JPWO2004060919A1 - Agonist antibodies against heteroreceptors - Google Patents

Abstract

After immunizing the animal with each of the A chain and B chain of the receptor, mRNA was extracted from the spleen cells of the animal, and RT-PCR was performed to determine the L chain and H chain using primers corresponding to the variable regions containing CDRs. The variable region was recovered. Single-chain Fv was synthesized by assembly PCR and a phage library was constructed. The antigen-binding antibody clone was concentrated and cloned by panning, and the single chain variable region was inserted between the signal sequence for animal cells and CH1-hinge-CH2-CH3 to prepare an scFv-CH1-Fc expression vector. Antibody clones were introduced into cells in various combinations to express antibodies, and antibody clones showing activity similar to ligands were selected.

Description

  The present invention relates to an agonist antibody against a heteroreceptor and a pharmaceutical composition containing the antibody as an active ingredient.

Antibodies are attracting attention as pharmaceuticals because of their high stability in blood and lack of antigenicity. Among them, a bispecific antibody that can simultaneously recognize two types of antigens has been proposed, but at present, the two types of antigens are merely linked. However, since an antibody binds to a specific epitope in an antigen, it is considered that two antigens can be coordinated at a desired distance and angle by a bispecific antibody if an appropriate combination of antibodies is selected.
Many cytokine receptors are thought to be able to transmit signals into cells by changing the distance and angle between chains forming a dimer by binding to a ligand. That is, a suitable anti-receptor antibody can mimic receptor dimerization by a ligand and can be an agonist antibody. MPL already composed of homodimers (see US Patent Application Publication No. 98/17364 and literature (“Blood”, 1998, Vol. 92, No. 6, p. 1981-1988)), EPO reception Monoclonal antibodies exhibiting agonistic effects on human and GH receptors have been reported.
However, in the case of a receptor that forms a heterodimer, it is necessary to form a complex of two types of receptor chains, and therefore an agonistic action cannot be expected with a general antibody. For this purpose, the above-mentioned bispecific antibody capable of recognizing two kinds of antigens with two arms is considered suitable, but no report has been made yet.

抗ＡＲ１鎖抗体がＡＲ１−２４の場合、パートナーとなる抗ＡＲ２鎖抗体は、ＡＲ２−１３、ＡＲ２−３１、あるいはＡＲ２−４４であることが好ましく、抗ＡＲ１鎖抗体がＡＲ１−４１の場合、パートナーとなる抗ＡＲ２鎖抗体は、ＡＲ２−１１、ＡＲ２−１３、ＡＲ２−１４、ＡＲ２−２２、ＡＲ２−３３、ＡＲ２−３７、ＡＲ２−４０、ＡＲ２−４３、ＡＲ２−４４、あるいはＡＲ２−４５であることが好ましい。ＡＲ２−１３およびＡＲ２−４４は、ＡＲ１−４１およびＡＲ１−２４の両方の抗体に対してパートナーとなることが可能である。上記のような対を形成する抗体もまた、本発明に含まれる。
また、本発明において、上記可変領域を含む抗体は、必ずしも可変領域の全長配列を有している必要はなく、ＣＤＲ配列が保存されていれば、ＦＲ配列は抗体をヒト化等する際に適宜変更してよい。
従って、本発明は、上記可変領域においてＣＤＲに対応するアミノ酸配列を有する抗体を含む。
上記可変領域の配列番号において、ＣＤＲに対応するアミノ酸番号を表２に示す。

