0199] (1)W固定浓度的肥R3-ECD融合蛋白(lug/ml)与不同浓度梯度的单抗混合, 37°C预先赔育30πΰη,100μ1/孔,加入化ISA板,37°C赔育比。 [0199] (1) W fixed concentration of fat R3-ECD fusion protein (lug / ml) with different concentrations of mAb gradient mixed, 37 ° C pre-incubated lose 30πΰη, 100μ1 / hole, added of ISA board, 37 ° C lose fertility ratio.

[0200] (2)PBST洗板10遍,拍干。 [0200] (2) PBST plate was washed 10 times, and pat dry. 用5%的脱脂牛奶PBST按1 :6000比例稀释兔抗hGH 多抗,100μ1/孔,37°C赔育比。 Dilution of rabbit polyclonal anti-hGH ratio 6000, 100μ1 / hole, 37 ° C incubation compensation ratio: Press 1 with 5% skim milk in PBST.

[0201] (3)PBST洗板10遍,拍干。 [0201] (3) PBST plate was washed 10 times, and pat dry. 用5%的脱脂牛奶PBST按1 :6000比例稀释HRP标记的羊抗兔多抗,100μ1/孔,37°C赔育比,用0. 5%〇PBST洗板10遍。 With 5% skim milk in PBST at 1: 6000 dilution ratio of HRP labeled goat anti-rabbit polyclonal antibody, 100μ1 / hole, 37 ° C incubation compensation ratio, with 0.5% 〇PBST washer 10 times.

[020引(4)显色;用DAB法显色,100μ1/孔,于室温反应5~lOmin。 [020 primer (4) color; chromogenic method with DAB, 100μ1 / hole, at room temperature 5 ~ lOmin.

[020引(5)终止;用2M/L硫酸溶液,100μ1/孔。 [020 primer (5) terminates; with 2M / L solution of sulfuric acid, 100μ1 / hole.

[0204] (6)测定0〇4加。 [0204] (6) Measurement of 0〇4 added.

[0205] 结果如图2所示,从图2中可W看出,鼠单抗抑制肥R3与配体HRG(参考文献, WallaschC,WeissFU,NiederfellnerG,etal.Heregulin-dependentregulation ofHER2/neuoncogenicsignalingbyheterodimerizationwithHERS[J].EMBO J. 1995, 14(17) :4267-4275.)结合的IC5。 [0205] The results shown in Figure 2, can be seen from the figure W 2, Antibody inhibition of HRG ligand fertilizer R3 (Reference, WallaschC, WeissFU, NiederfellnerG, etal.Heregulin-dependentregulation ofHER2 / neuoncogenicsignalingbyheterodimerizationwithHERS [J] .EMBO J. 1995, 14 (17):. 4267-4275) combined IC5. 分别为927 为1. 5nM,1044 为2. 3nM、933 为0. 3nM、 1050 为2.8nM。 927, respectively is 1. 5nM, 1044 is 2. 3nM, 933 to 0. 3nM, 1050 to 2.8nM. 阳20引连施例7、杭肥R3单杭对肿概牛长的抑制作巧 20 male lead connected Example 7, R3 single fertilizer Hang Hang inhibition of swelling almost as long bovine Qiao

[0207] (1)处于对数生长期的A549细胞(人非小细胞肺癌细胞系,购自中国科学院细胞库),0. 25%膜酶消化、收集并计数。 [0207] (1) A549 cells in the logarithmic growth phase (human non-small cell lung cancer cell line, purchased from the cell bank CAS), 0.25% membrane enzyme digestion, collected and counted.

[020引(2)无血清的培养液洗一遍,lOOOrmp室温离必5min。 [020 Primer (2) serum-free culture medium again washed, lOOOrmp from room temperature will 5min. 无血清培养液重悬。 Resuspended in serum-free culture. 按1X10^只的A549细胞接种的裸鼠背部皮下。 Press 1X10 ^ only in A549 cells in nude mice inoculated subcutaneously.

[0209] (3)两周左右能看到明显的肿瘤长出时,将裸鼠随机分组,每组8只,抗体按500μg/只的剂量进行腹腔注射治疗,WPBS作为对照,每隔Η天治疗一次并测量肿瘤体积,治疗持续一个月时间。 [0209] about (3) can be seen two weeks when palpable tumors grow, the mice were randomly divided into groups of eight, antibodies via intraperitoneal injection press 500μg / dose only, WPBS As a control, every day Η tumor volume was measured once and treatment, treatment time for a month. 裸鼠肿瘤体积大小;V= 0. 5XaXb2,a为肿瘤实体长直径,b为肿瘤实体短直径。 The size of the tumor volume in nude mice; V = 0. 5XaXb2, a longer diameter of tumor mass, b is the short diameter of the tumor mass.

[0210] (4)实验结束时将肿瘤取下,去皮后称重。 [0210] (4) at the end of the experiment tumors were removed, weighed peeled.

[0211] 结果如图3所示,图3A显示了结果如图3所示,图3A显示了持续治疗时各组的肿瘤体积,图3B显示了治疗结束后取出肿瘤的重量;从图3中可W看出抗体927、抗体993、抗体1044、抗体1050可W显著地抑制肿瘤的生长,4个抗体的肿瘤抑制率> 65% 。 [0211] The results shown in Figure 3, Figure 3A shows the results shown in Figure 3, Figure 3A shows the tumor volume of each group during continued treatment, Figure 3B shows the weight of the tumors removed after the end of treatment; FIG. 3 from W can be seen that antibody 927, an antibody 993, antibody 1044, antibody 1050 W can significantly inhibit tumor growth, tumor inhibition rate 4 antibody> 65%. 21引连施例8、肥R3单杭与西耍昔单杭协同抑制A431细胸牛长 Example 8 even lead 21, a single fertilizer R3 Hang Xi West playing single small chest Hang synergistic inhibition of bovine long A431

