JP4739521B2 - Polypeptides containing N-terminal choline-binding protein A truncated amino acids, vaccines derived therefrom and uses thereof - Google Patents

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Ｎ＝３ ＬＮｎｔ実験、各３ウェル
Ｎ＝２ シアリルラクトース実験、各３ウェル
【０１６１】
レクチン活性は細胞結合活性と相関する：
ヒト細胞は、炭水化物（糖蛋白質および糖脂質）を含む表面分子をもち、細菌は、非常に異なる蛋白質または脂質の骨組みにかかわらずこの炭水化物によってこれら糖共役物と結合する。したがって、in vitroでレクチン活性を有するポリペプチドをもつ細菌は、ヒトの細胞表面に付着することができる。in vitroレクチン活性と細胞結合作用との間の正比例関係は肺炎球菌について知られている。例えば、ＬＮｎｔは、ＴＮＦ活性化Ａ５４９ヒト肺細胞との肺炎球菌の結合を競合的に抑制し、in vivoで肺炎の進行を阻止する。ＣｂｐＡ切端形のレクチン活性は細胞結合活性を反映するということを確認するために、ＣｂｐＡおよび切端形を肺炎球菌と肺細胞との結合の抑制についてテストした（表２）。完全長ＣｂｐＡおよびポリペプチドＲ２は、肺炎球菌の肺細胞への付着をコントロールに対してそれぞれ５８％および６３％抑制した。ポリペプチドＲ１は有効ではなく、Ｒ２のＬｎＮＴ結合活性が必要とするものを示唆し、肺炎球菌の肺細胞との結合を説明する。
【０１６２】
表２

Ｎ＝２ 各々２又は３ウェルの実験
【０１６３】
ＬＮｎｔレクチン活性はＲ２に依存する：
ＣｂｐＡのＮ−末端領域は、各々約１１０アミノ酸の２つの繰り返しを含んでいる（図１参照、ポリペプチド２内の領域ＡおよびＣ）。この２つのドメインの生物活性に対する相対的寄与を調べるために、ドメインＡのみを含むＲ１をＲ２および完全長ＣｂｐＡと比較した。付着アッセイでテストしたとき、ポリペプチドＲ１はＬＮｎｔへの付着を全く抑制しなかった（野性型の９１、９２および１１２％）。しかしながら、ポリペプチドＲ１はシアリルラクトースへの結合に関してはある程度の抑制を示した（コントロールの６８および４０％）。これは、ポリペプチドＲ２はＬＮｎｔレクチン活性に必要で、Ｒ２はＬＮｎｔレクチンドメインの候補物質であることを示している。対照的にＲ１はシアリン酸の認識で活性を有するようである。
【０１６４】
ＣｂｐＡのＮ−末端ドメインに対する抗体は細胞結合を阻止する：
ＣｂｐＡのＮ−末端ドメインが細胞と結合するならば、Ｎ−末端ドメイン活性に関する干渉は、細胞または精製糖共役物と細菌の結合を防止または逆行させるであろう。そのような干渉メカニズムの１つは抗体である。
【０１６５】
表３ 抗ＣｂｐＡ Ｒ２抗体によるＬＮｎＴ被覆表面へのＲ６肺炎球菌の結合抑制

未希釈の５μｌのウサギ抗体＋５μｌの２×１０⁷ Ｒ６ｘを室温で３０分予備保温、続いてＬＮｎｔ被覆ウェルに加えて付着アッセイ。別個の２実験を示す。
【０１６６】
ＣｂｐＡ（Ｒ２）のリコンビナントＮ−末端ドメインに対して作製した抗血清を肺炎球菌のＬＮｎｔへの付着を阻止する能力についてテストした。ウサギのポリクローナル抗ＣｂｐＡ抗血清（５μｌ）＋５μｌの２ｘ１０⁷標識細菌を室温で３０分保温した。この混合物を固定ＬＮｎｔ上に３０分重層し、続いて３回ＰＢＳで洗浄して未結合細菌を除去した。プレートに結合した細菌を顕微鏡で数え、結果を平均値＋６ウェルの標準偏差として示す。表３に示した結果は、Ｒ２に対して作製した抗血清は肺炎球菌のＬＮｎｔへの結合を阻止することを明示している。図５は、肺炎球菌Ｒ６ｘのＬＮｎｔモデルレセプターへの結合抑制について免疫前と抗ＣｂｐＡＲ２抗体の定量曲線を示している。７０％以上の肺炎球菌の付着が、１：１００および１：２００の希釈で抗Ｒ２によって阻止された。さらに１：４００の希釈では活性が失われるということはこの作用の特異性を示している。
【０１６７】
表３および図５で示した抗血清を製造するために用いたＣｂｐＡは血清型４のＣｂｐＡであった。付着抑制アッセイで用いた肺炎球菌Ｒ６ｘ株は血清型２に由来している。異種血清型の細菌の付着を阻止する抗体の能力は、活性型を越える交差防御活性を示している。そのような活性は効果的なワクチン免疫のために極めて望ましい。
【０１６８】
ＣｂｐＡのＮ−末端の天然の構造に対する抗体の活性：
ＣｂｐＡは、Rosenowらが記載したように、その天然のホスト、肺炎球菌からコリンアフィニティーカラムで精製できる。また別には、ポリヒスチジンタグは、転写される蛋白質がいくつかのヒスチジン残基によって伸長されるように、当該遺伝子の末端の操作で付加できる。これらの残基は完全長ポリペプチドのニッケル親和性マトリックス精製を容易にし、この完全長ポリペプチドは、より短い切端形と異なり天然の三次元構造の保持に有利である。特に肺炎球菌だけでなく大腸菌または他のホスト細菌からこれら生化学的手段によって精製されたＣｂｐＡは、その天然の三次元構造を保持する。免疫原として用いた場合、自然の状態で折り畳まれたＣｂｐＡは、折り畳みが異なる可能性がある切端形による免疫で誘発される抗体とは潜在的に異なる抗体を産生する。同様に、治療薬として用いられるＣｂｐＡは、付着を阻止するその能力が改善された切端形とは異なる三次元構造を有するであろう。これらを斟酌するならば、その天然の三次元構造で折り畳むことを可能にする完全長蛋白質としてＣｂｐＡを生成し、続いて生化学的にＣ−末端（ＣＢＤ）を切断するのが有利であろう。例えば、ヒドロキシルアミンによる処置は、コリン結合プロテインＡの血清型Ｒ６ｘおよび血清型４のアミノ酸４７５位でＣｂｐＡを切断し、Ｎ−末端およびＣ−末端を分離させるであろう。その結果Ｎ−末端フラグメントは治療薬または免疫原として適切である。
【０１６９】
また別には、天然のＣｂｐＡは免疫原として用いることができ、活性な構造に対する抗血清が得られる。この混合物中の生物活性を有する抗Ｎ−末端抗体は吸収によりＢＤに対する抗体を除去することによって濃縮することができる。そのような抗体は２００μｌの血清を１ｘ１０⁸のＣｂｐＡ不完全細菌と一緒に室温で１時間保温して調製した。この変異体のその他のコリン結合蛋白質は抗ＣＢＤ抗体で吸収して除去し、遠心して細菌を除去することによって抗血清から除かれた。
吸収抗ＣｂｐＡ抗体の生物活性を明示させるために、ＬＮｎｔモデルレセプターへの肺炎球菌の付着を阻止する吸収抗血清の能力を調べた。Ｒ６ｘ肺炎球菌を１：６００の希釈の抗血清と保温し、ＬＮｎｔアルブミンで被覆したウェルに添加した。
【０１７０】
表４ 吸収抗ＣｂｐＡ抗血清は付着を阻害する

【０１７１】
これらの結果は、その天然の構造をもつＣｂｐ／ＡのＮ−末端ドメインに対する抗体は付着を強力に阻止することを示している。この活性は、図５の切端形の活性よりはるかに高い（後者は１：６００の希釈で活性を示さなかった）。吸収抗ＣｂｐＡ抗血清のこの活性は、図５の力価測定実験でもまた明瞭に示された。肺炎球菌タイプ４のＬＮｎｔ被覆ウェルに対するベースライン付着は三角記号で示されている。肺炎球菌と未吸収（四角記号）または吸収（ダイヤ型記号）抗血清との種々の希釈での予備保温によって、得られる付着が減少することが示された。両抗血清が同様な付着の減少を示すという事実は、ＣｂｐＡに対する抗体の阻止活性の大半はＮ−末端に存在することを示している（すなわち、吸収によるコリン結合ドメインに対する抗体の除去は生物活性を減少させない）。
【０１７２】
実施例３：抗Ｒ２抗血清による受動防御：
ウサギ免疫血清の作製：
ポリペプチドＲ２（ＣｂｐＡ切端形）およびＣｂｐＡに対するウサギ免疫血清をコバンス（Covance)(Denver, PA)で作製した。免疫前血清を採集した後、ニュージランド白色ウサギを、フロイントの完全アジュバント中の２５０μｇのＲ２（両方のアミノ末端繰り返しを含む、上記の調製物４８３：５８）で免疫した。２１日目にフロイントの不完全アジュバント中の１２５μｇのＲ２でウサギを追加免疫し３１日目に採血した。第二のウサギは精製ＣｂｐＡで同様に免疫した。
【０１７３】
マウスの受動防御：
Ｃ３Ｈ／ＨｅＪマウス（５匹／群）の腹腔に滅菌ＰＢＳ中の（免疫前および３１日目免疫血清）１００μｌの１：２希釈のウサギ抗Ｒ２血清または免疫前血清を投与し受動免疫を施した。血清投与後１時間で、マウスを１６００ＣＦＵの肺炎連鎖球菌血清型６Ｂ（ＳＰ３１７株）で攻撃した。マウスを１４日間生存についてモニターした。ポリペプチドＲ２に対して作製したウサギ免疫血清で免疫したマウスの８０％が攻撃に対して生存した（図４）。免疫前ウサギ血清で免疫したマウスは全て７日目までに死亡した。
この結果は、ＣｂｐＡに特異的な抗体は全身的肺炎球菌感染に対して防御作用を有することを示している。さらにこの結果は、コリン結合領域は防御に不要であることを示している。なぜならば、防御のためには、切端蛋白質であるポリペプチドＲ２（保存コリン結合繰り返しを欠く）に特異的な抗体で十分であるからである。さらに、ＣｂｐＡ血清型４に対して誘導された血清は血清型６Ｂによる攻撃に対して防御作用を示した。
【０１７４】
実施例４：抗Ｒ１抗血清による能動防御：
Ｃ３Ｈ／ＨｅＪマウス（１０匹／群）の腹腔にＣｂｐＡ切端形蛋白質Ｒ１（５０μｌのＰＢＳ中の１５μｇ、および５０μｌのフロイントの完全アジュバント）を投与し受動免疫を施した。１０匹のシャム免疫マウス群にはＰＢＳおよびアジュバントを投与した。第二の免疫は４週間後に、１５μｇの蛋白質をフロイントの不完全アジュバントとともに腹腔に投与して実施した（シャム免疫群はＰＢＳおよびフロイントの不完全アジュバント（ＩＦＡ）を投与）。免疫反応の分析のために３、６、および９週目に血液を採取した（眼窩後方採血）。１０匹のＣｂｐＡ免疫マウスから９週目に集めた血清の抗ＣｂｐＡ切端形のＥＬＩＳＡ終末点力価は４０９６０００であった。シャム免疫マウスから得た血清では抗体は検出されなかった。マウスを１０週目に５６０ＣＦＵの肺炎連鎖球菌血清型６Ｂ（ＳＰＳＪ２ｐ株、P. Flynn（St. Jude Children's Research Hospital, Memphis, TN）から提供）で攻撃した。マウスを１４日間生存についてモニターした。ＣｂｐＡ切端形蛋白質Ｒ１で免疫したマウスの８０％が攻撃に対して生存した。全てのシャム免疫マウスは８日目までに死亡した（図７）。
【０１７５】