また、本発明で開示されている可変領域を用いて全長抗体を作製する場合、定常領域は特に限定されず、当業者に公知の定常領域を用いることが可能であり、例えば、Ｓｅｑｕｅｎｃｅｓ ｏｆ ｐｒｏｔｅｉｎｓ ｏｆ ｉｍｍｕｎｏｌｏｇｉｃａｌ ｉｎｔｅｒｅｓｔ，（１９９１），Ｕ．Ｓ．Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｈｅａｌｔｈ ａｎｄ Ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ．Ｐｕｂｌｉｃ Ｈｅａｌｔｈ Ｓｅｒｖｉｃｅ Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈや、Ａｎ ｅｆｆｉｃｉｅｎｔ ｒｏｕｔｅ ｔｏ ｈｕｍａｎ ｂｉｓｐｅｃｉｆｉｃ ＩｇＧ，（１９９８）．Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ ｖｏｌ．１６，６７７−６８１、等に記載されている定常領域を用いることができる。
本発明の抗体はアゴニスト作用を有することから、該抗体が作用する受容体の活性（機能）低下に起因する疾病に対して、有効な薬剤となることが期待される。即ち本発明は、本発明の抗体を有効成分として含有する医薬組成物を提供する。例えば、本発明の抗体がサイトカイン受容体に対してアゴニスト活性を有する抗体である場合には、該抗体はサイトカイン様作用を有するものと考えられる。従って該抗体は抗ウイルス作用、抗腫瘍作用、細胞増殖・分化調節作用を有する医薬品（医薬組成物）となることが期待される。
治療または予防目的で使用される本発明の抗体を有効成分として含む医薬組成物は、必要に応じて、それらに対して不活性な適当な薬学的に許容される担体、媒体等と混和して製剤化することができる。例えば、滅菌水や生理食塩水、安定剤、賦形剤、酸化防止剤（アスコルビン酸等）、緩衝剤（リン酸、クエン酸、他の有機酸等）、防腐剤、界面活性剤（ＰＥＧ、Ｔｗｅｅｎ等）、キレート剤（ＥＤＴＡ等）、結合剤等を挙げることができる。また、その他の低分子量のポリペプチド、血清アルブミン、ゼラチンや免疫グロブリン等の蛋白質、グリシン、グルタミン、アスパラギン、アルギニン及びリシン等のアミノ酸、多糖及び単糖等の糖類や炭水化物、マンニトールやソルビトール等の糖アルコールを含んでいてもよい。注射用の水溶液とする場合には、例えば生理食塩水、ブドウ糖やその他の補助薬を含む等張液、例えば、Ｄ−ソルビトール、Ｄ−マンノース、Ｄ−マンニトール、塩化ナトリウムが挙げられ、適当な溶解補助剤、例えばアルコール（エタノール等）、ポリアルコール（プロピレングリコール、ＰＥＧ等）、非イオン性界面活性剤（ポリソルベート８０、ＨＣＯ−５０）等と併用してもよい。
また、必要に応じ本発明の抗体をマイクロカプセル（ヒドロキシメチルセルロース、ゼラチン、ポリ［メチルメタクリル酸］等のマイクロカプセル）に封入したり、コロイドドラッグデリバリーシステム（リポソーム、アルブミンミクロスフェア、マイクロエマルジョン、ナノ粒子及びナノカプセル等）とすることもできる（″Ｒｅｍｉｎｇｔｏｎ′ｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅ １６ｔｈ ｅｄｉｔｉｏｎ″，Ｏｓｌｏ Ｅｄ．（１９８０）等参照）。さらに、薬剤を徐放性の薬剤とする方法も公知であり、本発明の抗体に適用し得る（Ｌａｎｇｅｒ ｅｔ ａｌ．，Ｊ．Ｂｉｏｍｅｄ．Ｍａｔｅｒ．Ｒｅｓ．１５：１６７−２７７（１９８１）；Ｌａｎｇｅｒ，ｃｈｅｍ．Ｔｅｃｈ．１２：９８−１０５（１９８２）；米国特許第３，７７３，９１９号；欧州特許出願公開（ＥＰ）第５８，４８１号；Ｓｉｄｍａｎ ｅｔ ａｌ．，Ｂｉｏｐｏｌｙｍｅｒｓ ２２：５４７−５５６（１９８３）；ＥＰ第１３３，９８８号）。
本発明の医薬組成物の投与量は、剤型の種類、投与方法、患者の年齢や体重、患者の症状、進行の程度等を考慮して、最終的には医師の判断により適宜決定されるものであるが、一般に大人では、１日当たり、０．１〜２０００ｍｇを１〜数回に分けて経口投与することができる。より好ましくは１〜１０００ｍｇ／日、更により好ましくは５０〜５００ｍｇ／日、最も好ましくは１００〜３００ｍｇ／日である。これらの投与量は患者の体重や年齢、投与方法などにより変動するが、当業者であれば適当な投与量を適宜選択することが可能である。投与期間も、患者の治癒経過等に応じて適宜決定することが好ましい。
また、本発明の抗体をコードする遺伝子を遺伝子治療用ベクターに組込み、遺伝子治療を行うことも考えられる。投与方法としては、ｎａｋｅｄプラスミドによる直接投与の他、リポソーム等にパッケージングするか、レトロウイルスベクター、アデノウイルスベクター、ワクシニアウイルスベクター、ポックスウイルスベクター、アデノウイルス関連ベクター、ＨＶＪベクター等の各種ウイルスベクターとして形成するか（Ａｄｏｌｐｈ『ウイルスゲノム法』，ＣＲＣ Ｐｒｅｓｓ，Ｆｌｏｒｉｄ（１９９６）参照）、または、コロイド金粒子等のビーズ担体に被覆（ＷＯ９３／１７７０６等）して投与することができる。しかしながら、生体内において抗体が発現され、その作用を発揮できる限りいかなる方法により投与してもよい。好ましくは、適当な非経口経路（静脈内、腹腔内、皮下、皮内、脂肪組織内、乳腺組織内、吸入または筋肉内の経路を介して注射、注入、またはガス誘導性粒子衝撃法（電子銃等による）、添鼻薬等粘膜経路を介する方法等）により十分な量が投与される。ｅｘ ｖｉｖｏにおいてリポソームトランスフェクション、粒子衝撃法（米国特許第４，９４５，０５０号）、またはウイルス感染を利用して血液細胞及び骨髄由来細胞等に投与して、該細胞を動物に再導入することにより本発明の抗体をコードする遺伝子を投与してもよい。This invention is made | formed in view of such a condition, The objective is to provide the antibody which has an agonist effect with respect to the receptor containing a hetero chain.
The present inventors have intensively studied to solve the above problems. In screening for agonist antibodies, it is necessary to select a large number of antibodies (α, β) for each of the two types of chains (A, B) constituting the receptor, and test each one of the combinations of α, β. In order to produce bispecific antibodies, fusion between antibody-producing hybridomas or an antibody expression vector must be introduced into the cells. The present inventors have succeeded in producing a bispecific antibody having an agonistic action on a receptor consisting of a heterochain, for example, by the following method. More specifically, it was performed as follows.
Animals were immunized with the A and B chains of the receptor, respectively. MRNA is extracted from the spleen cells of this animal, and L- and H-chain variable regions are recovered by RT-PCR using primers corresponding to the variable regions including the complementarity determining region (CDR) of the antibody. did. Single-chain Fv (scFv) was synthesized by assembly PCR to construct a phage library. The antigen-binding antibody clone was concentrated and cloned by panning, and the single-chain variable region (scFv) was inserted between the signal sequence for animal cells and CH1-hinge-CH2-CH3, and the scFv-CH1-Fc expression vector was Produced. Various combinations were introduced into cells to express antibodies. This culture supernatant was added to cells that react with the target ligand, and antibody clones showing the same activity as the ligand were selected.
By the above method, an agonist antibody against type I interferon receptor consisting of two types of chains AR1 and AR2 was successfully separated. That is, the present inventors have succeeded in isolating a bispecific antibody having an agonistic action against a receptor consisting of a hetero chain for the first time, and completed the present invention.
The present invention relates to an antibody having an agonistic effect on a receptor containing a hetero chain, more specifically,
[1] an antibody having agonist activity against a receptor containing a heterochain,
[2] The antibody according to [1], wherein the receptor is a cytokine receptor,
[3] The antibody according to [2], wherein the cytokine receptor is an interferon receptor,
[4] The antibody according to [3], wherein the interferon receptor is a type I interferon receptor,
[5] The antibody according to [4], wherein the type I interferon receptor comprises an AR1 chain and an AR2 chain,
[6] The antibody according to [1], wherein the receptor is a multimer,
[7] The antibody according to [6], wherein the multimer is a dimer,
[8] The antibody according to any one of [1] to [7], which is a bispecific antibody,
[9] The antibody according to [5], comprising a variable region of an anti-AR1 chain antibody and a variable region of an anti-AR2 chain antibody,
[10] A variable region consisting of the following amino acid sequence (a) in an anti-AR1 chain antibody and a variable region consisting of the amino acid sequence described in any of (b1) to (b10) below in an anti-AR2 chain antibody: [9] the antibody according to
(A) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 1, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 2.
(B1) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 8.
(B2) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 9 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 10.
(B3) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 20.
(B4) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 13 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 14.
(B5) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 23, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 24
(B6) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 5 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 6.
(B7) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 17 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 18.
(B8) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 16.
(B9) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 21 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 22.
(B10) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 11 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 12.
[11] A variable region consisting of the following amino acid sequence (a) in an anti-AR1 chain antibody and a variable region consisting of the amino acid sequence described in any of (b1) to (b3) below in an anti-AR2 chain antibody: [9] the antibody according to
(A) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 3 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 4.
(B1) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 10.
(B2) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 25, and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 26
(B3) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 21 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 22.
[12] A pharmaceutical composition comprising the antibody according to any one of [1] to [11] as an active ingredient.
The present invention provides an antibody having agonist activity against a receptor containing a heterochain.
In the present invention, a receptor containing a hetero chain means that the receptor (multimer) is composed of two or more proteins (receptor chains) different from each other. Multimers are not limited by the number of proteins (receptor chains) such as dimers, trimers, and tetramers, but are preferably dimers. For example, when the receptor is a dimer, the heteroreceptor indicates that the two constituent proteins (receptor chains) are not identical.