[0213] 处于对数期生长的A431细胞(人头颈部表皮癌细胞系,购自中国科学院细胞库), 膜酶消化后计数,用含有1%FBS的DF/12培养基调整细胞密度到20000个细胞/ml,将稀释好的细胞W200ul/孔的量铺到96孔板中,37°C,5% (¾培养24小时。之后选取96孔板中间的8排4列32个孔,分成8组,每组4个重复,对应加入不同浓度的单抗,单独作用组, 肥R3单抗终浓度共六个梯度,分别为12. 5ug/ml,6. 25ug/ml,3. 12加g/ml;西妥昔单抗终浓度为加g/ml,协同作用组肥R3单抗与西妥昔单抗浓度减半混合。对照组中不加入任何抗体。37°C,5%C〇2培养4到5天,之后用CellCountingKit-8 (CCK-8)细胞增殖-毒性检测试剂盒(购自同仁化学)检测细胞活性,细胞活性与0D450读数正相关。 [0213] A431 cells in log phase growth (head and neck epidermoid carcinoma cell line, purchased from the cell bank CAS), the film after enzymatic digestion counted with a DF containing 1% FBS / 12 medium cell density was adjusted to 20,000 cells / ml, the diluted cells W200ul / well of plated into 96 well plates, 37 ° C, 5% (¾ for 24 hours. after selecting the middle row 96 8 4 32 holes, into 8 groups of 4 replicates, correspond to different concentrations of monoclonal antibody alone group, R3 monoclonal antibody at a final concentration of fat gradient a total of six, respectively 12. 5ug / ml, 6. 25ug / ml, 3. 12 plus g / ml; cetuximab plus a final concentration of g / ml, a synergistic effect with the monoclonal antibody R3 group fertilizer cetuximab concentration of the mixed half the control group without addition of any antibody .37 ° C, 5% C. 〇2 4-5 days culture, followed by proliferation CellCountingKit-8 (CCK-8) - cytotoxicity detection kit (available from Dojin Kagaku) ​​detecting cell viability, and cellular activity was positively correlated readings 0D450.

[0214] 实验结果如图4所示,从图中可W看出将肥R3抗体与西妥昔单抗联用,产生了协同效果,1044抗体与西妥昔单抗联用,与单用西妥昔相比,抑制细胞生长的活性提高了38%。 [0214] The results shown in Figure 4, can be seen from the figure the fertilizer W R3 antibody cetuximab in combination with, a synergistic effect, an antibody cetuximab 1044 in combination with, and alone compared cetuximab, cell growth inhibitory activity increased by 38%. 927抗体与西妥昔单抗联用,与单用西妥昔相比,抑制细胞生长的活性提高了32%。 927 antibody in combination with cetuximab, compared with cetuximab alone, cell growth inhibitory activity increased by 32%. 993抗体与西妥昔单抗联用,与单用西妥昔相比,抑制细胞生长的活性提高了16%。 993 antibody in combination with cetuximab, compared with cetuximab alone, cell growth inhibitory activity increased by 16%. 1050抗体与西妥昔单抗的协同效果较不明显。 1050 antibody cetuximab synergistic effect is less pronounced. 而且,使用本发明的抗体和西妥昔单抗联用可W显著降低西妥昔的用量。 Further, the present invention is the use of antibodies cetuximab and may be combined with W significantly reduce the amount of cetuximab. 邮1引连施例9、肥R3单杭可巧区編码序列的克降巧鉴定 Post 1 primer attached Example 9, the coding sequence grams fat R3 Hang happened that a single drop zone identification Qiao

[0216]W实施例1中筛选所得肥R3杂交瘤细胞株cDNA为模板,利用PCR技术,克隆肥R3 鼠源单抗可变区基因;经测序,选取无突变无终止密码子的序列,采用5'RACE技术克隆出功能性Vl和Vh基因。 Example The resulting cDNA Screening of the hybridoma cell line R3 fertilizer 1 [0216] W embodiment as a template by PCR, cloned murine R3 monoclonal antibody fat variable region gene; sequenced, mutated sequence selected no no termination codon, using cloning of a functional 5'RACE Vl and Vh gene.

[0217] 一、肥R3鼠源单抗可变区编码序列的克隆 [0217] First, the coding sequence of clone fertilizer R3 murine monoclonal antibody variable region

[0218](一)从杂交瘤细胞株中提取肥R3单抗的总RNA [0218] (a) extracting fat from the hybridoma cell line R3 mAb Total RNA

[0219] 用上海飞捷生物公司FAST1000试剂盒提取。 [0219] extracted with Shanghai Jetsgo biotech companies FAST1000 kit.

[0220] (1)取4X105杂交瘤细胞,100化pmX3min,弃上清。 [0220] (1) take 4X105 hybridoma cells, 100 of pmX3min, the supernatant was discarded. 用PBS洗涂一次。 Washcoated once with PBS. 将细胞重悬于100μ1PBS中,放入离必管内。 The cells were resuspended 100μ1PBS, the tube must be placed away.

[0221] (2)加入RB1液1ml,充分颠倒混匀直至完全溶解,室温放置5min。 [0221] (2) was added to RB1 1ml, sufficiently mix by inversion until complete dissolution room temperature for 5min.

[0222] (3)加入RB2液500μ1,充分颠倒混匀Imin。 [0222] (3) was added RB2 500μ1, sufficiently mix by inversion Imin. 将混匀后的液体吸入或直接倒入内套管中离必Imin。 The mixed liquid after suction or directly into the inner sleeve will leave Imin.

[0223] (4)弃去外套管中液体,内套管中加入500μ1洗液,离必Imin,再重复一次。 [0223] (4) was added 500μ1 discarded wash liquid jacket, the inner casing tube, will from Imin, repeated once.

[0224] (5)取出内套管,弃去外套管中液体,仍然套回内套管,不加洗液,离必Imin。 [0224] (5) remove the inner tube, the outer liquid discarded tubes, the inner tube is still set back, without lotion, will be from Imin.

[0225] (6)将内套管移入新的离必管中,在膜中央加入洗脱液40μ1,室温静置Imin,获得总RNA。 [0225] (6) into the inner sleeve will be from a new tube, was added in the center of the film eluent 40μ1, Imin allowed to stand at room temperature, to obtain total RNA.

[0226] (W上枪头、离必管和无菌水均用DEPC处理,经12rC灭菌20min。) [0226] (W on the tip, away from the pipe and will with DEPC-treated sterile water are, sterilized by 12rC 20min.)

[0227] (二)RT-PCR制备肥R3鼠源单抗cDNA [0227] (ii) RT-PCR R3 murine monoclonal antibody prepared fertilizer cDNA

[022引队总RNA为模板,Oligo(dT) 18为引物,RT-PCR扩增肥R3单抗cDNA。 [022 total RNA as a template primer team, Oligo (dT) 18 primer, RT-PCR amplification fertilizer R3 mAb cDNA. 反应体系和步骤如图3所示。 And the step of the reaction system as shown in FIG.