この結果は、ＣｂｐＡのリコンビナントフラグメントは、肺炎球菌の全身感染および致死に対して防御することができる特異的抗体の産生を誘発することを示している。さらにこの結果は、免疫原は切端蛋白質Ｒ１であるので、コリン結合領域は防御に不要であることを示している。さらにこの結果は、防御反応を誘発するためにはただ１回のアミノ末端繰り返しで十分であることを提唱している。リコンビナント肺炎球菌蛋白質が血清型４のＤＮＡ配列を基に作製され、血清型６Ｂ単離株による攻撃に続いて防御が認められたので、交差防御もまた明らかである。
【０１７６】
実施例５：幼獣ラットの鼻咽頭コロニー形成の予防：
ＣｂｐＡのＮ−末端ドメインは、in vitroで肺炎球菌の付着を競合的に抑制した。この活性を有するペプチドの治療的有用性を明らかにするために、幼獣ラットに切端形ペプチドを投与し、続いて肺炎球菌で攻撃して鼻咽頭のコロニー形成を調べた。
０．８μｇのポリペプチドＲ２またはＲ１を含む１０μｌのＰＢＳ、または蛋白質を含まない１０μｌのＰＢＳをラットに経鼻的に投与した。１５分後に、３型肺炎球菌（ＳＩＩＩ株）（１ｘ１０⁵ｃｆｅｒを含む１０μｌ）を経鼻的に導入した。競合的に肺炎球菌の付着およびコロニー形成を抑制するポリペプチドの能力を決定するために、鼻洗浄を７２時間に実施し、各群につき４匹の動物の各々について回収肺炎球菌数を定量した。ＳＩＩＩのみを与えられたラットは１０μｌにつき２２００、６５００、６９００および８７００（平均６０７５）コロニーを示した。切端形Ｒ２で処理した動物は、最大の減少（３６００、３５００、２５００、２１００）を示し、平均は１０μｌにつき２９２５の細菌（コントロールの４８％）であった。切端形Ｒ１で処置した動物もまたコロニー形成の減少を示し（５０００、４８００、３５００、１６００）、平均は３７２５（コントロールの６１％）であった。
この実験は、動物の治療実験デザインにおける本発明のペプチドの動物への投与で、その後の肺炎球菌の攻撃から動物を防御することができることを示した。
【０１７７】
考察：
これらの実験が示すように、ポリペプチドＲ２は：１）ワクチン抗原として投与されるとき、防御抗体を誘発し、ワクチン製剤として好ましい組成であり；２）ペプチドとして気道および／または鼻咽頭レセプターに輸送されるとき、肺炎球菌の付着を競合的に防止し、コロニー形成および侵襲性症状に対して予防薬および治療薬として好ましい組成物である。さらにまた、ＣｂｐＡ切端形は、ＣＢＤのないレクチンとして機能する。２つの炭水化物が認識される：図１の両方のＮ−末端繰り返し（ＡおよびＣ）を含むペプチドによってＬＮｎｔが、さらに最もＮ−末端側のＮ−末端のただ１つの繰り返しのみ（Ａ）を含むペプチドによってシアリン酸が認識される。Ｎ−末端繰り返しポリペプチドＲ１およびＲ２を含む切端形は細胞培養アッセイで同様にレクチン活性を示す。
【０１７８】
ポリペプチドＲ２活性の重要な特性には以下が含まれる：１）精製糖共役物レセプター類似体、肺細胞および動物モデルの認識についてポリペプチドＲ２および完全長ＣｂｐＡの生物活性の完全な相関関係。相関関係はまた、それらに対する抗体についても明らかである。２）４型由来菌および他の血清型（例えば６Ｂおよび２）を用いたin vitroアッセイの菌との間の交差防御。これは有用なワクチン、予防薬および治療薬には重要なことである。
【０１７９】
本発明は詳細に説明され、さらに種々の具体的な材料、方法および実施例を参考に詳述してきたが、本発明は特定の材料、材料の組合せ、およびその目的のために選択された方法に限定されないことは理解されよう。当業者には理解されるところであるが、多数の変型が暗示されている。同様に本発明の開示に関して本明細書に引用した一切の文献は参照により本明細書に含まれる。
【０１８０】
【図面の簡単な説明】
【図１】 図１は、コリン結合プロテインＡ（ＣｂｐＡ）、リコンビナント切端形Ｒ１（図２に示すＣｂｐＡのＮ−末端の約アミノ酸１６からアミノ酸３２１）、およびＲ２（図２に示すＣｂｐＡのＮ−末端の約アミノ酸１６からアミノ酸４４４）を模式的に示している。ドメインＡは、図２に示すＣｂｐＡアミノ酸配列のＮ−末端の約アミノ酸１５３からアミノ酸３２１である。ドメインＢは、図２に示すＣｂｐＡアミノ酸配列のＮ−末端の約アミノ酸２７０からアミノ酸３２６である。Ｃは、図２に示すＣｂｐＡアミノ酸配列のＮ−末端の約アミノ酸３２７からアミノ酸４３３である。
【図２】 図２Ａ−Ｂは、ＣｂｐＡのＮ−末端領域の核酸およびアミノ酸配列の多様な血清型における相同性の比較である。
【図３】 図３は、リコンビナントＲ１およびＲ２の発現および精製を示す。
【図４】 図４は、マウスので受動的防御の結果である。リコンビナントＲ２に対する免疫血清は肺炎連鎖球菌による致死性攻撃からマウスを防御した。
【図５】 図５は、ＬＮｎＴ−ＨＳＡ被覆プレートへのＲ６ｘ付着に対する抗Ｒ２抗体の力価測定を示す。
【図６】 図６は、ＬＮｎＴ−ＨＳＡ被覆プレートへの肺炎球菌吸着を阻止する活性について抗ＣｂｐＡ抗体および吸収抗ＣｂｐＡ抗体の力価を測定したものである。
【図７】 図７は、マウスでの能動的防御の結果を示す。リコンビナントＲ１に対する免疫血清は肺炎連鎖球菌による致死性攻撃からマウスを防御した（攻撃は血清型６Ｂで５６０ｃｆｕ）。
【配列表】

[0001]
In general, the present invention relates to N-terminal choline binding protein A truncated polypeptides. The invention also relates to vaccines that provide protection against or elicit protective antibodies against bacterial infections, particularly pneumococci, and antibodies and antagonists against said polypeptides for use in diagnostic and passive immunotherapy. The polypeptide and / or nucleic acid encoding the polypeptide is also useful as a competitive inhibitor of pneumococcal bacterial adhesion factor. The final object of the present invention is a therapeutic method using the polypeptide.
[0002]
Conventional technology
Streptococcus pneumoniae is a Gram-positive bacterium that is a major cause of invasive infections (eg sepsis, meningitis, otitis media and lobar pneumonia) (Tuomanen et al., NEJM 322: 1280-1284 (1995) )). Streptococcus pneumoniae strongly binds to cells in the upper and lower airways. Like most bacteria, pneumococcal adherence to human cells is achieved by the expression of bacterial surface proteins that bind in a lectin-like manner to eukaryotic carbohydrates (D. Cundell & E. Tuomanen, Microb. Pathog. 17: 361-374 (1994)). Streptococcus pneumoniae binds to non-inflammatory epithelium, one of the courses considered as an asymptomatic aspect. Changes to invasive disease involve local production of inflammatory factors that activate human cells and change the number and type of receptors available on human cells (D. Cundell et al., Nature 377: 435 -438 (1995)). Given the opportunity in this new setting, Streptococcus pneumoniae appears to utilize and mate with one of these unregulated receptors, the platelet activating factor (PAF) receptor (Cundell et al., Nature 377: 435). -438 (1995)). Within minutes after the appearance of the PAF receptor, the adsorption and invasion of pneumococci are enhanced. For example, suppression of bacterial binding to activated cells by soluble receptor analogs inhibits disease progression in animal models (I. Idanpaan-Heikkila, J. Infect. Dis. 176: 704-712 (1997)). Particularly effective in this regard are soluble carbohydrates containing sialic or non-added lacto-N-neotetraose, which prevent in vitro binding of pneumococci to human cells and prevent colonization in vivo in the lung. To do.