An antibody having agonist activity refers to an antibody having an agonistic action against a certain receptor. In general, when a ligand (factor) that is an agonist binds to a receptor, the three-dimensional structure of the receptor protein changes, and the receptor is activated (when the receptor is a membrane protein, usually cell proliferation, etc.) Signal). In the case of a receptor that forms a dimer, an agonist antibody can act like a ligand by dimerizing the receptor at an appropriate distance and angle. That is, a suitable anti-receptor antibody can mimic receptor dimerization with a ligand and can be an agonist antibody.
Physiological activities in which changes are induced by agonistic action include, for example, proliferation activity, survival activity, differentiation activity, transcription activity, membrane transport activity, binding activity, proteolytic activity phosphorylation / dephosphorylation activity, redox activity, Examples include, but are not limited to, metastasis activity, nucleolytic activity, dehydration activity, cell death-inducing activity, and apoptosis-inducing activity.
In a preferred embodiment of the present invention, a cytokine receptor can be mentioned as the receptor of the present invention. Cytokines are generally used as a general term for physiologically active proteins that control the proliferation and differentiation of various blood cells, and may also refer to growth factors and growth inhibitory factors of cells including non-immune cells. Therefore, cytokines are a general term for protein factors that are released from cells and mediate cell-to-cell interactions such as immunity, control of inflammatory response, antiviral action, antitumor action, and regulation of cell proliferation / differentiation.
Specific examples of cytokines include interleukins 1 to 15, colony stimulating factor (G-CSF, M-CSF, GM-CSF, etc.), interferon (IFN-α, IFN-β, IFN-γ, etc.), Chemokines, tumor necrosis factor (TNF), lymphotoxin, erythropoietin, epidermal growth factor, fibroblast growth factor and the like can be mentioned. Interferon is preferred, and type I interferon is particularly preferred.
Specific examples of cytokine receptors include interferon receptor family (IFN-α receptor, IFN-β receptor, IFN-γ receptor, IL-10 receptor, etc.), interleukin receptor family (IL- 2 receptor, IL-3 receptor, IL-6 receptor, GM-CSF receptor, etc.), serine-threonine kinase type receptor family (BMP receptor, TGF-β receptor, activin receptor, etc.) Tyrosine kinase type receptors (EGF receptor, PDGF receptor, VEGF receptor, c-kit receptor, c-fms receptor, etc.), immunoglobulin receptor family (IL-1 receptor, etc.), cells Death receptor family (TNF receptor, Fas receptor, NGF receptor, etc.), 7-transmembrane receptor family (IL-8 receptor, chemokine) Receptor, etc.) and the like, preferably interferon receptor family, more preferably a type I interferon receptor.
Interferons include IFN-α, IFN-β, IFN-γ, IFN-τ, and the like. Since IFN-α and IFN-β are highly homologous, these two IFNs can react via the same receptor. Interferon α and interferon β are classified as type I interferons.
As a preferred example of the type I interferon receptor, the AR1 chain (GenBank ACCESSION No: J03171, literature: Uze G, Lutfalla G, Gresser I. Genetic transfer of a functional human interferon [alpha]. of it cDNA.Cell (1990) 60, 225-234.) and AR2 chain (GenBank ACCESSION No: U29584, literature: Domanski P, Witte M, Kellum M, Rubinstein M, Hackett R, Pitha P, et al. expression of a long form of the [[beta]] subunit of the interferon [[alpha]] / [[beta]] receptor what is required for signaling, J Biol Chem16. SJ, Cinato E, Monneron-D., Reboul J, Rogers NC, et al. ne cluster.EMBO J (1995) 14,5100-5108.).
A multispecific antibody refers to an antibody that can specifically bind to different types of antigens. That is, a multispecific antibody is an antibody having specificity for at least two different antigens (for example, when the antigen is a heteroreceptor, the multispecific antibody has different domains constituting the heteroreceptor). recognize). Usually, such a molecule binds to two antigens (bispecific antibody: bispecific antibody), but has specificity for more (eg, three types) antigens. Also good.
The antibody against the heteroreceptor of the present invention is not particularly limited, but is preferably a monoclonal antibody. In addition, a recombinant antibody produced using a gene recombination technique is preferable. (See, for example, Borrebaeck, C.A.K. and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom MACMILLAN PUBLISHERS LTD, 19). A recombinant antibody is obtained by cloning DNA encoding the antibody from a hybridoma or antibody-producing cells such as sensitized lymphocytes that produce the antibody, incorporating the DNA into an appropriate vector, and introducing the vector into a host for production. be able to.
Furthermore, the antibody of the present invention may be an antibody fragment, an antibody modification product, a low molecular weight antibody or the like. For example, antibody fragments include Fab, F (ab ′) ₂ , Fv and the like. Here, the “Fv” fragment is the smallest antibody fragment and contains a complete antigen recognition site and a binding site. This region is a dimer in which the variable regions of one heavy chain and light chain are strongly linked by non-covalent bonds (VH-VL dimer). Three CDRs in each variable region interact to form an antigen binding site on the surface of the VH-VL dimer. Six CDRs confer antigen binding sites on the antibody. However, even one variable region (or half of an Fv containing only three CDRs specific for the antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to the antigen. .
The Fab fragment (also referred to as F (ab)) further comprises a light chain constant region and a heavy chain constant region (CH1). Fab ′ fragments differ from Fab fragments in that they additionally have several residues from the carboxy terminus of the heavy chain CH1 region including one or more cysteines from the antibody hinge region. Fab′-SH refers to Fab ′ in which one or more cysteine residues in the constant region have a free thiol group. The F (ab ′) fragment is F (ab ′) ₂ Produced by cleavage of the disulfide bond at the cysteine at the hinge of the pepsin digest. Other chemically conjugated antibody fragments are known to those skilled in the art.
Low molecular weight antibodies include single chain Fv (scFv), diabody, linear antibodies, single chain antibody molecules, and the like.
Diabody (Db) refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)). EP 404,097, WO 93/11161, etc.). A diabody is a dimer composed of two polypeptide chains, each of which is in a position where the light chain variable region (VL) and the heavy chain variable region (VH) cannot bind to each other in the same chain. They are linked by a short linker, for example, about 5 residues. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain V region fragment due to a short linker between them, a diabody forms two antigen-binding sites. Will have. At this time, if VL and VH for two different antigens (a, b) are combined with a linker of about 5 residues in the combination of VLa-VHb and VLb-VHa, they are secreted as bispecific Db. The
An scFv or scFv antibody fragment includes the VH and VL regions of an antibody, and these regions are present in a single polypeptide chain (Huston, JS et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). In general, the scFv polypeptide further comprises V _H And V _L It contains a polypeptide linker between the regions, which allows the scFv to form the structure required for antigen binding (for a review of scFv, see Puckthun “The Pharmacology of Monoclonal Antibodies” Vol. 113 (Rosenburg). and Moore ed (Springer Verlag, New York) pp. 269-315, 1994). The linker in the present invention is not particularly limited as long as it does not inhibit the expression of the antibody variable region linked to both ends thereof.