[0229] (Η)肥R3鼠源单抗可变区基因的克隆 Cloning [0229] (Η) fertilizer R3 murine monoclonal antibody variable region genes

[0230]A、兼并引物的合成 Synthesis [0230] A, the degenerate primers

[0231] 根据抗体信号肤及骨架区基因的保守性,设计并合成W下兼并引物(W下引物中W=A/T,K=G/T,R=A/G,Y=C/T,Μ=A/C,S=C/G,Ν=C/G/T,V=A/C/G): [0231] primers based on the conserved, design and synthesis of W at degenerate primers antibody signal peptide and the framework region gene under (W in W = A / T, K = G / T, R = A / G, Y = C / T , Μ = A / C, S = C / G, Ν = C / G / T, V = A / C / G):

[0232] (1)轻链上游可变区兼并引物:根据信号肤序列设计(5' -3') [0232] (1) a light chain variable region upstream degenerate primer: The signal peptide sequence was designed (5'-3 ')

[0233] [0233]

Figure CN105367657AD00211

[0234] 根据FR1保守序列设计巧'-3') [0234] The conserved sequences FR1 clever design '3')

[023引MKac-FwdGAYATTGTGMTSACMCARWCTMCA(SEQIDNO. :78) [023 cited MKac-FwdGAYATTGTGMTSACMCARWCTMCA (SEQIDNO:. 78)

[023引似轻链下游兼并引物巧' -3') [023 like the light chain the downstream primers degenerate primers clever '3')

[0237]MKac-RevGGATACAGTTGGTGCAGCATC(SEQIDNO. :63) [0237] MKac-RevGGATACAGTTGGTGCAGCATC (SEQIDNO:. 63)

[023引(3)重链上游兼并引物;根据信号肤序列设计(5' -3') [023 Primer (3) upstream of the heavy chain degenerate primer; signal peptide sequence designed according to (5 '3')

[0239] [0239]

Figure CN105367657AD00212

[0240] (4)重链上游兼并引物;根据FR1保守序列设计巧'-3') [0240] (4) upstream of the heavy chain degenerate primer; designed according to conserved sequences FR1 clever '3')

[0241] [0241]

Figure CN105367657AD00213

Figure CN105367657AD00221

[0242] (5)重链下游兼并引物 [0242] (5) downstream of the heavy chain degenerate primers

[0243]Μ肥C-RevATAGACAGATGGGGGTGTCGTTTTGGC(沈QIDNO. :79) [0243] Μ fat C-RevATAGACAGATGGGGGTGTCGTTTTGGC (Shen QIDNO:. 79)

[0244] B、可变区基因的克隆 [0244] B, cloned variable region genes

[024引W上述兼并引物和已制备肥R3单抗cDNA为模板,PCR扩增肥R3鼠源单抗可变区基因。 [024 cited above degenerate primers W and R3 mAb have been prepared fertilizer murine cDNA Amplification fertilizer R3 monoclonal antibody variable region gene as a template, PCR.

[0246] (1) PCR体系和参数设置如下: [0246] (1) PCR systems and parameters are as follows:

[0247] [0247]

Figure CN105367657AD00222

[024引PCR参数设置;95°C,预变性,5min;30轮如下循环;95°C变性,0. 5min,65°C复性, 0. 5min,72°C延伸,0. 5min;72°C延伸,lOmin。 [024 PCR primer parameters; 95 ° C, denaturation, 5min; 30 cycles of wheel;. 95 ° C denaturation, 0 5min, 65 ° C annealing, 0. 5min, 72 ° C extension, 0 5min;. 72 extending ° C, lOmin.

[0249] (2)用1 %的琼脂糖凝胶电泳分析PCR扩增结果,并与DM分子量标记LD2000判断扩增片段的大小(如图4所示)。 [0249] (2) with a 1% agarose gel electrophoresis analysis of PCR amplification, and labeled with a molecular weight of DM LD2000 Analyzing the size of the amplified fragment (Figure 4). 结果显示;分别有5条兼并引物扩增出轻链可变区基因,有4条兼并引物扩增出重链可变区基因,其大小约为330bp左右,条带单一,与轻/重链可变区基因片段的理论大小基本一致。 The results are shown; respectively 5 degenerate primers to amplify a light chain variable region genes, there are four degenerate primers to amplify a heavy chain variable region gene, is about 330bp in size, a single band, and light / heavy chain theoretical substantially uniform size variable region gene fragments.

[0250] 采用博大泰克公司的胶回收试剂盒回收330bp处的单抗可变区PCR扩增片段,并连接于pMDlST克隆载体(购自Takara公司)上,转化入D册α大肠杆菌感受态细胞中,进行藍白斑筛选,将阳性克隆送Invitrogen公司测序验证。 [0250] The broad Tektronix gel extraction kit recovered at 330bp PCR amplified mAb variable region fragment, and ligated to pMDlST cloning vector (commercially available from Takara) and transformed into competent E. coli α D register cells , make the blue-white screening, positive clones will send Invitrogen Corporation sequencing.

[0巧1] 根据NCBII浊LAST化ttp://www.ncbi.nlm.nih.gov/)免疫球蛋白基因比对分析结果,筛选出功能性抗体可变区基因,设计抗体轻链和重链可变区下游引物: [Qiao 1 0] The LAST NCBII turbidity of ttp: //www.ncbi.nlm.nih.gov/) than the immunoglobulin gene on the analysis results, selected functional antibody variable region gene, an antibody light chain and heavy design chain variable region downstream primer:

[0252]采用5' RACE扩增出可变区5'端的序列。 [0252] The sequence of 5 'RACE to amplify the variable region 5' end. 最终获得肥R3单抗的功能性可变区基因。 Finally obtained fat R3 Mab functional variable region genes.

[0巧3] 得到的抗体基因序列及对应氨基酸序列如下。 [Qiao 3 0] gene sequence and the corresponding amino acid sequence of the antibody obtained as follows. 其中下划线部分表示CDR区。 Wherein the underlined portions represent CDR regions.

[0巧4] 927H可变区基因序列如(SEQIDNO. :1所示。 [Qiao 0 4] The variable region gene sequences 927H (SEQIDNO:. 1 FIG.

[0巧5] 927H可变区氨基酸序列为阳256] EVQLQQSGPELVKPGASVKISCKASGYSFTSYYIHWVKQRPGQGLEWIGWIFPRSGHTNYNEKFKGKAT LTADTSSSTAYMQVS化TS邸SAVYFCARS畑YYGTNAMDYWGQGTSVTVSS(SEQ ID NO. :2)。 [Qiao 0 5] 927H variable region amino acid sequences of a male 256] EVQLQQSGPELVKPGASVKISCKASGYSFTSYYIHWVKQRPGQGLEWIGWIFPRSGHTNYNEKFKGKAT LTADTSSSTAYMQVS the TS Di SAVYFCARS Hata YYGTNAMDYWGQGTSVTVSS (SEQ ID NO:. 2).