[0003]
Choline-binding protein: Adhesion factor candidate structural gene:
Streptococcus pneumoniae produces surface proteins that can bind to the bacterial surface by non-covalent binding to cell wall teichoic acid or lipoteicolic acid. The surface of Streptococcus pneumoniae is modified with choline binding proteins (CBPs) that bind non-covalently to phosphorylcholine. cbpA is a 75 kD surface-exposed choline binding protein that exhibits a chimeric structure. There is a C-terminal domain composed of a unique N-terminal domain proline-rich region followed by 10 repeat regions required for choline binding.
[0004]
cbpA is an adhesion factor (ligand) for a glycoconjugate-containing receptor present on the surface of a eukaryotic cell. Mutants lacking cbpA showed reduced toxicity in nasopharyngeal colonization in a cub model. This binding targets the choline determinant that modifies teicoyl acid and is mediated by the symbolic choline binding domain present in each of the same proteins. This choline-binding domain was discovered and completely characterized by Lopez et al. In experiments on autolysis (Ronda et al., Eur. J. Biochem. 164: 621-624 (1987)). Other proteins containing this domain include pneumococcal phage autolysin and its protective antigen, pneumococcal surface protein A (PspA) (C. Ronda et al., Eur. J. Biochem. 164: 621- 624 (1987); LS McDaniel et al., Microb. Pathog. 13: 261-269 (1992)).
[0005]
CbpA, which shares the C-terminus with others of the same type but whose binding activity to human cells arises from its unique N-terminal domain, cannot colonize in the nasopharyngeal region. Since the colony formation process and development depends on the binding of pneumococci to human cells as a first step, disruption of N-terminal domain function by cross-reacting antibodies or competitive inhibition of peptides mimicking this domain prevents disease. It will be extremely important. Choline binding proteins for anti-pneumococcal vaccines are discussed in international application PCT / US97 / 07198, which is hereby incorporated by reference. Current vaccines against Streptococcus pneumoniae take advantage of this very common 23 serotype of capsular purified carbohydrates, but such vaccines are only 50% protective (Shapiro et al. ., NJEM 325: 1453 (1991)) under 2 years old is not immunogenic. Furthermore, in the infection of multidrug resistant microorganisms, therapeutic peptides may optionally be used for therapy. Thus, the present invention addresses what has long been sought by providing protective vaccines.
[0006]
Problems to be solved by the invention
The present invention provides an isolated polypeptide comprising an N-terminal choline binding protein A truncated amino acid sequence. This polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, 3-7 or 9-11 (including fragments, variants, variants, analogs or derivatives thereof). Furthermore, the present invention provides an isolated polypeptide comprising an N-terminal choline-binding protein A truncated amino acid sequence having the amino acid sequence shown in SEQ ID NO: 24 representing its three-dimensional structure, and a method for producing such a polypeptide. To do. The isolated polypeptide is suitable for immunization of animals and humans against bacterial infections, preferably pneumococci.
Another object of the invention is an N-terminal choline-binding protein A truncated form with lectin activity and no choline binding activity. The invention further provides an immunogenic N-terminal choline binding protein A truncated form or fragment thereof.
[0007]
The invention also relates to isolated nucleic acids, such as recombinant DNA or cloned genes, or degenerate variants, mutants, analogs, fragments thereof. These either encode the isolated polypeptide or competitively suppress the activity of the polypeptide. The isolated nucleic acid comprising a degenerate, variant, variant, analog or fragment thereof preferably has the sequence shown in SEQ ID NO: 12, 14-17, 19-22 or 23. In another embodiment of the invention, the recombinant DNA molecule or the complete DNA sequence of such a cloned gene is operably linked to expression control sequences and introduced into a suitable host. Accordingly, the present invention encompasses a single cell host transformed with a cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present invention, more specifically a DNA sequence determined from the sequence set forth above or a fragment thereof. .
[0008]
The antibody against the isolated polypeptide includes an antibody produced in a natural state and an antibody produced by gene recombination. These include both polyclonal antibodies and monoclonal antibodies produced by known genetic techniques, as well as bispecific (chimeric) antibodies, as well as other suitable for diagnostic applications along with the ability to regulate bacterial attachment. Also included are antibodies that also include a function, such as, but not limited to, a competitor.
[0009]
Yet another object of the present invention is to treat a mammal to control the amount or activity of a bacterium or its subunits, treat an invasive, accidental or idiopathic condition, or treat their unintended consequences. Provide a workaround. The present invention provides pharmaceutical compositions for use in therapy. These pharmaceutical compositions are based on the present isolated polypeptides, their subunits or their binding partners.
Finally, the present invention provides pharmaceutical compositions, vaccines and diagnostic agents and therapeutic methods using them.
[0010]
Means to solve the problem
An object of the present invention is an isolated polypeptide comprising an N-terminal choline binding protein A truncated amino acid sequence. The polypeptide is suitable for use in immunizing animals against pneumococcal infection. These polypeptides or fragments thereof can be used as protective vaccines against pneumococci and other bacteria with cross-reactive proteins when formulated with a suitable adjuvant.
[0011]
The present invention provides an isolated polypeptide comprising an N-terminal choline binding protein A truncated amino acid sequence. In certain embodiments, the polypeptide has the amino acid sequence shown in any of SEQ ID NOs: 1, 3-5, 7 or 9-11 (including fragments, variants, variants, analogs or derivatives thereof). In another embodiment, the polypeptide has amino acid KXXXE (SEQ ID NO: 6).
[0012]
The present invention provides an isolated polypeptide comprising the N-terminal choline binding protein A truncated amino acid sequence shown in FIG. In one embodiment, the polypeptide has an amino acid sequence that is a conserved region shown in FIG. For example, conserved regions include amino acid sequences 158 to 210; 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; It is not limited to these. FIG. 2 shows the serotype homology of the nucleic acid and amino acid sequences of the N-terminal region of CbpA encompassed by the present invention.
[0013]
Furthermore, the present invention provides an isolated polypeptide comprising an N-terminal choline-binding protein A truncated amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 24, which represents its three-dimensional structure. In certain embodiments, the polypeptide is an analog, fragment, variant or variant thereof. The variants included are shown in FIG. The present invention also provides an amino acid sequence of an N-terminal choline-binding protein A truncated form having amino acids from about position 16 to about 475 of serotype 4 shown in FIG. 2, or an amino acid corresponding to serotype 4 shown in FIG. An isolated polypeptide comprising is provided. In some embodiments, the three-dimensional structure is consistent with that present in the native protein.
[0014]
A method for producing this polypeptide is, for example, as follows: full-length choline-binding protein A is cleaved with hydroxylamine. In this case, hydroxylamine cleaves choline-binding protein A at amino acid asparagine (N) at position 475 of serotype R6x and serotype 4, or serotype R6x or serotype in the case of the different serotypes shown in FIG. Cleavage at the amino acid corresponding to 4, thereby producing an N-terminal choline-binding protein A truncated form. Alternative methods of creating choline-binding protein A truncated forms or fragments thereof and retaining the natural three-dimensional structure (ie, the structure of full-length choline-binding protein A) are also contemplated and will be apparent to those skilled in the art. is there. Since the polypeptide retains its three-dimensional structure, the isolated polypeptide is suitable for use as an immunogen when immunizing animals and humans against bacterial infections, preferably pneumococci.
[0015]
The polypeptide comprising the amino acid sequence of serotype 4 of choline binding protein A (CbpA) is as follows:
ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKVEEAKKKAEDQKE
EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE
VESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPAEQPQPAPAPKAE
KPAPAPKPEN (SEQ ID NO: 24).
[0016]
“Polypeptide R2” refers to a polypeptide comprising the amino acid sequence from positions 16 to 444 of the N-terminal truncated form of choline binding protein A (CbpA) serotype 4 (see FIG. 1) having the following sequence:
ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKVEEAKKKAEDQKE
EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE
VESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPA (SEQ ID NO: 1).
[0017]
The DNA sequence encoding choline binding protein A (CbpA) serotype 4 N-terminal truncated polypeptide R2 is as follows:
GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA
AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA
GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG
AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA
GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA
ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA
AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA
GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC
AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG
AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA
CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG
ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA
CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG
AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG
AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG
GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC
ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG
AAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGA
AGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAA
GAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACT
TGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAG
TAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAGC
AAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAAA
ATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAG
CAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO: 12).
[0018]
The amino acid sequence of serotype 4 CbpA is as follows:
ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKVEEAKKKAEDQKE
EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE
VESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPAEQPQPAPAPKAE
KPAPAPKPENPAEQPKAEKPADQQAEEDYARRSEEEYNRLTQQQPPKTEKPA
QPSTPKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNSNGAMATG
WLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANGSMATGWLQYNGS
WYYLNANGSMATGWLQYNGSWYYLNANGDMATGWVKDGDTWYYLEAS
GAMKASQWFKVSDKWYYVNGSGALAVNTTVDGYGVNANGEWVN (SEQ ID NO: 2).
[0019]
The DNA sequence encoding the amino acid sequence of serotype 4 CbpA is as follows:
GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA
AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA
GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG
AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA
GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA
ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA
AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA
GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC
AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG
AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA
CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG
ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA
CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG
AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG
AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG
GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC
ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG
AAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGA
AGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAA
GAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACT
TGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGgCTTGAACTA
GTAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAG
CAAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAA
AATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCA
GCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTGAACAACCACAACCAG
CGCCGGCTCCAAAAGCAGAAAAACCAGCTCCAGCTCCAAAACCAGAGAA
TCCAGCTGAACAACCAAAAGCAGAAAAACCAGCTGATCAACAAGCTGAA
GAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACA
GCAACCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACA
GGCTGGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTC
AATGGCGACAGGATGGCTCCAAAACAAtGGCTCAtGGTAcTACcTCAACAG
CAATGGCGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACT
ATCTAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGT
TCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAGGATGGCTCCA
ATACAATGGCTCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAG
GATGGCTCCAATACAATGGCTCATGGTACTACCTAAACGCTAATGGTGAT
ATGGCGACAGGTTGGGTGAAAGATGGAGATACCTGGTACTATCTTGAAGC
ATCAGGTGCTATGAAAGCAAGCCAATGGTTCAAAGTATCAGATAAATGGT
ACTATGTCAATGGCTCAGGTGCCCTTGCAGTCAACACAACTGTAGATGGC
TATGGAGTCAATGCCAATGGTGAATGGGTAAACTAA (SEQ ID NO: 13).