These antibody fragments and low molecular weight antibodies can be produced by treating antibodies with enzymes such as papain or pepsin to generate antibody fragments, or constructing genes encoding these antibody fragments and introducing them into expression vectors And then expressed in a suitable host cell (eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol). (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; , J.e al, Methods Enzymol (1986) 121,663-669;.. Bird, R.E.and Walker, B.W., see Trends Biotechnol (1991) 9,132-137)..
Examples of the modified antibody include an antibody bound to various molecules such as polyethylene glycol (PEG), and an antibody bound to a cytotoxic substance, endotoxin, radioactive substance and the like. In the modified antibody of the present invention, the substance to be bound is not limited. Such a modified antibody can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.
The origin of the antibody of the present invention is not limited to human antibodies, mouse antibodies, rat antibodies and the like. Further, it may be a genetically modified antibody such as a chimeric antibody or a humanized antibody.
Methods for obtaining human antibodies are already known. For example, a target human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a target antigen (International Patent Application Publication) Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).
Genetically modified antibodies can be produced using known methods. Specifically, for example, a chimeric antibody is an antibody comprising the heavy and light chain variable regions of an immunized animal antibody and the heavy and light chain constant regions of a human antibody. A chimeric antibody can be obtained by ligating DNA encoding the variable region of an antibody derived from an immunized animal with DNA encoding the constant region of a human antibody, incorporating it into an expression vector, introducing it into a host, and producing it. .
A humanized antibody is a modified antibody also referred to as a reshaped human antibody. Humanized antibodies are constructed by grafting CDRs of antibodies derived from immunized animals into the complementarity determining regions of human antibodies. The general gene recombination technique is also known.
Specifically, several oligonucleotides were prepared so that a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody was linked to the terminal portion. It is synthesized from nucleotides by PCR. The obtained DNA is obtained by ligating with DNA encoding the constant region of a human antibody, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication Number EP 239400, International Patent Application Publication Number). WO 96/02576). As the FR of a human antibody linked through CDR, a complementarity determining region that forms a favorable antigen binding site is selected. If necessary, amino acid in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site (Sato, K. et al., Cancer). Res. (1993) 53, 851-856).
The method for obtaining the agonist antibody of the present invention is not particularly limited, and may be obtained by any method. For example, when obtaining an agonist antibody against a heteroreceptor composed of two types of chains (A chain and B chain), an immunized animal is immunized with each of the two types of chains (A, B) constituting the receptor, and a plurality of anti-antibodies Obtain an A chain antibody and a plurality of anti-B chain antibodies. Thereafter, a bispecific antibody comprising the H chain and L chain of the anti-A chain antibody and the H chain and L chain of the anti-B chain antibody is prepared. Here, since a plurality of types of anti-A chain antibodies and anti-B chain antibodies are obtained, it is preferable to prepare bispecific antibodies in as many combinations as possible. After producing the bispecific antibody, an antibody having agonist activity is selected.
In one embodiment of the invention, the antibody of the invention is a bispecific antibody. The preparation of the two-characteristic antibody can be performed by a known method such as fusion between antibody-producing hybridomas or introduction of an antibody expression vector into a cell. For example, an animal is immunized with each of the A chain and B chain of the receptor. MRNA is extracted from the spleen cells of this animal, and the variable regions of the L chain and H chain are recovered by RT-PCR using primers corresponding to the variable regions containing CDRs. A single-chain Fv (scFv) is synthesized by assembly PCR to construct a phage library. The antigen-binding antibody clone was concentrated and cloned by panning, and the single chain variable region (scFv) was inserted between the signal sequence for animal cells and CH1-hinge-CH2-CH3, and the scFv-CH1-Fc expression vector was inserted. Make it. A bispecific antibody can be obtained by introducing a vector encoding an anti-A chain antibody and a vector encoding an anti-B chain antibody into the same cell and expressing the antibody.
The selection of an antibody having agonist activity can be performed, for example, by the following method.
(1) By adding an antibody at the time of culturing a cell that proliferates in a factor-dependent manner, it is used as an index whether or not the cell proliferates like the factor. When the cell grows, the test multispecific antibody is determined to have an agonistic action.
(2) Whether or not a reaction similar to the factor is exhibited by adding at the time of culturing a cell line showing the original activity (not necessarily proliferation) of the factor is used as an index. An antibody is determined to have an agonistic action when it exhibits a reaction similar to a factor.
The cell normally expresses on the cell surface a receptor to which the antibody of the present invention can act as an agonist, and emits a signal by binding to a ligand (for example, an agonist antibody) of the receptor. Therefore, the cells used in the above method are preferably cells (factor-dependent proliferating cells) that can proliferate depending on the ligand (factor) of the receptor. Moreover, it is preferable that the said receptor is what normally emits a cell proliferation signal by couple | bonding with a ligand. However, when the receptor is of a type that does not emit a cell proliferation signal, it is used in the above method by fusing the receptor with a receptor of a type that emits a cell proliferation signal to form a so-called chimeric receptor. can do. The chimeric receptor emits a cell proliferation signal by binding to a ligand. The receptor suitable for constructing a chimeric receptor by fusing with the receptor is not particularly limited as long as it is a receptor that emits a cell proliferation signal, but is usually a membrane protein, more preferably extracellular. Is a receptor with a ligand receptor chain and a intracellular receptor chain. Specific examples include G-CSF receptor, mpl, neu, GM-CSF receptor, EPO receptor, c-Kit, and FLT-3. As a preferable example of the above-described factor-dependent proliferating cells in the present invention, specifically, factor dependency in which a chimeric receptor in which extracellular is a ligand receptor chain and intracellular is a G-CSF receptor chain is expressed. Proliferating cells BaF3 can be shown. In addition, examples of cells that can be used in the above method include NFS60, FDCP-1, FDCP-2, CTLL-2, DA-1, and KT-3.
In addition, examples of a method for selecting an antibody having agonist activity include methods using various quantitative and / or qualitative changes as indicators. For example, a cell-free assay, a cell-based assay, a tissue index, or a biological index can be used.