[0巧7] 92化可变区基因序列如沈QIDNO. :3所示。 [Qiao 0 7] variable region gene sequences, such as sink 92 QIDNO:. 3 shown in FIG.

[0巧引92化可变区氨基酸序列为阳259] DIVMTQSPS化TVTAGEKVTMSCKSSQSLFNSGNQKNYLTWYQ服PGQPP化LIYWASTRESGVPDRFT GSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR(沈QIDNO. :4)。 [Qiao primer 92 0 variable region amino acid sequences of a male 259] DIVMTQSPS of TVTAGEKVTMSCKSSQSLFNSGNQKNYLTWYQ service PGQPP of LIYWASTRESGVPDRFT GSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR (Shen QIDNO:. 4).

[0260] 927抗体的的重链可变区中,各FR和CDR如下: [0260] 927 antibody heavy chain variable region, each of the FR and CDR follows:

[0261] [0261]

Figure CN105367657AD00231

[026引927抗体轻链可变区中,各FR和CDR如下: [026 Primer 927 antibody light chain variable region, each of the FR and CDR follows:

[0263] [0263]

Figure CN105367657AD00232

[0264] [0264]

[026引1044H可变区基因序列如沈QIDNO. :5所示。 [026 primer 1044H variable region gene sequence such as Shen QIDNO:. 5 FIG.

[0266] 1044H可变区氨基酸序列为 [0266] 1044H variable region amino acid sequence

[0267]EVQLQQSGTELMKPGASVKISCKATGGTFSNYWIDWVKQRPG服LEWIGEILPGSGGTDY肥KFKGKAT FTADTSSNTAYMQLS化TS邸SAVYYCAR迎迎YEMWGQGTLVTVSS(SEQIDNO. : 6)。 [0267] EVQLQQSGTELMKPGASVKISCKATGGTFSNYWIDWVKQRPG service LEWIGEILPGSGGTDY fertilizer KFKGKAT FTADTSSNTAYMQLS the TS Di SAVYYCAR Yingying YEMWGQGTLVTVSS (SEQIDNO:. 6).

[026引104化可变区基因序列如沈QIDNO. : 7所示。 [026 primer variable region gene sequences, such as sink 104 QIDNO:. 7 shown in FIG.

[0269] 104化可变区氨基酸序列: [0269] 104 humanized variable region amino acid sequences:

[0270] DIVMTQAAFSNPVTLGTSASISCRSSK化L服NGITYLYWYLQKPGQSPQ化IYQMSNLASGVPDRFSS SGSGTDFTLRISRVEAEDVGVYYCAQ边玉LEI1FGGGTKLEIKR(沈Q ID NO. : 8)。 [0270] DIVMTQAAFSNPVTLGTSASISCRSSK of L service NGITYLYWYLQKPGQSPQ IYQMSNLASGVPDRFSS SGSGTDFTLRISRVEAEDVGVYYCAQ side of jade LEI1FGGGTKLEIKR (Shen Q ID NO:. 8).

[027。 [027. 1044抗体的的重链可变区中,各FR和CDR如下: 1044 heavy chain variable region of an antibody, each FR and CDR follows:

[0272] [0272]

Figure CN105367657AD00241

[027引1044抗体轻链可变区中,各FR和CDR如下: [1044 primer 027 antibody light chain variable region, each of the FR and CDR follows:

[0274] [0274]

Figure CN105367657AD00242

[027引993H可变区基因序列如沈Q ID NO. :9所示。 [027 primer 993H variable region gene sequences, such as Shen Q ID NO:. 9 shown in FIG.

[0276] 993H可变区氨基酸序列: 阳277] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYSLSWVRQTP邸化EWVASITFGGTAYYSDSVKGRFTI S畑NARNILYLQMSSLK沈DTAMYYCVRGDGYEDPMDYWGQGTSVTVSS(沈Q ID NO. :10)。 [0276] 993H variable region amino acid sequence: male 277] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYSLSWVRQTP Di of EWVASITFGGTAYYSDSVKGRFTI S Hata NARNILYLQMSSLK Shen DTAMYYCVRGDGYEDPMDYWGQGTSVTVSS (Shen Q ID NO:. 10).

[027引99化可变区基因序列如沈Q ID NO. : 11所示。 [027 99 primers of the variable region gene sequences, such as Shen Q ID NO:. 11 FIG.

[0279] 99化可变区氨基酸序列: 阳280] DIVMTQTTVSLAV化GQRATISCRA沈SVDSYGKSFMHWYQQKPGQPP化LIYRASNLESGIPARFSGS GSRTDFTITINPVEA孤VSTYYCQQS肥DPYTFGGGTKLEIR(沈Q ID NO. :12)〇[02引]993抗体的的重链可变区中,各FR和CDR如下: [0279] 99 amino acid sequence of variable regions: male 280] DIVMTQTTVSLAV of GQRATISCRA sink SVDSYGKSFMHWYQQKPGQPP of LIYRASNLESGIPARFSGS GSRTDFTITINPVEA isolated VSTYYCQQS fertilizer DPYTFGGGTKLEIR (Shen Q ID NO:. 12) square [02 primer] 993 antibody heavy chain variable region each FR and CDR are as follows:

[0282] [0282]

Figure CN105367657AD00251

[0283] 993抗体轻链可变区中,各FR和CDR如下: [0283] 993 antibody light chain variable region, each of the FR and CDR follows:

[02841 [02841

[028引1050H可变区基因序列如沈QIDNO. [028 primer 1050H variable region gene sequences, such as heavy QIDNO.

Figure CN105367657AD00252

:13所示。 : 13.

[0286] 1050H可变区氨基酸序列: 阳287] QVQLQQSGPELVKPGASVKISCKASGYSFTSYYIHWVKQRPGQ化EWIGWIFPGSGHTKC肥NFKAKAT LTADTSSSTAYMQLS化TS邸SAVYFCARS畑YYGSNAVDYWGQGTSVTVSS(沈QIDNO. :14)〇[028引105化可变区基因序列如沈QIDNO. : 15所示。 [0286] The amino acid sequence of the variable region 1050H: male 287] QVQLQQSGPELVKPGASVKISCKASGYSFTSYYIHWVKQRPGQ EWIGWIFPGSGHTKC fertilizer NFKAKAT LTADTSSSTAYMQLS of the TS Di SAVYFCARS Hata YYGSNAVDYWGQGTSVTVSS (Shen QIDNO:. 14) square [028 lead 105 of the variable region gene sequences, such as Shen QIDNO:. 15 Suo shows.