[0020]
“Polypeptide R1” refers to a polypeptide comprising the amino acid sequence from position 16 to position 321 of N-terminal truncated / choline binding protein A (CbpA) serotype 4 having the following sequence:
ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKSSDSSVGEETL (SEQ ID NO: 3).
[0021]
The DNA sequence encoding polypeptide R1 is as follows:
GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA
AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA
GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG
AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA
GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA
ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA
AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA
GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC
AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG
AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA
CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG
ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA
CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG
AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG
AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG
GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC
ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG
AAGAAACTCTTC (SEQ ID NO: 14).
[0022]
“Polypeptide C / R2” refers to a polypeptide comprising repeat region C within R2, where repeat region C is from position 327 to position 433 of serotype 4 N-terminal choline binding protein A (CbpA). It has an amino acid sequence, which has the following sequence:
KPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDVEVK
KAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEEEAK
RKA (SEQ ID NO: 4).
[0023]
The DNA sequence of polypeptide C / R2 is as follows:
AAACCAGAAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTA
AGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAAC
CAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAG
TTAAAAAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCG
AAACGAGGAAAAAGTTAAGCAAGCAAAAGCGGAAGTTGAGAGTAAAAAA
GCTGAGGCTACAAGGTTAGAAAAAATCAAGACAGATCGTAAAAAAGCAG
AAGAAGAAGCTAAACGAAAAGCA (SEQ ID NO: 15).
[0024]
“Polypeptide A / R2” refers to a polypeptide comprising repeat region A within R2, where repeat region A is from position 153 to position 269 of serotype 4 N-terminal choline binding protein A (CbpA). It has an amino acid sequence, which has the following sequence:
TEPGEKVAEAKKKVEEAEKKAKDQKEEDRRNYPTITYKTLELEIAESDVEVK
KAELELVKVKANEPRDEQKIKQAEAEVESKQAEATRLKKIKTDREEAEEEAK
RRADA (SEQ ID NO: 5).
As shown in FIG. 1, region A of polypeptide R2 is the same as region A in R1.
[0025]
The DNA sequence encoding polypeptide A / R2 is as follows:
ACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAA
GCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAACTACC
CAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTG
GAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAACGAAC
CTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAA
ACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTGAAGAA
GCAGAAGAAGAAGCTAAACGAAGAGCAGATGCT (SEQ ID NO: 16).
[0026]
The identity or position of one or more amino acid residues can be altered or modified to include variations such as deletions, substitutions and additions. Deletions include those with fewer residues than all the residues unique to the protein, substitutions replace one or more specific residues with other residues, and additions add one or Two or more amino acid residues are added to the terminal or central part of the polypeptide (see FIG. 2). These molecules include: preferred codon incorporation for expression by selected non-mammalian hosts; provision of a site for cleavage by a restriction endonuclease enzyme; initiation, termination or intermediate that facilitates construction of an easy-to-express vector Provision of additional DNA sequences. In particular, examples of serotype 4 amino acid substitutions include (but are not limited to): E at position 154 substituted with K; P at position 155 substituted with L; G at position 156 substituted with E E at position 157 is replaced with K; K at position 181 is replaced with E; D at position 182 is replaced with A; R at position 187 is replaced with Y, H or L; I at position 194 is replaced with N; E at position 200 is replaced with D; E at position 202 is replaced with D; E at position 209 is replaced with K; K at position 212 is replaced with E; V at position 218 is replaced with L; V at position 220 is K at position 221 is replaced by E; N at position 223 is replaced with D or K; P at position 225 is replaced with S, T or R; D at position 227 is replaced with N; E at position 228 Is substituted with K; Q at position 229 is replaced with E, G or D; K at position 230 is replaced with T; K at position 232 is replaced with N; E at position 235 Substitute with K; A at position 236 is replaced with E; E at position 237 is replaced with K; S at position 240 is replaced with N; K at position 241 is replaced with E; Q at position 242 is replaced with K; K in position is replaced with E; K in position 250 is replaced with N; E in position 257 is replaced with Q or K; A in position 263 is replaced with L; K in position 264 is replaced with E; R in position 265 Is substituted with N; R at position 266 is replaced with I; A at position 267 is replaced with K or V; D at position 258 is replaced with T; A at position 269 is replaced with D; A at position 291 is T; Substitution with V, P, G or X; G at position 294 is substituted with G, A or E; V at position 295 is substituted with D or A; P at position 295 is substituted with L or F; L at position 299 is Substitution with P or Q; P at position 328 with S; E at position 329 with G; E at position 340 with A; K at position 343 with E or D E at position 347 is replaced with K; D at position 349 is replaced with A; R at position 354 is replaced with H; E at position 366 is replaced with D; E at position 375 is replaced with K; K at position 378 is E substituted at 390; E substituted at G; P at 391 substituted with S; N at 393 substituted with D; V at 397 substituted with I; and K at 408 substituted with Q.
[0027]
"Polypeptide R2 serotype R6x" refers to a polypeptide comprising the amino acid sequence from position 16 to position 444 of the N-terminal truncated form of choline binding protein A (CbpA) serotype R6x having the following sequence:
ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL
NIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVA
EAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVK
EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLK
EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSSSLK
SGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVKE
AELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKR
KAAEEDKVKEKPA (SEQ ID NO: 7).
[0028]
The DNA sequence encoding polypeptide R2 serotype R6x is as follows:
GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC
AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA
ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC
CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA
ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA
AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG
AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA
GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA
CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG
CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGC
ACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTA
CAAGGTTAGAAAACAtCAAGACAGAtCGTAAAAAAGCAGAAGAAGAAGCT
AAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCAG
AtCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTA
GCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGT
AGGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAG
CAGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCA
AAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTG
ACCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAA
CTAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGC
AAGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGA
AAACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAA
GCAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO: 17).
[0029]
Amino acid sequence of CbpA of serotype R6x:
ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL
NIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVA
EAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVK
EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLK
EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSSSLK
SGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVKE
AELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKR
KAAEEDKVKEKPAEQPQPAPATQPEKPAPKPEKPAEQPKAEKTDDQQAEEDY
ARRSEEEYNRLTQQQPPKTEKPAQPSTPKTGWKQENGMWYFYNTDGSMAT
GWLQNNGSWYYLNANGAMATGWLQNNGSWYYLNANGSMATGWLQNNG
SWYYLNANGAMATGWLQYNGSWYYLNSNGAMATGWLQYNGSWYYLNA
NGDMATGWLQNNGSWYYLNANGDMATGWLQYNGSWYYLNANGDMATG
WVKDGDTWYYLEASGAMKASQWFKVSDKWYYVNGSGALAVNTTVDGYG
VNANGEWVN (SEQ ID NO: 8).
[0030]
DNA sequence encoding the amino acid sequence of CbpA of serotype R6x:
GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC
AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA
ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC
CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA
ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA
AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG
AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA
GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA
CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG
CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGC
ACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTA
CAAGGTTAGAAAACAtCAAGACAGAtCGTAAAAAAGCAGAAGAAGAAGCT
AAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTtCAGA
tCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAG
CAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTA
GGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAGC
AGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAA
AAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGA
CCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAAC
TAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGCA
AGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAA
AACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAG
CAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTGAACAACCACAACC
AGCGCCGGCTACTCAACCAGAAAAACCAGCTCCAAAACCAGAGAAGCCA
GCTGAACAACCAAAAGCAGAAAAAACAGATGATCAACAAGCTGAAGAAG
ACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACAGCAA
CCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACAGGCT
GGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTCAATG
GCAACAGGATGGCTCCAAAACAACGGTTCATGGTACTATCTAAACGCTAA
TGGTGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACTATC
TAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGTTCA
TGGTACTACCTAAACGCTAATGGTGCTATGGCGACAGGATGGCTCCAATA
CAATGGTTCATGGTACTACCTAAACAGCAATGGCGCTATGGCGACAGGAT
GGCTCCAATACAATGGCTCATGGTACTACCTCAACGCTAATGGTGATATG
GCGACAGGATGGCTCCAAAACAACGGTTCATGGTACTACCTCAACGCTAA
TGGTGATATGGCGACAGGATGGCTCCAATACAACGGTTCATGGTATTACC
TCAACGCTAATGGTGATATGGCGACAGGTTGGGTGAAAGATGGAGATACC
TGGTACTATCTTGAAGCATCAGGTGCTATGAAAGCAAGCCAATGGTTCAA
AGTATCAGATAAATGGTACTATGTCAATGGCTCAGGTGCCCTTGCAGTCA
ACACAACTGTAGATGGCTATGGAGTCAATGCCAATGGTGAATGGGTAAAC
TAA (SEQ ID NO: 18).
[0031]
Polypeptide R1 serotype R6x refers to a polypeptide comprising the amino acid sequence from position 16 to position 321 of the N-terminal truncated / truncated form of choline binding protein A (CbpA) serotype R6x having the following sequence:
ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL
NIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVA
EAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVK
EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLK
EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETL (SEQ ID NO: 9).
[0032]
The DNA sequence encoding polypeptide R1 is as follows:
GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC
AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA
ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC
CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA
ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA
AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG
AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA
GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA
CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG
CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAATCTCGAAACGAGGG
CACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCT
ACAAGGTTAGAAAACAtCAAGACAGATCGTAAAAAAGCAGAAGAAGAAG
CTAAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCA
GATCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGC
TAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGC
GTAGGTGAAGAAACTCTTC (SEQ ID NO: 19).
[0033]
"Polypeptide C / R2 serotype R6x" contains a repetitive region C having an amino acid sequence from position 327 to 433 at the N-terminus of choline-binding protein A (CbpA) serotype R6x having the following sequence in R2 Refers to a polypeptide (see FIG. 2):
KSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVK
EAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAK
RKA (SEQ ID NO: 10).