As an index of a cell-free system, enzymatic reaction and quantitative and / or qualitative changes of protein, DNA, and RNA can be used. As the enzyme reaction, for example, amino acid transfer reaction, sugar transfer reaction, dehydration reaction, dehydrogenation reaction, substrate cleavage reaction and the like can be used. In addition, protein phosphorylation, dephosphorylation, dimerization, multimerization, degradation, dissociation, and the like, DNA, RNA amplification, cleavage, and extension can be used. For example, phosphorylation of a protein existing downstream in the signal transduction pathway can be used as a detection index.
As a cell system indicator, changes in cell phenotype, for example, quantitative and / or qualitative changes of the produced substance, changes in proliferation activity, changes in cell number, changes in morphology, changes in characteristics, etc. it can. As the production substance, secreted proteins, surface antigens, intracellular proteins, mRNA and the like can be used. Changes in morphology include protrusion formation and / or change in number of protrusions, change in flatness, change in elongation / aspect ratio, change in cell size, change in internal structure, and deformity / uniformity as a cell population Changes in sex and cell density can be used. These morphological changes can be confirmed by observation under a microscope. As changes in properties, anchorage dependence, cytokine dependence responsiveness, hormone dependence, drug resistance, cell motility, cell migration activity, pulsatility, changes in intracellular substances, and the like can be used. Cell motility includes cell invasion activity and cell migration activity. Examples of changes in intracellular substances include enzyme activity, mRNA amount, Ca ²⁺ The amount of intracellular signaling substances such as cAMP and the amount of intracellular proteins can be used. In the case of a cell membrane receptor, a change in cell proliferation activity induced by receptor stimulation can be used as an index.
As an index of the tissue system, a change in function according to the tissue used can be used as a detection index. Biological indicators include tissue weight changes, blood system changes such as blood cell number changes, protein content, enzyme activity, electrolysis mass changes, and circulatory system changes such as blood pressure and heart rate changes. Etc. can be used.
The method for measuring these detection indexes is not particularly limited, and is as follows: absorption, emission, color development, fluorescence, radioactivity, fluorescence polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption spectrum, light scattering, fluorescence Resonant energy transfer, etc. can be used. These measurement methods are well known to those skilled in the art, and can be appropriately selected according to the purpose.
For example, the absorption spectrum can be measured with a commonly used photometer, plate reader, etc., emission can be measured with a luminometer, and fluorescence can be measured with a fluorometer. Mass can be measured using a mass spectrometer. Radioactivity is measured by measuring equipment such as a gamma counter according to the type of radiation, the degree of fluorescence polarization is measured by BEACON (Takara Shuzo), the surface plasmon resonance signal is measured by BIACORE, time-resolved fluorescence, fluorescence resonance energy transfer is measured by ARVO, etc. it can. Furthermore, a flow cytometer or the like can also be used for measurement. These measurement methods may measure two or more types of detection indices with one measurement method. If simple, more detections can be made by measuring two or more types simultaneously and / or successively. It is also possible to measure an indicator. For example, fluorescence and fluorescence resonance energy transfer can be measured simultaneously with a fluorometer.
Antibodies against the receptor can be obtained by methods known to those skilled in the art. For example, it can be prepared by immunizing an immunized animal with an antigen. Antigens for immunizing animals include complete antigens having immunogenicity and incomplete antigens (including haptens) having no immunogenicity. In the present invention, a receptor that is considered that the agonist antibody of the present invention acts as a ligand is used as the antigen (immunogen). The receptor in the present invention is not particularly limited, but is preferably a heterodimer. As an animal to be immunized, for example, mouse, hamster, rhesus monkey, or the like can be used. Those skilled in the art can immunize these animals with antigens by well-known methods. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline or the like, mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. The mammal is dosed several times every 4-21 days. Thus, after immunizing a mammal and confirming that a desired antibody level rises in serum, immune cells are collected from the mammal.
In the present invention, preferably, the variable regions of the L and H chains of the antibody are recovered from the immunized animal or cells of the animal. This operation can be performed by those skilled in the art using generally known techniques. Animals immunized with an antigen express antibodies against the antigen, especially in spleen cells. Therefore, for example, mRNA can be prepared from spleen cells of an immunized animal, and the variable regions of the L chain and H chain can be recovered by RT-PCR using primers corresponding to the CDRs of the animal. Here, the CDR refers to three regions (CDR1, CDR2, CDR3) that are present in the hypervariable region in the variable region of an antibody and directly bind to each other in a complementary manner. As a primer corresponding to CDR, for example, a primer corresponding to a framework having lower diversity than CDR, or a primer corresponding to a signal sequence and CH1, CL portions can be used. It is also possible to immunize lymphocytes in vitro. Thereafter, a DNA probe encoding an antibody contained in the spleen or lymphocyte of the immunized animal can be specifically bound to a conventional method, for example, a gene encoding an antibody heavy chain and light chain. It isolates by the method using etc.
The receptor used as an immunogen may be the whole protein constituting the receptor or a partial peptide of the protein. In addition, as an immunogen used for immunizing an animal, an antigen that is an antigen may be bound to another molecule to form a soluble antigen, or a fragment thereof may be used in some cases. When a transmembrane molecule such as a receptor is used as an antigen, it is preferable to use these fragments (for example, the extracellular region of the receptor). Alternatively, a cell that expresses a transmembrane molecule on the cell surface can be used as an immunogen. Such cells may be cells derived from nature (such as tumor cell lines) or configured to express transmembrane molecules by recombinant techniques.
The obtained antibody can be purified to homogeneity. For separation and purification of antibodies, separation and purification methods used for ordinary proteins may be used. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratories, 1988), but is not limited thereto. Examples of columns used for affinity chromatography include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like.
When the antibody of the present invention is an antibody having agonistic activity against, for example, a type I interferon receptor containing an AR1 chain and an AR2 chain, preferably the variable region in the anti-AR1 chain antibody and the anti-AR2 chain antibody And a variable region. Examples of the antibody include, but are not limited to, an antibody comprising any of the following variable regions of an anti-AR1 chain antibody and any of the following variable regions of an anti-AR2 chain antibody. it can.
-Variable region of anti-AR1 chain antibody: AR1-41, AR1-24
-Variable region of anti-AR2 chain antibody: AR2-37, AR2-11, AR2-13, AR2-45, AR2-22, AR2-43, AR2-40, AR2-14, AR2-44, AR2-33, AR2 -31
The amino acid sequences of VH and VL of each of the above variable regions are shown in SEQ ID NOs: 1 to 26 (the relationship between VH and VL of each variable region and SEQ ID NO: is shown in Table 1).