[0289] 105化可变区氨基酸序列: 阳290] DIVMTQSPS化TVTAGEKVTMSCKSSQS化NSGNQKNYLTWYQ服PGQPP化LIYWASTRESGVPDRFS GSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR(沈QIDNO. :16) [0289] 105 amino acid sequence of variable regions: male 290] DIVMTQSPS of TVTAGEKVTMSCKSSQS of NSGNQKNYLTWYQ service PGQPP of LIYWASTRESGVPDRFS GSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR (Shen QIDNO:. 16)

[02W] 1050抗体的的重链可变区中,各FR和CDR如下: [02W] 1050 heavy chain variable region of an antibody, each FR and CDR follows:

[0292] [0292]

Figure CN105367657AD00253

Figure CN105367657AD00261

阳29引连施例10、肥R3人-鼠嵌合单克降杭体直核表汰裁体的构律 10 male 29 even applied primer, fat people embodiment R3 - mouse chimeric monoclonal configuration law Hang drop of straight cutting jig body checklist

[0296] 利用重叠PCR技术将轻链\基因与人Ig的基因进行拼接,构成轻链嵌合基因;轻链5'端引入BamHI限制性内切酶位点,轻链3'端引入EcoRI限制性内切酶位点。 [0296] Overlapping PCR technique using the human Ig light chain genes and gene \ splicing, a chimeric gene composed of the light chain; light chain 5 'end within the BamHI endonuclease restriction site, the light chain 3' end EcoRI restriction of the restriction enzyme sites. 同理,构建重链嵌合基因。 Similarly, the heavy chain chimeric gene construct. 分别将上述轻链/重链基因插入PCDNA3. 1(+/-)表达载体(购自Invitrogen公司)的单克隆酶切位点,构建肥R3人-鼠嵌合抗体的表达载体。 Respectively, above the light / heavy chain gene expression vector (Invitrogen Corp.) restriction site was inserted monoclonal PCDNA3 1 (+/-), Construction of human fat R3 - mouse chimeric antibody expression vector.

[0297] (1)按常规方法设计上下游物,利用PCR技术分别于重链/轻链可变区基因5'端引入BamHI单酶切位点,在重链/轻链恒定区基因3'端引入EcoRI单酶切位点。 [0297] (1) designed in a conventional manner on the downstream thereof, respectively, using PCR heavy chain / light chain variable region gene 5 'end single BamHI restriction site, the gene in the heavy / light chain constant region 3' end of the single EcoRI restriction site is introduced. BamHI和EcoRI双酶切重链/轻链嵌合基因,切胶回收目的片段。 BamHI and EcoRI digested heavy chain / light chain chimeric gene, Gel Extraction fragment.

[0298] (2) PCDNA3. 1(+/-)真核表达载体的处理;BamHI和EcoRI双酶切PCDNA3. 1(+/-) 载体,切胶回收目的片段(~5400bp)。 (+/-) processing cores adenovirus vector [0298] (2) PCDNA3 1;.. BamHI and EcoRI double digestion PCDNA3 1 (+/-) vector, Gel Extraction fragment (~ 5400bp).

[029引做将(1)中抗体重链/轻链基因分别克隆到似中PCDNA3. 1 (+/-)载体的BamHI 和EcoRI位点。 [029 do lead to (1) antibody heavy-chain / light-chain genes were cloned in the similar PCDNA3. 1 BamHI and EcoRI sites (+/-) vector.

[0300] (4)用上述连接产物转化畑5α感受态细胞,小量抽提重组质粒DNA。 [0300] (4) Conversion Hata 5α competent cells with the above ligation product, mini preps recombinant plasmid DNA. 挑选插入目的片段的阳性克隆送上海Invitrogen公司测序鉴定。 Choose positive insert fragment of clone sent Shanghai Invitrogen Corporation sequencing.

[0301] 经酶切及测序鉴定,验证本发明构建的肥R3单抗重链/轻链的重组表达载体其序列正确。 [0301] by restriction analysis and sequencing verified the recombinant expression construct of the present invention fat R3 mAb heavy chain / light chain vector correct sequence.

[0302] 本实施例中的肥R3人-鼠嵌合单克隆抗体的核酸序列和氨基酸序列如下。 [0302] R3 human fat present in the embodiment - a nucleic acid sequence and amino acid sequences of murine chimeric monoclonal antibodies as follows.

[0303] 人-鼠嵌合序列927重链氨基酸序列为: [0303] human - mouse chimeric heavy chain sequence of 927 amino acid sequence:

Figure CN105367657AD00271

[030引人-鼠嵌合序列927重链核酸酸序列如SEQIDNO. : 50所示。 [030 introduced - mouse chimeric nucleic acid sequences of the heavy chain sequence 927 as SEQIDNO: 50 shown in FIG.

[0306] 人-鼠嵌合序列993重链氨基酸序列为: [0306] human - mouse chimeric heavy chain sequence of 993 amino acid sequence:

Figure CN105367657AD00272

[030引人-鼠嵌合序列993重链核酸序列如SEQIDNO. : 51所示。 [030 introduced - mouse chimeric 993 heavy chain sequence of a nucleic acid sequence as SEQIDNO: 51 shown in FIG.

[0309] 人-鼠嵌合序列1044重链氨基酸序列: [0309] human - mouse chimeric heavy chain amino acid sequence of sequence 1044:

Figure CN105367657AD00273

[031。 [031. 人-鼠嵌合序列1044重链核酸序列如SEQIDNO. : 52所示。 Human - mouse chimeric heavy chain sequence of a nucleic acid sequence as 1044 SEQIDNO: 52 shown in FIG.

[0312] 人-鼠嵌合序列1050重链氨基酸序列: [0312] human - mouse chimeric heavy chain amino acid sequence of sequence 1050:

Figure CN105367657AD00274

[0314] 人-鼠嵌合序列1050重链核酸序列如SEQIDNO. : 53所示。 [0314] human - mouse chimeric heavy chain sequence of a nucleic acid sequence as 1050 SEQIDNO: 53 shown in FIG.

[0315] 人-鼠嵌合序列927轻链氨基酸序列: [0315] human - mouse chimeric light chain sequence of 927 amino acid sequence:

Figure CN105367657AD00281

[0317] 人-鼠嵌合序列927轻链核酸序列如SEQIDNO. : 54所示。 [0317] human - mouse chimeric light chain sequence of the nucleic acid sequence such as 927 SEQIDNO: 54 shown in FIG.