[0034]
Polypeptide C / R2 serotype R6x DNA sequence:
AAATCAGGAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTG
AGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAAC
CAATACTTACAAAACGCTTGACCTTGAAATTGCTGAGTCCGATGTGAAAG
TTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCG
AGACGAGGAAAAAATTAAGCAAGCAAAAGCGAAAGTTGAGAGTAAAAAA
GCTGAGGCTACAAGGTTAGAAAACATCAAGACAGATCGTAAAAAAGCAG
AAGAAGAAGCTAAACGAAAAGCA (SEQ ID NO: 20).
[0035]
“Polypeptide A / R2 serotype R6x” includes a repetitive region A within R2 having an amino acid sequence from position 155 to position 265 at the N-terminus of choline-binding protein A (CbpA) serotype R6x having the following sequence: Refers to a polypeptide (see FIG. 2):
PGEKVAEAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKE
AELELVKEEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKR
KADA (SEQ ID NO: 11).
[0036]
DNA sequence encoding polypeptide A / R2 serotype R6x:
CCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGA
AAAAAGCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAA
TACTTACAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTA
AAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAAC
GAGGGCACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTG
AGGCTACAAGGTTAGAAAACAtCAAGACAGATCGTAAAAAAGCAGAAGA
AGAAGCTAAACGAAAAGCAGATGCT (SEQ ID NO: 21).
[0037]
The present invention is directed to an isolated polypeptide, which consists of the amino acid sequence shown in SEQ ID NO: 22 or 23 (including fragments, variants, variants, analogs or derivatives thereof).
SPSLKPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDV
EVKKAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEE
EAKRKAAEEDKVKEKPA (SEQ ID NO: 22, serotype 4, sites 323-434); or
PSSSLKSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESD
VKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAE
EEAKRKAAEEDKVKEKRA (SEQ ID NO: 23; serotype R6x; positions 322-434).
[0038]
“Polypeptide B / R2” refers to a polypeptide comprising the amino acid sequence from position 270 to position 326 of the N-terminal truncated form of choline-binding protein A (CbpA) serotype 4 shown in FIG. “Polypeptide B / R2 serotype R6x” refers to a polypeptide comprising the amino acid sequence from position 264 to position 326 of the N-terminal truncated form of choline-binding protein A (CbpA) serotype R6x shown in FIG. The present invention contemplates a polypeptide having the amino acid sequence of regions A, B, C, A + B, B + C, A + C as shown in FIG.
The present invention further provides an isolated polypeptide comprising an N-terminal choline binding protein A truncated amino acid sequence, wherein the polypeptide has amino acid KXXXE (SEQ ID NO: 6).
[0039]
The present invention is directed to a polypeptide comprising the N-terminal choline-binding protein A truncated amino acid sequence shown in FIG. In one embodiment, the polypeptide has an amino acid sequence that is a conserved region shown in FIG. For example, conserved regions include the amino acid sequences of 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; Not. FIG. 2 shows the nucleic acid and amino acid sequence homology of the N-terminal regions of various serotypes of CbpA encompassed by the present invention.
[0040]
The present invention provides an isolated polypeptide comprising an N-terminal choline-binding protein A truncated amino acid sequence that has lectin activity but does not bind to choline. In certain embodiments, the polypeptide has an amino acid sequence set forth in any of SEQ ID NOs: 1, 3-5, 7, or 9-11 (including fragments, variants, variants, analog derivatives thereof).
[0041]
As used herein, “polypeptide having lectin activity” refers to a polypeptide, peptide or protein that binds non-covalently to a carbohydrate. As defined herein, “adhesion” refers to non-covalent binding between bacteria and human cells, or secretions that are sufficiently stable and resistant to washing. As defined herein, “binding to LNnT” means binding to a lacto-N-neotetraose coated substrate over an albumin control.
[0042]
The present invention provides an isolated immunogenic polypeptide comprising an N-terminal choline binding protein A truncated amino acid sequence. As intended by the present invention, the immunogenic polypeptide comprises the amino acid sequence shown in any of SEQ ID NOs: 1, 3-7, or 9-11 (fragments, variants, variants, analogs or derivatives thereof) ). The present invention provides an isolated polypeptide comprising the N-terminal choline binding protein A truncated amino acid sequence shown in FIG. In one embodiment, the polypeptide has an amino acid sequence that is a conserved region shown in FIG.
The object of the present invention is an analog of a polypeptide comprising the amino acid sequence described above. In the analog polypeptide, an N-terminal methionine or N-terminal polyhistidine may optionally be linked to the N- or COOH-terminus of the polypeptide containing the amino acid sequence.
[0043]
In another embodiment, the present invention provides peptide fragments derived from proteolytic digestion products of the aforementioned polypeptides. In another embodiment, the polypeptide derivative has one or more chemical molecular moieties attached thereto. In another embodiment, the chemical moiety is a water soluble polymer. In another embodiment, the chemical molecular moiety is polyethylene glycol. In another embodiment, the chemical moiety is mono-, di-, tri- or tetrapegylated. In another embodiment, the chemical moiety is monopegylated at the N-terminus.
[0044]
The attachment of polyethylene glycol (PEG) to compounds is particularly useful because PEG is very toxic in mammals (Carpenter et al., (1971)). For example, the adenosine deaminase PEG adduct has been approved for use in humans for the treatment of severe combined immunodeficiency syndrome in the United States. A second advantage obtained by PEG conjugation is that it effectively reduces the immunogenicity and antigenicity of the heterologous compound. For example, human protein PEG adducts may be useful in the treatment of disease without the risk of eliciting a severe immune response in other species of mammals. The compounds of the present invention can be subjected to drug delivery by microencapsulation means to reduce or prevent a host immune response against the compound or cells producing the compound. The compounds of the invention can also be transported by microencapsulation within a membrane (eg, a liposome).
[0045]
A number of PEG activated forms suitable for direct reaction with proteins have been reported. PEG reagents useful for reaction with amino groups of proteins include active esters or carbonate derivatives of carboxylic acids, particularly those with leaving groups of N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2- Those that are nitrobenzene-4-sulfonates are included. PEG derivatives containing maleimide or haloacetyl groups are useful reagents for the modification of protein free sulfhydryls. Similarly, PEG reagents containing aminohydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.
[0046]
In certain embodiments, the amino acid residues of the polypeptides disclosed herein are preferably in the “L” isomeric form. In another embodiment, residues of the “D” isomeric form are replaced with any of the L-amino acid residues as long as the polypeptide maintains the desired functional properties for lectin activity. NH₂Refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group at the carboxy terminus of a polypeptide. Abbreviations used herein follow standard polypeptide nomenclature (J. Biol. Chem. 243: 3552-59 (1969)).
Note that all amino acid residue sequences herein are represented by the formula in which the left-to-right direction is the normal direction from the amino terminus to the carboxy terminus. Furthermore, the leading or trailing dash in an amino acid residue sequence indicates a peptide bond or another sequence having one or more amino acid residues.
[0047]
Synthetic polypeptides (produced using solid phase, liquid phase well known techniques or peptide condensation techniques, or any combination thereof) can include natural or unnatural amino acids. Amino acids used in peptide synthesis are standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin (standard Maryland solid phase method standard deprotection, neutralization, conjugation and wash protocol). (Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963))) or a readily basic Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acid (first Carpino & Han (J Org. Chem. 37: 3403-3409 (1972)). Thus, the polypeptides of the present invention include D-amino acids, combinations of D- and L-amino acids, and various “designer” amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids. Can convey certain characteristics. Synthetic amino acids include ornithine instead of lysine, fluorophenylalanine instead of phenylalanine, and norleucine instead of leucine or isoleucine. In addition, specific amino acids can be assigned to specific conjugation steps to produce α-helical, β-turn, β-sheet, γ-turn and cyclic peptides.
[0048]
In one aspect of the invention, the polypeptide can include a special amino acid at the C-terminus. This special amino acid is used to stimulate free glycine or glycine-amide groups.₂H or CONH₂Contains either side chain. Another case of interest for this particular residue would be as a D or L amino acid analog with a side chain consisting of a linker or bead binding. In certain embodiments, the pseudo-free C-terminal residue will have a D or L type optical structure. In another embodiment, a racemic mixture of D and L isomers can be used.
[0049]
In yet another embodiment, pyroglutamate is included as the N-terminal residue of the peptide. Pyroglutamate is not amenable to continuous reaction by Edman degradation, but by limiting the substitution to only 50% of the peptide on one bead containing N-terminal pyroglutamate, sufficient non-pyrrole in the bead for sequencing The glutamate peptide will remain. One skilled in the art will readily appreciate that this technique can be used to sequence any peptide containing an Edman degradation resistant residue at the N-terminus. Other methods for examining the properties of individual peptides exhibiting the desired activity are described in detail below. The specific activity of a peptide containing a blocked N-terminal group (eg pyroglutamate) does not block the activity of a completely (100%) blocked peptide when a particular N-terminal group is present in 50% of the peptide. It will be readily apparent by comparison with (0%) peptide.
[0050]
In addition, the present invention uses peptide-like linkages (eg, ester linkages) to produce peptides with more precisely defined structural properties, and to use peptide mimetics, as well as to produce peptides with novel properties. I intend to. In another embodiment, a reduced peptide bond (ie R₁-CH₂-NH-R₂Can be produced. Where R₁And R₂Is an amino acid residue or sequence. The reduced peptide bond may be introduced as a dipeptide subunit. Such molecules will be resistant to peptide bond hydrolysis (eg, protease activity). Such peptides will provide ligands with inherent functions and activities (eg, increased half-life in vivo by resistance to metabolic degradation or protease activity). Furthermore, it is well known that constrained peptides exhibit enhanced functional activity in certain systems (Hruby, Life Sciences 31: 189-199 (1982); Hruby et al., Biochem. J 268: 249-262 (1990)). The present invention provides a method for producing a constrained peptide incorporating an arbitrary sequence at all other positions.