When the anti-AR1 chain antibody is AR1-24, the partner anti-AR2 chain antibody is preferably AR2-13, AR2-31, or AR2-44, and when the anti-AR1 chain antibody is AR1-41, the partner The anti-AR2 chain antibody is AR2-11, AR2-13, AR2-14, AR2-22, AR2-33, AR2-37, AR2-40, AR2-43, AR2-44, or AR2-45. It is preferable. AR2-13 and AR2-44 can be partners to both AR1-41 and AR1-24 antibodies. Antibodies that form pairs as described above are also included in the present invention.
In the present invention, the antibody containing the variable region does not necessarily have the full length sequence of the variable region. If the CDR sequence is conserved, the FR sequence is appropriately used when the antibody is humanized. You may change it.
Therefore, the present invention includes an antibody having an amino acid sequence corresponding to CDR in the variable region.
Table 2 shows amino acid numbers corresponding to CDRs in the variable region SEQ ID NOs.

In the case of producing a full-length antibody using the variable region disclosed in the present invention, the constant region is not particularly limited, and a constant region known to those skilled in the art can be used. For example, Sequences of proteins of immunological interest, (1991), U.S. Pat. S. Department of Health and Human Services. Public Health Service National Institutes of Health and An efficient route to human bispecific IgG, (1998). Nature Biotechnology vol. 16, 677-681, etc. can be used.
Since the antibody of the present invention has an agonistic action, it is expected to be an effective drug against a disease caused by a decrease in activity (function) of a receptor on which the antibody acts. That is, the present invention provides a pharmaceutical composition containing the antibody of the present invention as an active ingredient. For example, when the antibody of the present invention is an antibody having agonist activity against a cytokine receptor, the antibody is considered to have a cytokine-like action. Accordingly, the antibody is expected to be a pharmaceutical product (pharmaceutical composition) having an antiviral action, an antitumor action, and a cell growth / differentiation regulating action.
A pharmaceutical composition containing the antibody of the present invention used as an active ingredient for therapeutic or prophylactic purposes is mixed with an appropriate pharmaceutically acceptable carrier, vehicle, etc. inert to them as necessary. It can be formulated. For example, sterile water, physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders and the like. Other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugars such as mannitol and sorbitol Alcohol may be included. In the case of an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and appropriate dissolution You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc.
If necessary, the antibody of the present invention can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles). And nanocapsules etc.) (see “Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980), etc.). Furthermore, a method of making a drug a sustained-release drug is also known and can be applied to the antibody of the present invention (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem.Tech.12: 98-105 (1982); U.S. Patent 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers 22: 547-556 (1983). EP 133,988).
The dosage of the pharmaceutical composition of the present invention is finally determined as appropriate based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, degree of progression, etc. However, in general, for adults, 0.1 to 2000 mg per day can be orally administered in 1 to several divided doses. More preferred is 1-1000 mg / day, even more preferred is 50-500 mg / day, and most preferred is 100-300 mg / day. These doses vary depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. The administration period is also preferably determined appropriately according to the patient's healing progress and the like.
It is also conceivable to perform gene therapy by incorporating a gene encoding the antibody of the present invention into a gene therapy vector. As the administration method, in addition to direct administration with naked plasmid, it can be packaged in liposomes or as various virus vectors such as retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector, adenovirus related vector, HVJ vector, etc. (See Adolph “Virus Genome Method”, CRC Press, Florida (1996)), or coated on a bead carrier such as colloidal gold particles (WO93 / 17706, etc.). However, the antibody may be administered by any method as long as the antibody is expressed in vivo and can exert its action. Preferably, by suitable parenteral routes (intravenous, intraperitoneal, subcutaneous, intradermal, adipose tissue, intramammary tissue, inhalation or intramuscular route, injection, infusion, or gas-induced particle bombardment (electronic A sufficient amount is administered by a method such as a gun), a method via a mucosal route such as an nasal drug, etc. Ex vivo administration to liposomes, blood cells, bone marrow-derived cells, etc. using liposome transfection, particle bombardment (US Pat. No. 4,945,050), or viral infection, and reintroducing the cells into animals The gene encoding the antibody of the present invention may be administered by