[031引人-鼠嵌合序列993轻链氨基酸序列: [031 introduced - mouse chimeric light chain sequence of 993 amino acid sequence:

Figure CN105367657AD00282

[0320] 人-鼠嵌合序列993轻链核酸序列如SEQ ID NO. : 55所示。 [0320] human - mouse chimeric light chain sequence of 993 nucleic acid sequence as SEQ ID NO: 55 shown in FIG.

[0321] 人-鼠嵌合序列1044轻链氨基酸序列: [0321] human - mouse chimeric light chain sequence of 1044 amino acid sequence:

Figure CN105367657AD00283

[0323] 人-鼠嵌合序列1044轻链核酸序列如SEQIDNO. : 56所示。 . [0323] human - mouse chimeric light chain sequence of the nucleic acid sequence as 1044 SEQIDNO: 56 shown in FIG.

[0324] 人-鼠嵌合序列1050轻链氨基酸序列: [0324] human - mouse chimeric light chain sequence of 1050 amino acid sequence:

Figure CN105367657AD00284

[0326] 人-鼠嵌合序列1050轻链核酸序列如SEQIDNO. : 57所示。 [0326] human - mouse chimeric light chain sequence of the nucleic acid sequence as 1050 SEQIDNO: 57 shown in FIG. 防327] 连施例11、肥R3人-鼠嵌合杭体布CH0细胸中的表汰及鉴定 Anti-327] Example 11 is connected, fat R3 human - mouse chimeric Hang A cloth chest table eliminating fine CH0 and Identification

[032引采用脂质体法将本发明的肥R3人-鼠嵌合抗体重链/轻链表达载体共转染邸0/ 化化细胞(购自ATCC),W未转染质粒的空细胞对照。 [032 cited liposome method of the present invention R3 fat - mouse chimeric antibody heavy chain / light chain expression vectors were co-transfected Di 0 / of cells (purchased from ATCC), W is not null cells transfected plasmid control. 培养7化后,采用常规化ISA法,用HRP标记的羊抗人IgG抗体检测肥R3人-鼠嵌合抗体的表达及嵌合抗体对肥R3抗原的特异性识别。 After incubation of 7, of the conventional ISA method using HRP-labeled goat anti-human IgG antibody fat R3 human - mouse chimeric antibody and expression of chimeric antibodies specifically recognizing the antigen R3 fat.

[0329] 鉴定结果如下表所示: [0329] The results identified in the following table:

[0330] [0330]

Figure CN105367657AD00291

[033。 [033. ELISA法检测了培养7化后细胞培养上清的0D450值,上表中。 ELISA assay of cell culture supernatant after culture 0D450 value of 7, the above table. + "表示阳性、 表示阴性。 + "Indicates positive, negative representation.

[0332] 结果显示,共转染表达质粒载体的C册细胞成功表达嵌合抗体,能有效识别肥R3 抗原,而未转染表达质粒的C册细胞培养上清不能识别肥R3抗原。 [0332] The results show that co-transfection of plasmid vectors expressing cells C volumes successful expression of chimeric antibodies, can effectively identify fat R3 antigen, but not cells transfected with a plasmid expressing the C register does not recognize the culture supernatant fertilizer R3 antigen. 即瞬时转染C册成功表达了能特异性识别肥R3抗原的人-鼠嵌合抗体。 That is transiently transfected C registered successful expression of the antigen can specifically recognize R3 fat people - mouse chimeric antibody.

[0333] 在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考郝样。 [0333] The present application are incorporated in all documents mentioned herein incorporated by reference, as if each reference were individually incorporated by reference Hao like. 此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可W对本发明作各种改动或修改,送些等价形式同样落于本申请所附权利要求书所限定的范围。 Furthermore, it should be understood that, after reading the above teachings of the present invention, those skilled in the art of the present invention may be W that various changes or modifications, to send more equivalents of the present application to the same extent fall within the appended claims as defined.

Claims (18)