[0051]
A constrained peptide, cyclic peptide or regidized peptide is an amino acid or amino acid analog that provides a chemical functional group that can form a cross-link to constrain, cyclize or stiffen the peptide after the cross-linking process. It can manufacture by a synthesis | combination on the condition that it inserts in at least two places. Cyclization is promoted when turn-inducing amino acids are incorporated. Examples of amino acids that can crosslink peptides include cysteines that form disulfides, aspartic acid that forms lactones or lactases, and chelators that chelate transition metals to form bridges (eg, γ-carboxyl-glutamic acid (Gla) (Bachem )). Protected γ-carboxylglutamic acid can be produced by modifying the synthesis described by Zee-Cheng & Olson (Biophys. Res. Commun. 94: 1128-1132 (1980)). Peptides containing at least two amino acids whose peptide sequence can form a bridge, for example, oxidize cysteine residues to form disulfide bonds, or treat with addition of metal ions to form chelates, thereby Peptides can be cross-linked to produce constrained, cyclic or rigid peptides.
[0052]
The present invention provides a method for systematically forming crosslinks. For example, if four cysteine residues are incorporated into the peptide sequence, different protecting groups can be used (Hiskey, in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross and Meienhofer, eds., Academic Press : New York, pp. 137-167 (1981); Ponsanti et al., Tetrahedron 46: 8255-8266 (1990)). The first cysteine pair can be deprotected and further oxidized, followed by deprotection and oxidation of the second set. In this way, a constant disulfide bridge set can be formed. Alternatively, cysteine pairs and matching amino acid analog pairs can be incorporated so that the crosslinks can have different chemical properties.
[0053]
The following non-classical amino acids can be incorporated into peptides to introduce specific constitutive motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., J. Am. Chem Soc. 113: 2275-2283 (1991)); (2S, 3S) -methyl-phenylalanine, (2S, 3R) -methyl-phenylalanine, (2R, 3S) -methyl-phenylalanine and (2R, 3R) -methyl Phenylalanine (Kazmierski & Hruby, Tetrahedron Lett. (1991)); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, Ph.D. Thesis, University of Arizona (1989)); hydroxy-1,2,3 4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J. Takeda Res. Labs. 43: 53-76 (1989)); β-carboline (D and L) (Kazmierski, Ph.D. Thesis, University of Arizona (19 88)); HIC (histidine isoquinolinecarboxylic acid) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); and HIC (histidine cyclic urea) (Dharanipragada).
[0054]
The following amino acid analogs and peptidomimetics can be introduced into a peptide to induce or promote specific secondary structure: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), β-turn-induced dipeptide analogs (Kemp et al., J. Org. Chem. 50: 5834-5838 (1985)); β-sheet-induced analogs (Kemp et al., Tetrahedron Lett. 29: 5081-5082 (1988) ); Β-turn-inducing analogs (Kemp et al., Tetrahedron Lett. 29: 5057-5060 (1988)); α-helix-inducing analogs (Kemp et al., Tetrahedron Lett. 29: 4935-4938 (1988)); γ-turn-inducing analogs (Kemp et al., J. Org. Chem. 54: 109-115 (1989)); and analogs provided by the following references (Nagai & Sato, Tetrahedron Lett. 26: 647- 650 (1985); DiMaio et al., J. Chem. Soc. Perkin Trans. P.1687 (1989)); Gly-Ala turn analogs (Kahn et al., Tetrahedron Lett. 30: 2317 (1989)); Amide bond isotopes (Jones et al., Tetrahedro n Lett. 29: 3853-3856 (1988)); tetrasol (Zabrocki et al., J. Am. Chem. Soc. 110: 5875-5880 (1988)); DTC (Samanen et al., Int. J. Protein) Pep. Res. 35: 501-509 (1990)); and analogs disclosed by the following references (Olson et al., J. Am. Chem. Soi. 112: 323-333 (1990); Garvey et al., J. Org. Chem. 56: 436 (1990)). Constitutive mimics of β-turns and β-overhangs and peptides containing them are described in US Pat. No. 5,444,0013 (Kahn, Aug. 8, 1995).
[0055]
The present invention further provides modification or derivation of the polypeptides or peptides of the present invention. Peptide modifications are well known to those of skill in the art and include phosphorylation, carboxymethylation and acylation. The modification can be performed by chemical or enzymatic means. In another aspect, glycated or fatty acylated peptide derivatives can be produced. The production of glycated or fatty acylated peptides is well known in the art. Fatty acyl peptide derivatives can also be produced. For example, free amino groups (N-terminal or lysyl) can be acylated (eg, myristoylated). In another embodiment, the structure is (CH₂)_nCH_ThreeAn amino acid containing an aliphatic side chain of can be incorporated into a peptide. Such and other peptide-fatty acid conjugates suitable for use in the present invention are disclosed in British patent GB-8809162.4, international patent application PCT / AU89 / 00166 and reference 5 (supra). Yes.
[0056]
Mutations can be performed on a nucleic acid encoding a polypeptide by changing specific codons to codons for different amino acids. In general, such mutations are performed by allowing the fewest nucleotide changes. This type of substitution mutation causes the amino acids of the resulting protein to change in a non-conservative manner (ie, by changing codons from amino acids belonging to a group of amino acids having a particular size or property to amino acids belonging to another group). ) Or in a conservative manner (ie by changing codons from amino acids belonging to a group of amino acids having a particular size or property to amino acids belonging to the same group). In general, such conservative changes result in little change in the structure and function of the resulting protein.
[0057]
Non-conservative changes further change the structure, activity or function of the resulting protein. The present invention is believed to include sequences containing conservative changes that do not significantly change the activity or binding properties of the resulting protein. Substitution of an amino acid in the sequence can be selected from other amino acids in the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing an aromatic ring structure are phenylalanine, tryptophan and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes will not affect the apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point.
[0058]
Particularly preferred substitutions are as follows:
Positive charge can be maintained with Lys instead of Arg and vice versa;
Negative charge can be maintained with Glu instead of Asp and vice versa;
-Ser in place of Thr, free -OH can be maintained;
-Free NH with Gln instead of Asn₂Is maintained.
[0059]
Synthetic DNA sequences allow convenient construction of genes that express analogs or “muteins”. A general method for regiospecific incorporation of unnatural amino acids into proteins has been described by Noren et al. (Science 244: 182-188 (1989)). This method can be used to make analogs containing unnatural amino acids.
In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA technology within the art can be used. Such techniques are explained fully in the literature. See, for example: Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology", Vol. I-III (RM Ausubel, ed. (1994)); "Cell Biology: A Laboratory Handbook ", Vol.I-III (JE Celis, ed. (1994));" Current Protocols in Immunology ", Vol.I-III (JE Coligan, ed. (1994));" Oligonucleotide Synthesis " , (MJ Gait, ed. (1984)); "Nucleic Acid Hybridization" (BD Hames & SJ Higgins, ed. (1985)); "Transcription and Translation" (BD Hames & SJ Higgins, ed. (1984)); "Animal Cell Culture" (RI Freshney, ed. (1986)); "Immobilized Cells and Enzymes" (IRL Press, (1986)); B. Perbal, "A Practical Guide to Molecular Cloning" (1984).
[0060]
In another embodiment, pyroglutamate can be included as the N-terminal residue of the peptide. Pyroglutamate is not amenable to continuous reaction by Edman degradation, but by limiting the substitution to only 50% of the peptide on one bead with N-terminal pyroglutamate, sufficient non-pyroglutamate peptide on the bead is due to sequencing. Will remain. One skilled in the art will appreciate that this technique can be used to sequence any peptide that incorporates a residue resistant to Edman degradation at the N-terminus. Other methods for examining the properties of individual peptides exhibiting the desired activity are described in detail below. The specific activity of a peptide containing a blocked N-terminal group (eg pyroglutamate) is shown to be less than that of a fully (100%) blocked peptide if the individual N-terminal group is present in 50% of the peptide (0 %) Will be readily revealed by comparison with the peptide.
[0061]
Derivative chemical moiety: The chemical moiety suitable for induction can be selected from water-soluble polymers. The polymer selected must be water soluble so that the component to which it is attached does not precipitate in an aqueous environment (eg, a physiological environment). Preferably, the polymer will be pharmaceutically acceptable in order to use the final product therapeutically. One skilled in the art can select the desired polymer taking into account whether the polymer / component conjugate is used for therapy, and if so, the desired dose, circulation time, resistance to proteolysis and other considerations. For this component, these can be confirmed using the assays disclosed herein.
[0062]
Examples of the water-soluble polymer include polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / anhydrous malein. From acid copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, pro-propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols and polyvinyl alcohol Selected from the group consisting of Polyethylene glycol propionaldehyde may be advantageous for production due to its stability in water.
[0063]
The molecular weight of the polymer may be any, and the polymer may be branched or unbranched. In the case of polyethylene glycol, the preferred molecular weight is about 2 kDa to about 100 kDa for ease of handling and production (the term “about” is the production of polyethylene glycol, some molecules are heavier than the aforementioned molecular weight, Indicates that the molecule is lighter than that). Desired therapeutic profile (e.g., desired release duration, impact if there is an effect on biological activity, ease of handling, degree or lack of antigenicity, and other properties of polyethylene glycol for therapeutic proteins or analogs) Other sizes can also be used according to known effects.
[0064]
The number of polymer molecules to be attached varies, and those skilled in the art can confirm the influence on the function. Mono-, di-, tri-, or some combination of derivatives can be prepared using the same or different chemical molecular moieties (eg, polyethylene glycols of different molecular weights in the polymer, for example). The ratio of polymer molecules to component molecules will vary and will depend on their ratio in the reaction mixture. In general, the optimal ratio (in terms of reaction efficiency in the absence of excess unreacted components and polymer) is determined by, for example, the desired degree of induction (eg, mono, di, tri, etc.), the molecular weight of the polymer selected, the polymer branching It is further determined by requirements such as reaction conditions.