FIG. 1 shows the growth inhibitory activity of antibodies against Daudi cells. A dose-dependent agonist activity could be confirmed.
FIG. 2 shows the ability of the antibody to activate ISRE against pISRE-Luc-introduced K562 cells. The result of the luciferase activity measurement of AR1-41 / AR2-13 is shown. □ indicates IFN-α2a, and ● indicates an AR1-41 / AR2-13 bispecific antibody.
FIG. 3 shows the ability of the antibody to activate ISRE against pISRE-Luc-introduced K562 cells. The result of the luciferase activity measurement of AR1-41 / AR2-13 is shown. □ indicates IFN-α2a, and ● indicates an AR1-41 / AR2-14 bispecific antibody.
FIG. 4 shows the ISRE activation ability of antibodies against pISRE-Luc-introduced K562 cells. The result of the luciferase activity measurement of AR1-41 / AR2-13 is shown. □ indicates IFN-α2a, and ● indicates an AR1-24 / AR2-13 bispecific antibody.
FIG. 5 shows the ability of the antibody to activate ISRE against pISRE-Luc-introduced K562 cells. The result of the luciferase activity measurement of AR1-41 / AR2-13 is shown. □ indicates IFN-α2a, and ● indicates an AR1-24 / AR2-31 bispecific antibody.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[Example 1] Antigen and immunity A soluble receptor expression vector having a FLAG or His6 tag added to the C-terminus of the extracellular region of each of human AR1 and AR2 was introduced into CHO cells, and affinity was obtained from the culture supernatant. Purified using a column. A high expression cell was established by introducing an expression vector of a chimeric molecule of the extracellular region of human AR1 and the G-CSF receptor into the mouse pro B cell line BaF3. Similarly, a high-expression cell of a chimeric molecule between the extracellular region of human AR2 and the G-CSF receptor was established. Each cell was immunized into the peritoneal cavity of BALB / c. AR1His or AR2His was intravenously injected 3 days before the removal of the spleen.
[Example 2] Separation of antibody from scFv display library (a) Panning of phage library PolyA (+) RNA was extracted from the spleen of an immunized mouse, and scFv was synthesized by RT-PCR. A plasmid library that was expressed as a fusion protein with gene3 was constructed (J. Immun. Methods, 201, (1997), 35-55). Library E. coli (2 × 10 ⁹ cfu) was inoculated into 50 mL 2 × YTAG (100 μg / mL ampicillin, 2 × TY containing 2% glucose) and cultured at 37 ° C. to an OD 600 of 0.4 to 0.5. 4 × 10 ¹¹ helper phage VCSM13 was added and left at 37 ° C. for 15 minutes for infection. 450 mL 2 × YTAG, 25 μL 1 mol / L IPTG was added thereto, and cultured at 26 ° C. for 10 hours. The culture supernatant was collected by centrifugation, mixed with 100 mL PEG-NaCl (10% polyethylene glycol 8000, 2.5 mol / L NaCl), and allowed to stand at 4 ° C. for 60 minutes. Phage was precipitated by centrifugation at 10,800 × g for 30 minutes, the precipitate was suspended in 40 mL of water, mixed with 8 mL PEG-NaCl, and allowed to stand at 4 ° C. for 20 minutes. Phage was precipitated by centrifugation at 10,800 × g for 30 minutes and suspended in 5 mL PBS. AR1FLAG and AR2FLAG were labeled with biotin using No-Weighed Premeasured NHS-PEO4-Biotin Microtubes (Pierce). 100 pmol of biotin-labeled AR1FLAG or AR2FLAG was added to the phage library and contacted with the antigen for 60 minutes. 600 μL of Streptavidin MagneSphere (Promega) washed with 5% M-PBS (PBS containing 5% skim milk) was added and allowed to bind for 15 minutes. The beads were washed 3 times each with 1 mL PBST (PBS containing 0.1% Tween-20) and PBS. The beads were suspended in 0.8 mL of 0.1 mol / L glycine / HCl (pH 2.2) for 5 minutes to elute the phages. 45 μL 2 mol / L Tris was added to the collected phage solution for neutralization, added to 10 mL of logarithmic growth phase (OD 600 0.4 to 0.5) XL1-Blue, and left to stand at 37 ° C. for 30 minutes for infection. I let you. This was spread on 2 × YTAG plates and cultured at 30 ° C. Colonies were collected, inoculated into 2xYTAG, and cultured at 37 ° C until OD 600 0.4-0.5. 5 μL of 1 mol / L IPTG and 10 ¹¹ pfu helper phage (VCSM13) were added to 10 mL of the culture solution and allowed to stand at 37 ° C. for 30 minutes. After centrifugation, the cells were resuspended in 100 mL of 2 × YTAG containing 25 μg / mL kanamycin and cultured at 30 ° C. for 10 hours. The culture supernatant was collected by centrifugation, mixed with 20 mL PEG-NaCl, and allowed to stand at 4 ° C. for 20 minutes. Phage was precipitated by centrifugation at 10,800 × g for 30 minutes, and suspended in 2 mL PBS for the next panning. In the second panning, the beads were washed 5 times with PBST and PBS. A clone producing an AR-binding phage was selected by ELISA from E. coli obtained by infecting the eluted phage.
(B) Phage ELISA
The single colony was inoculated into 150 μL 2 × YTAG and cultured at 30 ° C. overnight. After inoculating 5 μL of this in 500 μL 2 × YTAG and culturing at 37 ° C. for 2 hours, 100 μL of 2 × YTAG containing 2.5 × 10 ⁹ pfu of helper phage and 0.3 μL 1 mol / L IPTG was added and allowed to stand at 37 ° C. for 30 minutes. Subsequently, the cells were cultured overnight at 30 ° C., and the centrifuged supernatant was subjected to ELISA. A StreptaWell 96 microtiter plate (Roche) was coated overnight with 100 μL of PBS containing 1.0 μg / mL biotin-labeled AR1FLAG or AR2FLAG. After washing with PBST to remove the antigen, blocking was performed overnight with 200 μL of 2% M-PBS. 2% M-PBS was removed, the culture supernatant was added here, and the mixture was allowed to stand for 40 minutes to bind the antibody. After washing, the bound phage was detected with HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 2% M-PBS and BM blue POD substrate (Roche), and the reaction was stopped by addition of sulfuric acid. Was measured.
(C) Sequencing and clone selection Primers PBG3-F1 (5′-CAGCTATGAAATACCTATTGCC-3 ′ / SEQ ID NO: 27) and PBG3-R1 (5′-CTTTTCATATAATCAAAATCACCGG-3 ′) from the phage solution of clones that were positive by ELISA / SEQ ID NO: 28) was used to amplify the scFv region by PCR, and its nucleotide sequence was determined. 1 μL of phage solution, 2 μL of 10 × KOD Dash buffer solution, 0.5 μL of 10 μmol / L primer, and 20 μL of PCR reaction solution containing 0.3 μL of KOD Dash polymerase (TOYOBO, 2.5 U / μL) at 96 ° C. with Perkin Elmer 9700 Amplification was carried out for 10 seconds, 55 ° C., 10 seconds, 72 ° C., 30 seconds and 30 cycles. After PCR, 3 μL of ExoSAP-IT (Amersham) was added to 5 μL of the reaction solution, and incubated at 37 ° C. for 20 minutes, then at 80 ° C. for 15 minutes. For this sample, BigDye Terminator Cycle Sequencing kit (PIG3-Terminator Cycle sequencing kit) using PBG3-F2 (5'-ATTGCCTACGGCAGCCGCT-3 '/ SEQ ID NO: 29) or PBG3-R2 (5'-AAATACCCGGAACCAGAGCC-3' / SEQ ID NO: 30) as a primer. The reaction was carried out using an Applied Biosystems PRISM 3700 DNA Sequencer. Forty-five clones for anti-AR1 and anti-AR2 were selected for clones having different CDR3 of the amino acid sequence deduced from the base sequence.
[Example 3] Bispecific antibody expression Via a SfiI site between a human signal sequence driven by the CAGG promoter and intron-CH1-Fc (human IgG4 cDNA) for expression as scFv-CH1-Fc An expression vector pCAGGss-g4CH hetero IgG4 into which scFv can be inserted was constructed. In order to express it as a hetero molecule, amino acid substitution to IgG3 CH3 part with reference to IgG1 knobs-into-holes (Protein Engineering vol.9, 617-621, 1996, Nature Biotechnology vol. 16, 677-681, 1998) Created the body. Type A is a substitute for Y349C, T366W. Type B is a substitution product of E356C, T366S, L368A, Y407V. A substitution (-ppcpScp- → -ppcpPcp-) was also introduced into both hinge portions. In addition, a human IL-3 signal sequence was constructed for the A type, and a human IL-6 signal sequence was constructed for the B type (pCAGG-IL3ss-g4CHPa, pCAGG-IL6ss-g4CHPb). The scFv region PCR product of the clone selected from the nucleotide sequence was treated with SfiI, and the anti-AR1 clone was subcloned into pCAGG-IL3ss-g4CHPa and the anti-AR2 clone was subcloned into pCAGG-IL3ss-g4CHPb. Expression vectors were transfected into HEK293 cells using Lipofectamine 2000 for a total of 2025 combinations of anti-AR1 and anti-AR2 clones 45 × 45, and the culture supernatant after 3 days was collected.
Example 4 Isolation of Agonist Bispecific Antibodies (a) BaF3 Proliferation Assay BaF3-ARG is an extracellular region of AR1 and AR2 and an intracellular region of G-CSF receptor in mouse IL-3-dependent proliferating cells BaF3. And introduced an expression vector for the chimeric molecule. BaF3-ARG proliferated depending on IFNα. A 96-well plate was seeded with 0.1 mL of medium containing 1 × 10 ³ cells and samples washed three times. After culturing for 4 days, 10 μL of viable cell count measuring reagent SF (nacalai test) was added and incubated at 37 ° C. for 2 hours, and then A450 was measured.
(B) Daudi Cell Growth Inhibition Assay Daudi cells are a human B cell line that is highly sensitive to IFN. 6.25 × 10 ³ cells per well were seeded in a 96-well plate with 0.1 mL of medium containing the sample. After culturing for 4 days, 10 μL of viable cell count measuring reagent SF (nacalai test) was added and incubated at 37 ° C. for 2 hours, and then A450 was measured.
(C) Sequence of agonist bispecific antibody The amino acid sequences of the variable regions of the antibodies selected by the above screening are shown in SEQ ID NOs: 1 to 26. The relationship between the name of each antibody and the sequence number is shown in Table 1 above.
(D) Reporter gene assay using ISRE To 100 mL of DMRIE-C (Invitrogen) added to 3 mL of OPTI-MEM I and stirred, 40 μg of pISRE-Luc was added and allowed to stand at room temperature for 20 minutes after stirring. did. This 8x10 ⁶ / 2mL OPTI-MEMI addition to human cell K562 adjusted to, after 4 hours incubation at 37 ° C., the cells were further cultured overnight by adding 15% FCS-RPMI1640 of 10 mL. The next day, K562 recovered by centrifugation was resuspended in 10.5 mL of 10% FCS-RPMI 1640 and seeded on a 96-well flat bottom plate at 70 μL / well.
The concentration of bispecific scFv-CH in the antibody gene-introduced HEK293 culture supernatant was adjusted to 12.5 ng / mL in terms of IgG, and a 5-fold dilution series was prepared. This was added at 30 μL / well to the cells into which the reporter plasmid was introduced. As a positive control well, 30 μL / well of a 5-fold dilution series of IFN-α2a was dispensed. After culturing at 37 ° C. for 24 hours, 50 μL / mL of Bright-Glo Luciferase Assay System (Promega) was added and allowed to stand at room temperature for 10 minutes, and then luciferase activity was measured with Analyst HT (LJL) (FIGS. 1 and 2). 3 and 4).