  1. 1. 一种抗体的重链可变区,其特征在于,所述的重链可变区包括以下三个互补决定区CDR: (1) 互补决定区⑶R1 所述互补决定区CDR1 选自:SEQIDNO. :17、SEQIDNO. :23、SEQIDNO. :29 和SEQID NO. : 35 ; (2) 互补决定区⑶R2 所述互补决定区CDR2 选自:SEQIDNO. :18、SEQIDNO. :24、SEQIDNO. :30 和SEQID NO. : 36 ; (3) 互补决定区⑶R3 所述互补决定区CDR3 选自:SEQIDNO. :19、SEQIDNO. :25、SEQIDNO. :31 和SEQID NO. :37。 A heavy chain variable region of an antibody, wherein the heavy chain variable region comprising three complementarity determining regions CDR: (1) the ⑶R1 complementary determining regions CDR1 complementarity determining region is selected from: SEQIDNO .: 17, SEQIDNO: 23, SEQIDNO: 29 and SEQID NO: 35; (2) the complementarity determining regions ⑶R2 complementarity determining region CDR2 selected from:... SEQIDNO: 18, SEQIDNO: 24, SEQIDNO: 30... and SEQID NO:. 36; (3) the complementary determining regions ⑶R3 CDR3 complementarity determining region is selected from: SEQIDNO: 19, SEQIDNO: 25, SEQIDNO: 31 and SEQID NO: 37.....
  2. 2. 如权利要求1所述的抗体的重链可变区,其中,所述重链可变区的三个互补决定区CDR1、CDR2、CDR3 的氨基酸序列分别如SEQIDNO. : 17、SEQIDNO. : 18 和SEQIDNO. : 19 所示;或者所述重链可变区的三个互补决定区⑶R1、⑶R2、⑶R3的氨基酸序列分别如SEQID NO. : 23、SEQIDNO. : 24 和SEQIDNO. : 25 所示;或者所述重链可变区的三个互补决定区⑶R1、⑶R2、⑶R3的氨基酸序列分别如SEQID NO. : 29、SEQIDNO. : 30 和SEQIDNO. : 31 所示;或者所述重链可变区的三个互补决定区⑶R1、⑶R2、⑶R3的氨基酸序列分别如SEQID NO. : 35、SEQIDNO. : 36 和SEQIDNO. : 37 所示; 优选地,所述重链可变区具有SEQIDNO. :2、6、10或14所示的氨基酸序列。 2. A heavy chain variable region of the antibody of claim 1, wherein three of said heavy chain variable region complementarity determining regions CDR1, the amino acid sequence of CDR2, CDR3 are as SEQIDNO:. 17, SEQIDNO.: 18 and SEQIDNO: 19 below; or a heavy chain variable region of three complementarity determining regions ⑶R1, the amino acid sequence ⑶R2, ⑶R3 respectively as SEQID NO:.... 23, SEQIDNO: 24, and SEQIDNO: 25 shown in FIG. ; or the three complementarity determining regions of the heavy chain variable region ⑶R1, the amino acid sequence ⑶R2, ⑶R3 respectively as SEQID NO: 29, SEQIDNO: 30, and SEQIDNO:... 31 below; or a heavy chain variable three complementarity determining region amino acid sequences ⑶R1, ⑶R2, ⑶R3 respectively as SEQID NO: 35, SEQIDNO: 36, and SEQIDNO: 37 as shown; preferably, the heavy chain variable region having SEQIDNO:... 2. , the amino acid sequence shown in 6,10 14.
  3. 3. -种抗体重链,其特征在于,所述抗体重链具有如权利要求1所述的抗体的重链可变区;优选地,所述抗体重链具有SEQIDNO. :42、43、44或45所示的氨基酸序列。 3 - heavy chain antibodies, wherein said antibody heavy chain having a heavy chain variable region of an antibody according to claim 1; Preferably, the antibody heavy chain has SEQIDNO: 42,43,44. 45 or the amino acid sequence.
  4. 4. 一种抗体的轻链可变区,其特征在于,所述的轻链可变区包括以下三个互补决定区CDR: (1) 互补决定区⑶R1' 所述互补决定区CDR1' 选自:SEQIDNO. :20、SEQIDNO. :26、SEQIDNO. :32 和SEQ IDNO. : 38 ; (2) 互补决定区⑶R2' 所述互补决定区CDR2' 选自:SEQIDNO. :21、SEQIDNO. : 27、SEQIDNO. :33 和SEQ IDNO. : 39 ; (3) 互补决定区⑶R3' 所述互补决定区CDR3' 选自:SEQIDNO. : 22、SEQIDNO. : 28、SEQIDNO. :34 和SEQ IDNO. :40。 A light chain variable region of an antibody, wherein the light chain variable region comprising three complementarity determining regions CDR: (1) complementarity determining region ⑶R1 'the complementarity determining regions CDR1' is selected from : SEQIDNO: 20, SEQIDNO: 26, SEQIDNO: 32 and SEQ IDNO: 38; (2) complementarity determining regions ⑶R2 'the complementarity determining region CDR2' is selected from:.... SEQIDNO: 21, SEQIDNO: 27,.. . SEQIDNO: 33 and SEQ IDNO:. 39; (3) complementarity determining regions ⑶R3 'the complementary determining regions CDR3' is selected from: SEQIDNO: 22, SEQIDNO: 28, SEQIDNO: 34 and SEQ IDNO: 40.....
  5. 5. 如权利要求4所述的抗体的轻链可变区,其中,所述轻链可变区的三个互补决定区CDR1'、CDR2'、CDR3' 的氨基酸序列分别如SEQIDNO. :20、SEQIDN0. :21 和SEQIDNO. :22 所示;或者所述轻链可变区的三个互补决定区⑶R1'、⑶R2'、⑶R3'的氨基酸序列分别如SEQID NO. : 26、SEQIDNO. : 27 和SEQIDNO. : 28 所示;或者所述轻链可变区的三个互补决定区⑶Rl'、⑶R2'、⑶R3'的氨基酸序列分别如SEQID NO. : 32、SEQIDNO. : 33 和SEQIDNO. : 34 所示;或者所述轻链可变区的三个互补决定区⑶R1'、⑶R2'、⑶R3'的氨基酸序列分别如SEQID NO. : 38、SEQIDNO. : 39 和SEQIDNO. : 40 所示; 优选地,所述轻链可变区具有SEQIDNO. :4、8、12或16所示的氨基酸序列。 5. The amino acid sequence of the light chain variable region of the antibody of claim 4, wherein the three complementarity determining regions of the light chain variable region CDR1 ', CDR2', CDR3 'are as SEQIDNO:. 20, . SEQIDN0: 21, and SEQIDNO: 22 shown; three complementarity determining regions, or the light chain variable region ⑶R1 ', ⑶R2', ⑶R3 'amino acid sequences are set forth in SEQID NO:. 26, SEQIDNO: 27 and. SEQIDNO: 28 shown; three complementarity determining regions, or the light chain variable region ⑶Rl ', ⑶R2', ⑶R3 'amino acid sequences are set forth in SEQID NO:.... 32, SEQIDNO: 33, and SEQIDNO: 34 Suo shown; three complementarity determining regions, or the light chain variable region ⑶R1 ', ⑶R2', ⑶R3 'amino acid sequences are set forth in SEQID NO: 38, SEQIDNO: 39, and SEQIDNO: 40 as shown; preferably,... the light chain variable region having SEQIDNO:. 8, 12, or the amino acid sequence shown in 16.
  6. 6. -种抗体的轻链,其特征在于,所述的轻链具有如权利要求3所述的抗体的轻链可变区;优选地,所述抗体轻链具有SEQIDNO. :46、47、48或49所示的氨基酸序列。 6. - antibodies light chain, wherein said light chain having a light chain variable region of an antibody according to claim 3; Preferably, the antibody light chain has SEQIDNO: 46,47,. amino acid sequence 48 or 49.
  7. 7. -种特异性结合HER3的单克隆抗体,其特征在于,所述单克隆抗体具有以下特性: (1) 与HER3蛋白亲和力常数Μ彡1X101。 7. - species specific monoclonal antibodies bind to HER3, wherein said monoclonal antibody has the following characteristics: (1) HER3 protein with an affinity constant Μ San 1X101. ; (2) 结合HER3胞外区的DI或Dili结构域。 ; (2) binds the extracellular domain of HER3 or Dili DI domain.