[0065]
A polyethylene glycol molecule (or other chemical molecule moiety) must be attached to a component in view of its effect on the functional or antigenic domain of the protein. There are a number of coupling methods available to those skilled in the art. For example, EP 401384 (which is incorporated herein by reference) describes the coupling of PEG to G-CSF, and Malik et al. (Exp. Hematol. 20: 1028-1035 (1992)) described tresyl chloride. The PEGylation of GM-CSF used is reported. For example, polyethylene glycol is covalently linked to an amino acid residue via a reactive group (eg, a free amino or carboxyl group). A reactive group is a group to which an activated polyethylene glycol molecule binds. Amino acid residues having a free amino group include lysine residues and the N-terminal amino acid residue. Amino acid residues having a free carboxyl group include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
[0066]
The present invention provides an isolated nucleic acid encoding a polypeptide comprising an N-terminal choline binding protein A truncated amino acid sequence. The present invention provides an isolated nucleic acid encoding a polypeptide comprising the N-terminal choline binding protein A truncated amino acid sequence shown in FIG. In certain embodiments, the nucleic acid is as shown in any of SEQ ID NOs: 12, 14-17, or 19-21, including fragments, variants, variants, analogs or derivatives thereof. The nucleic acid is DNA, cDNA, genomic DNA, or RNA. Furthermore, the isolated nucleic acid can be operably linked to an RNA transcription promoter. It is also contemplated that the nucleic acid is used to competitively inhibit lectin activity.
[0067]
A “vector” is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment can be linked to replicate the binding segment.
“DNA” refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine), either single-stranded or double-stranded. The term refers to the primary and secondary structure of the molecule and is not limited to any particular tertiary structure. Thus, this term includes double-stranded DNA, viruses, plasmids, and chromosomes found as linear DNA molecules (eg, restriction fragments), among others. When considering the structure of a specific double-stranded DNA molecule, the sequence herein refers only to the sequence in the 5 ′ to 3 ′ direction of a non-transcribed DNA strand (ie, a strand having a sequence homologous to mRNA). It is described in the usual way.
[0068]
When an expression control sequence controls transcription and translation of a DNA sequence, the DNA sequence is “operably linked” to the expression control sequence. The term “functionally linked” has an appropriate initiation signal (eg, ATG) in front of the DNA sequence to be expressed and further expresses the DNA sequence under the control of the expression control sequence, Maintaining an accurate reading frame to produce the desired product encoded by. If the gene desired to be inserted into the recombinant DNA molecule does not contain an appropriate start signal, such start signal can be inserted before the gene.
[0069]
Furthermore, this invention provides the vector containing said nucleic acid molecule. The promoter may be or be the same as a bacterial, yeast, insect or mammalian promoter. Further, the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC), bacterial phage, or eukaryotic cell viral DNA.
Many other vector backbones known in the art as useful for protein expression can be used. Such vectors include adenovirus (AV), adeno-associated virus (AAV), simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), moloney murine leukemia virus, DNA delivery system ( Ie, liposomes), and expression plasmid delivery systems, including but not limited to. In addition, one class of vectors contains DNA elements derived from viruses such as bovine papillomavirus, polyomavirus, baculovirus, retrovirus or Semliki Forest virus. Such vectors may be obtained commercially or the described sequences may be assembled by methods well known in the art.
[0070]
The present invention also provides a host vector system for producing a polypeptide comprising a suitable host cell vector. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells such as bacterial cells (including gram positive bacteria), yeast cells, fungal cells, insect cells and animal cells. As a host, for example, mouse fibroblast NIH3T3, CHO cell, HeLa cell, Ltk^-A large number of mammalian cells can be used, including but not limited to cells, Cos cells.
[0071]
A variety of host / expression vector combinations may be used to express the DNA sequences of this invention. Useful expression vectors will consist of, for example, chromosomal segments, non-chromosomal DNA sequences and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids such as E. coli plasmid colE1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as PR4; phage DNA such as multiple phage lambda derivatives NM989 and other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids, such as 2μ plasmids or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; plasmids and phages Vectors derived from DNA combinations, such as plasmids modified to utilize phage DNA or other expression control sequences, are included.
[0072]
Any of a variety of expression control sequences (sequences that control the expression of a DNA sequence operably linked to the sequence) can be used in these vectors to express the DNA sequences of the present invention. Such useful expression control sequences include, for example, SV40, CMV, vaccinia, polyoma or adenovirus early or late promoter, lac system, trp system, TAC system, TRC system, LTR system, phage λ major operators and promoters Region, regulatory region of fd coat protein, promoter for 3-phosphoglycylate kinase or other glycolytic enzymes, promoter of acid phosphatase (eg Pho5), promoter of yeast α-mating factor, prokaryotic or eukaryotic cell or virus Other sequences known to control the expression of these genes and various combinations thereof are included.
[0073]
A variety of unicellular hosts are also useful for expression of the DNA sequences of the present invention. These hosts include well-known eukaryotic and prokaryotic cells such as E. coli strains, Pseudomonas, Bacillus, Streptomyces, fungi (eg yeast), and animal cells (eg CHO, R1.1, BW and LM cells, African green monkey kidney cells (eg, COS1, COS7, BSC1, BSC40 and BMT10), insect cells (eg, Sf9), human cells and tissue culture plant cells.
[0074]
It will be appreciated that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of the present invention. Furthermore, not all hosts function equally well with the same expression system. However, one skilled in the art can select the appropriate vector, expression control sequence and host without undue experimentation and achieve the desired expression without departing from the scope of the present invention. For example, when choosing a vector, the host must be considered because the vector functions in it. Also considered are the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector (eg, antibiotic markers).
[0075]
A variety of factors are usually considered when choosing an expression control sequence. These include, for example, the relative strength of the system, its controllability, and compatibility (particularly with respect to potential secondary structure) with the individual DNA sequences or genes to be expressed. Suitable unicellular hosts are generated, for example, by the compatibility of selected vectors with their hosts, their secretion properties, their precise protein folding ability, and their fermentation requirements, as well as the DNA sequence to be expressed. It is selected considering the toxicity of the product to the host and the ease of purification of the expression product.
[0076]
The present invention further provides a method for producing a polypeptide. The method includes growing the host vector system described above under suitable conditions that permit production of the polypeptide, and recovering the polypeptide so produced.
The present invention further provides an antibody capable of specifically recognizing or binding to the isolated polypeptide. The antibody may be a monoclonal antibody or a polyclonal antibody. Further, the antibody can be labeled with a detectable marker that is either a radioactive marker, a colorimetric marker, a fluorescent marker, or a chemiluminescent marker. The labeled antibody may be a monoclonal antibody or a polyclonal antibody. In certain embodiments, the labeled antibody is a purified labeled antibody. Methods for labeling antibodies are well known in the art.
[0077]
The term “antibody” includes, for example, both naturally occurring and non-naturally occurring antibodies. In particular, the term “antibody” includes polyclonal antibodies and monoclonal antibodies as well as fragments thereof. Furthermore, the term “antibody” includes chimeric and fully synthetic antibodies and fragments thereof. Such antibodies include, but are not limited to, polyclonal, monoclonal, single chain, Fab fragments and Fab expression libraries.
[0078]
Various methods known in the art can be used for the production of polyclonal antibodies against a polypeptide or a derivative or analog thereof (see, eg, Antibodies--A Laboratory Manual, Harlow & Lane, eds Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 1988). In order to produce antibodies, various host animals can be immunized by injecting the truncated CbpA or derivative thereof (eg, a fragment or fusion protein). These host animals include rabbits, mice, rats, sheep, goats and the like. In certain embodiments, the polypeptide can be conjugated to an immunogenic carrier such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants can be used to enhance the immunological response depending on the host species.
[0079]
For the production of monoclonal antibodies or fragments, analogs or derivatives thereof, any technique that produces antibody molecules by continuous cell line culture can be used (see, eg, Antibodies--A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 1988). In addition to the hybridoma technology originally reported by Kohler & Milstein (Nature 256: 495-497 (1975)), these technologies include trioma technology and human B cell hybridoma technology (Kozbor et al., Immunology Today 4:72). (1983)) and EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), including It is not limited.
[0080]
In yet another embodiment of the invention, monoclonal antibodies can be produced in sterile animals using modern techniques (PCT / US90 / 02545). According to the present invention, human antibodies can be used, whether human antibodies are human hybridomas (Cote et al., Proc. Natl. Acad. Sci. USA, 80: 2026-2030 (1983)), or Human B cells can be obtained by in vitro transformation with EBV virus (Cole et al., In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96 (1985)). Indeed, according to the present invention, for the production of a “chimeric antibody” that splices a gene of a mouse antibody molecule specific for a polypeptide together with a gene derived from a human antibody molecule with appropriate biological activity. (Morrison et al., J. Bacteriol. 159-870 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985) )), And such antibodies are within the scope of the present invention. Such human antibodies or humanized chimeric antibodies are preferred for use in the treatment of human diseases (described below) because they induce much less immune responses, particularly allergic reactions, than exogenous antibodies. In yet another embodiment of the invention, techniques for the construction of Fab expression libraries (Huse et al., Science 246: 1275-1281 (1989)) are utilized. This technique allows the rapid and easy identification of monoclonal Fab fragments with the desired specificity for the polypeptide or derivative or analog thereof.
[0081]
Antibody fragments that contain the idiotype of the antibody molecule can be generated by known techniques. For example, such a fragment includes F (ab ′)₂Fragments (which can be produced by pepsin digestion of antibody molecules); Fab ′ fragments (F (ab ′)₂Including, but not limited to, Fab fragments (which can be produced by treating antibody molecules with papain and a reducing agent).
[0082]
In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art such as: radioimmunoassay, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassay, immunoradiation Assay, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay (eg using colloidal gold, enzyme or radioisotope labeling), Western blot, precipitation reaction, agglutination assay (eg gel aggregation assay, hemagglutination assay) ), Complement binding assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays. In certain embodiments, the primary antibody is detected by detecting binding of the secondary antibody or reagent to the primary antibody. In yet another embodiment, the secondary antibody is labeled. Many means for detecting binding in an immunoassay are known in the art and are within the scope of the present invention.
[0083]
For in vivo detection, the antibody is labeled with, for example: enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidal gold, latex particles, and chemiluminescent agents. Alternatively, the antibody is labeled for in vivo detection, for example with: a radioisotope (preferably technetium or iodine), a magnetic resonance shift reagent (eg gadolinium and manganese), or radioactivity Non-permeating reagent.