Industrial applicability

  The present invention provides an antibody having an agonistic effect on a receptor containing a hetero chain. The antibody of the present invention is highly expected to be a pharmaceutical because it is considered to have high stability in blood and no antigenicity.

[Sequence Listing]

Claims (12)

An antibody having agonistic activity against a receptor containing a heterochain. The antibody according to claim 1, wherein the receptor is a cytokine receptor. The antibody according to claim 2, wherein the cytokine receptor is an interferon receptor. The antibody according to claim 3, wherein the interferon receptor is a type I interferon receptor. The antibody according to claim 4, wherein the type I interferon receptor comprises an AR1 chain and an AR2 chain. The antibody according to claim 1, wherein the receptor is a multimer. The antibody according to claim 6, wherein the multimer is a dimer. The antibody according to any one of claims 1 to 7, which is a bispecific antibody. 6. The antibody of claim 5, comprising a variable region of an anti-AR1 chain antibody and a variable region of an anti-AR2 chain antibody. The variable region which consists of the amino acid sequence of the following (a) in an anti-AR1 chain antibody, and the variable region which consists of the amino acid sequence in any one of the following (b1)-(b10) in an anti-AR2 chain antibody. The antibody according to 1.
(A) The heavy chain variable region is the amino acid sequence described in SEQ ID NO: 1, the light chain variable region is the amino acid sequence described in SEQ ID NO: 2, and (b1) the heavy chain variable region is the amino acid described in SEQ ID NO: 7. The L chain variable region is the amino acid sequence described in SEQ ID NO: 8 (b2) the H chain variable region is the amino acid sequence described in SEQ ID NO: 9, and the L chain variable region is described in SEQ ID NO: 10. The amino acid sequence (b3) heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region is the amino acid sequence (b4) heavy chain variable region described in SEQ ID NO: 20. The L chain variable region is the amino acid sequence described in SEQ ID NO: 14 (b5) The H chain variable region is the amino acid sequence described in SEQ ID NO: 23, and the L chain variable region is SEQ ID NO: 24. The amino acid sequence described (b6 ) The H chain variable region is the amino acid sequence set forth in SEQ ID NO: 5, the L chain variable region is the amino acid sequence set forth in SEQ ID NO: 6 (b7) The H chain variable region is the amino acid sequence set forth in SEQ ID NO: 17 The L chain variable region is the amino acid sequence described in SEQ ID NO: 18 (b8) the H chain variable region is the amino acid sequence described in SEQ ID NO: 15, and the L chain variable region is the amino acid sequence described in SEQ ID NO: 16. (B9) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 21, the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 22, and (b10) the heavy chain variable region is the amino acid set forth in SEQ ID NO: 11 The amino acid sequence of which the L chain variable region is represented by SEQ ID NO: 12 The variable region which consists of an amino acid sequence of the following (a) in an anti-AR1 chain antibody, and the variable region which consists of the amino acid sequence in any one of the following (b1)-(b3) in an anti-AR2 chain antibody. The antibody according to 1.
(A) The heavy chain variable region is the amino acid sequence described in SEQ ID NO: 3, the light chain variable region is the amino acid sequence described in SEQ ID NO: 4, and (b1) the heavy chain variable region is the amino acid described in SEQ ID NO: 9. The L chain variable region is the amino acid sequence described in SEQ ID NO: 10 (b2), the H chain variable region is the amino acid sequence described in SEQ ID NO: 25, and the L chain variable region is described in SEQ ID NO: 26. Amino acid sequence (b3) The heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 21 and the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 22. The pharmaceutical composition which contains the antibody in any one of Claims 1-11 as an active ingredient.

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