  8. 8. 如权利要求7所述的单克隆抗体,其中,所述单克隆抗体具有: (1) 如权利要求1所述的重链可变区,和/或(2) 如权利要求3所述的轻链可变区;或者所述单克隆抗体具有: (1) 如权利要求2所述的重链,和/或(2) 如权利要求4所述的轻链。 8. The monoclonal antibody according to claim 7, wherein said monoclonal antibody has: (1) a heavy chain variable region as claimed in claim 1, and / or (2) as claimed in claim 3 light chain variable region; or the monoclonal antibody having: (1) a heavy chain as claimed in claim 2, and / or (2) a light chain as claimed in claim 4.
  9. 9. 一种重组蛋白,其特征在于,所述的重组蛋白具有: (i) 如权利要求1所述的抗体的重链可变区、如权利要求2所述的抗体重链、如权利要求3所述的抗体的轻链可变区、如权利要求4所述的抗体轻链、或如权利要求5所述的单克隆抗体;以及(ii) 任选的协助表达和/或纯化的标签序列。 A recombinant protein, wherein said recombinant protein has: (i) a heavy chain variable region of the antibody as claimed in claim 1, an antibody heavy chain according to claim 2, as claimed in claim (ii) optionally assist the expression and / or purification tag and; the 3 light chain variable region of an antibody, such as an antibody light chain according to claim 4, or a monoclonal antibody as claimed in claim 5, wherein sequence. 在另一优选例中,所述的标签序列包括6His标签。 In another preferred embodiment, the tag sequence comprises a 6His tag. 在另一优选例中,所述的重组蛋白特异性抗HER3。 In another preferred embodiment, the recombinant protein specific anti-HER3. 在另一优选例中,所述的重组蛋白选自下组: (a) 具有SEQIDNO. :42-49所示的氨基酸序列的多肽; (b) 将(a)中的氨基酸序列经过一个或多个(如1-20个)氨基酸残基的取代、缺失或添加而形成的、由(a)衍生的且特异性抗HER3的多肽。 In another preferred embodiment, the recombinant protein is selected from the group consisting of: (a) having SEQIDNO: polypeptide amino acid sequence shown 42-49; (b) amino acid sequence (a) is subjected to one or more. a substituted (e.g. 1-20) amino acid residues, the deletion or addition, of (a) and specific anti-HER3-derived polypeptide.
  10. 10. -种免疫偶联物,其特征在于,该免疫偶联物含有: (a) 载体部分,所述载体部分含有如权利要求1所述的抗体的重链可变区、如权利要求3所述的抗体重链、如权利要求4所述的抗体的轻链可变区、如权利要求6所述的抗体轻链、 或如权利要求7所述的单克隆抗体或权利要求9所述的重组蛋白;和(b) 选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。 10. - Species immunoconjugate, wherein the immunoconjugate comprises: (a) a carrier portion, the carrier portion of the heavy chain variable region comprising the antibody according to claim 1, as claimed in claim 3 the antibody heavy chain, a light chain variable region 4 as the antibody of claim antibody light chain according to claim 6, or a monoclonal antibody as claimed in claim 7 or as claimed in claim in claim 9 recombinant protein; and (b) coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  11. 11. 一种多核苷酸,其特征在于,它编码选自下组的蛋白质: 如权利要求1所述的抗体的重链可变区、如权利要求3所述的抗体重链、如权利要求4 所述的抗体的轻链可变区、如权利要求6所述的抗体轻链、或如权利要求7所述的单克隆抗体或权利要求9所述的重组蛋白。 11. A polynucleotide characterized in that it codes for a protein selected from the group consisting of: a heavy chain variable region as recited in claim 1 antibody as claimed in claim antibody heavy chain according to claim 3, 4 of the light chain variable region of an antibody, such as an antibody light chain according to claim 6, or a monoclonal antibody as claimed in claim 7 or claim recombinant protein according to claim 9.
  12. 12. -种载体,其特征在于,它含有权利要求11所述的多核苷酸。 12. - kind of support, characterized in that it comprises the polynucleotide of claim 11.
  13. 13. -种遗传工程化的宿主细胞,其特征在于,它含有权利要求12所述的载体或基因组中整合有权利要求11所述的多核苷酸。 13. - genetic engineering of host cells, characterized in that it contains a vector as claimed in claim 12 or of the genome of incorporating a polynucleotide according to claim 11.
  14. 14. 一种制备重组多肽的制备方法,其特征在于,该方法包含: (a)在适合表达的条件下,培养权利要求13所述的宿主细胞; (b) 从培养物中分离出重组多肽,所述的重组多肽是权利要求7所述的单克隆抗体或权利要求9所述的重组蛋白。 14. A recombinant polypeptide prepared as described, wherein the method comprises: (a) under conditions suitable for expression, culturing the host cell according to claim 13; (b) separating the recombinant polypeptide from the culture said recombinant polypeptide is a monoclonal antibody according to claim 7 or claim recombinant protein according to claim 9.
  15. 15. -种药物组合物,其特在于,所述组合物中含有: (i) 如权利要求1所述的抗体的重链可变区、如权利要求3所述的抗体重链、如权利要求4所述的抗体的轻链可变区、如权利要求6所述的抗体轻链、或如权利要求7所述的单克隆抗体或权利要求9所述的重组蛋白;以及(ii) 药学上可接受的载体。 15. - pharmaceutical compositions, wherein Laid thereof, the composition comprising: (i) a heavy chain variable region of the antibody as claimed in claim 1, an antibody heavy chain according to claim 3, claim in claim 4, wherein the light chain variable region of an antibody, an antibody light chain according to claim 6, or a monoclonal antibody according to claim 7 or as claimed in claim in claim 9 said recombinant protein; and (ii) a pharmaceutically acceptable acceptable carrier.
  16. 16.如权利要求16所述的药物组合物,其特在于,所述组合物还包含西妥昔单抗。 16. A pharmaceutical composition according to claim 16, which Laid wherein said composition further comprises cetuximab.
  17. 17.如权利要求1所述的抗体的重链可变区、如权利要求3所述的抗体重链、如权利要求4所述的抗体的轻链可变区、如权利要求6所述的抗体轻链、或如权利要求7所述的单克隆抗体或权利要求9所述的重组蛋白,用于: (a)细胞的分离、制备、提取、检测;或(b) 制备用于分离、制备、提取、检测细胞的产品。 17. The light chain variable region of the heavy chain variable region of an antibody as claimed in claim 1, an antibody heavy chain according to claim 3, said antibody as claimed in claim 4, as claimed in claim 6 antibody light chains, as claimed in claim 7, or a monoclonal antibody or recombinant protein according to claim 9, for: (a) separation of cells, preparation, extraction, detection; or (b) preparing for separating, preparation, extraction, detection of cell products.
  18. 18. -种检测样品中是否含有HER3蛋白的方法,其特征在于,所述方法包括步骤: (1) 将样品与权利要求5所述的单克隆抗体接触; (2)检测是否形成抗原-抗体复合物,其中形成复合物就表示样品中存在HER3蛋白。 18. - The method of detecting whether the sample contains species HER3 protein, characterized in that, said method comprising the steps of: (a) contacting the monoclonal antibody with the sample 5 according to claim 1; (2) detecting formation of antigen - antibody complex, wherein the complex is formed on the sample indicates the presence of HER3 protein.
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