[0084]
Very common labels used in these experiments are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others. Many fluorescent materials are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas red, AMCA blue and lucifer yellow. A specific detection substance is an anti-rabbit antibody prepared with a goat and conjugated with fluorescein by isothiocyanate. Polypeptides can also be labeled with radioactive elements or enzymes. Radioactive labels can be detected by any currently available measurement method. The preferred isotope is^ThreeH,¹⁴C,³²P,³⁵S,³⁶Cl,⁵¹Cr,⁵⁷Co,⁵⁸Co,⁵⁹Fe,⁹⁰Y,¹²⁵I,¹³¹I and¹⁸⁶Rc can be selected.
[0085]
Enzyme labels are equally useful and can be detected by any currently available colorimetric, spectroscopic, fluorescent spectroscopic, amperometric, or gas pressure measuring methods. The enzyme is conjugated by reaction of the selected particles with a cross-linking molecule (eg, carbodiimide, diisocyanate, glutaraldehyde, etc.). Many enzymes that can be used in these methods are known and can be used. Preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase and peroxidase, and alkaline phosphatase. For example, US Pat. Nos. 3,654,090, 3,850,752, and 4016043 disclose alternative labeling materials and methods.
[0086]
In another embodiment of the present invention, a commercial test kit is produced that can be used by medical professionals to determine the presence or ability of a predetermined binding activity of a suspected target cell. In accordance with the testing techniques described above, certain types of such kits include at least a labeled polypeptide or a binding partner specific for it (eg, an antibody specific for the polypeptide) and instructions (eg, “competition”), According to the selected method such as “sandwich”, “DASP”). The kit also includes peripheral reagents (eg, buffers, stabilizers, etc.).
[0087]
Thus, a test kit comprising the following can be manufactured to display the presence of a predetermined bacterial binding activity or the ability of a cell to have that activity:
(A) a predetermined amount of at least one labeled immunochemically reactive component obtained by direct or indirect binding of a polypeptide of the invention or a binding partner specific for a detectable label; ;
(B) other reagents; and
(C) Instructions for using the kit.
[0088]
The present invention provides antagonists or inhibitors including, but not limited to: peptide fragments, mimetics, nucleic acid molecules, ribozymes, polypeptides, small molecules, carbohydrate molecules, carbon sugars, oligosaccharides, or antibody. Agents that competitively block or inhibit pneumococci are also contemplated by the present invention. The present invention provides an agent comprising an inorganic compound, nucleic acid molecule, oligonucleotide, organic compound, peptide, peptidomimetic compound or protein that inhibits the polypeptide.
[0089]
The present invention provides a vaccine comprising a polypeptide having the amino acid sequence shown in any of SEQ ID NOs: 1, 3-7, 9-11, 22 and 23 and a pharmaceutically acceptable adjuvant or carrier. The polypeptide can comprise the N-terminal choline binding protein A truncated amino acid sequence shown in FIG. The present invention provides a vaccine comprising a polypeptide having an amino acid sequence comprising the conserved region shown in FIG. 2 and a pharmaceutically acceptable adjuvant or carrier. For example, conserved regions include, but are not limited to, the amino acid sequences 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; Not. The present invention provides a vaccine comprising an isolated nucleic acid encoding the polypeptide and a pharmaceutically acceptable adjuvant or carrier.
[0090]
Active immunity against gram positive bacteria (especially pneumococci) can be induced by immunization (vaccination) with an immunizing amount of the polypeptide or peptide derivative or fragment thereof and an adjuvant. In this case, the polypeptide or antigenic derivative or fragment thereof is the antigenic component of the vaccine. The polypeptides of the invention or derivatives or fragments thereof are produced as a mixture with an adjuvant to produce a vaccine. Preferably, the derivative or fragment of the polypeptide of the invention used as the antigenic component of a vaccine is an adhesion factor. More preferably, a derivative or fragment of a polypeptide of the invention used as an antigenic component of a vaccine is an antigen common to all or many strains of Gram positive bacteria or is common to related species of fungi It is an antigen. Most preferably, the antigenic component of the vaccine is an attachment factor that is a common antigen.
[0091]
A vector containing the nucleic acid-based vaccine of the present invention can be obtained by methods known in the art (eg, nucleic acid infection (transfection), electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), Use of a gene gun, or DNA vector transporter (see, eg, Wu et al., J. Biol. Chem. 267: 963-967 (1992); Wu & Wu, J. Biol. Chem. 263: 14621-14624 (1988); Hartmut et al., Canadian Patent Application 2012311 (filed 15 March 1990)).
[0092]
The vaccine may be administered by any parenteral route. These routes include, but are not limited to, intramuscular, intraperitoneal, intravenous, and the like. Since the desired result of vaccination is to elicit an immune response against the antigen and thus the pathogenic organism, preferably administration is direct (also by induction or selection of a viral vector to the target) or indirectly ( Desirable is lymphoid tissue (eg administration to lymph nodes or spleen). Because immune cells are continuously replicating, they are ideal targets for retroviral based nucleic acid vaccines (because retroviruses require replicating cells)
[0093]
Passive immunization is a method in which infection with a gram-positive bacterium (preferably streptococci, more preferably pneumococci) is suspected by administering an antiserum, polyclonal antibody or neutralizing monoclonal antibody against the polypeptide of the present invention to a subject patient. Given to animals. Because passive immunity does not confer long-term protection, passive immunity is a useful tool for treating bacterial infections in subjects who have not been vaccinated. Passive immunity is particularly important for the treatment of antibiotic-resistant gram-positive bacterial strains because there are no other treatments available. Preferably, the antibody administered for passive immunotherapy is an autologous antibody. For example, if the subject is a human, the antibody is preferably human-derived or “humanized” to minimize the likelihood of an immune response to the antibody. Administration of active or passive vaccines or attachment factors of the present invention can be used to protect target animals from infection with gram positive bacteria (preferably streptococci, more preferably pneumococci).
[0094]
The present invention provides a pharmaceutical composition comprising an amount of a polypeptide of the present invention and a pharmaceutically acceptable carrier or diluent.
For example, such pharmaceutical compositions that prevent pneumococcal adherence to mucosal surfaces can include antibodies to lectin domains and / or soluble excess lectin domain proteins. Either mechanism blocks the attachment and inhibits the first step of infection, thereby reducing colonization. This in turn reduces human-to-human transmission and prevents the progression of symptoms.
[0095]
The present invention provides a method of eliciting an immune response in a subject exposed to or infected with pneumococci. This method involves administering to a subject an amount of a pharmaceutical composition, thereby eliciting an immune response.
The present invention provides a method for preventing infection by pneumococci in a subject. The method includes administering to the subject an amount of a pharmaceutical composition effective to protect against pneumococcal adherence, thereby preventing pneumococcal infection.
[0096]
The present invention provides a method for preventing infection by pneumococci in a subject. This method involves administering to a subject an amount of a pharmaceutical composition comprising an antibody and a pharmaceutically acceptable carrier or diluent, thereby preventing pneumococcal infection.
The present invention provides a method of treating a subject exposed to or infected with pneumococci. The method includes administering to the subject a therapeutically effective amount of a vaccine of the invention, thereby treating the subject.
[0097]
The present invention provides a method of inhibiting host cell colony formation in a subject exposed to or infected with pneumococci. This method involves administering to a subject an amount of a pharmaceutical composition comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3-5, 7 or 9-11, thereby eliciting an immune response Including that. Therapeutic peptides that inhibit colony formation are transported by the respiratory mucosa. The pharmaceutical composition comprises a polypeptide consisting of the amino acid sequence shown in FIG.
[0098]
As used herein, a “pharmaceutical composition” refers to a therapeutically effective amount of a polypeptide product of the invention that confers a therapeutic effect or benefit that prevents, for example, pneumococcal colonization. By means of suitable diluents, preservatives, stabilizers, emulsifiers, adjuvants and / or carriers are meant. As used herein, “therapeutically effective amount” refers to an amount that provides a therapeutic effect for a condition and dosing regimen. Such compositions are liquid, or lyophilized, or otherwise dry, to prevent various buffer components (eg, Tris HCl, acetate, phosphate), pH and ionic strength, surface absorption Additives such as albumin or gelatin, detergents (eg Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (eg glycerol, polyethylene glycerol), antioxidants (eg ascorbic acid, sodium metabisulfite) Preservatives (eg thimerosal, benzyl alcohol, parabens), swelling agents or tonicity modifiers (eg lactose, mannitol), covalent attachment of polymers (eg polyethylene glycol) to proteins, complexation with metal ions, or polymer compounds (Eg, polylactic acid, polyglycolic acid, hydro Including particulated preparations or liposomes Le etc.), micro-emulsions, micelles, unilamellar or multilamellar vesicles, incorporation of the material into erythrocyte ghosts, or spheroplasts.
[0099]
Such compositions will affect the physiological state, solubility, stability, in vivo release rate, and in vivo clearance rate of the therapeutic agent. The choice of composition will depend on the physical and chemical properties of the protein having therapeutic activity. For example, products derived from membrane-bound forms of active substances require detergent-containing formulations. Controlled or sustained release compositions include lipophilic depot (eg fatty acids, waxes, oils) formulations. Further contemplated by the present invention is an activity conjugated to a polymer (eg, poloxamer or poloxamine) and a tissue specific receptor, ligand or antibody directed against an antigen or a tissue specific receptor ligand. A particulate composition coated with a substance. In certain embodiments, the compositions of the present invention comprise particles with protective coatings, protease inhibitors, or penetration enhancers for various routes of administration, including parenteral, pulmonary, nasal, and oral administration. Includes chemical forms.
[0100]
Further, as used herein, “pharmaceutically acceptable carrier” is well known to those skilled in the art and is 0.01-0.1M, preferably 0.05M phosphate buffer or 0.8% saline. Contains water. Furthermore, such pharmaceutically acceptable carriers will be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil (eg olive oil), injectable organic esters (eg ethyl oleate). Aqueous carriers include water, aqueous alcohol solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, those based on Ringer's dextrose), and the like. Preservatives and other additives such as antibacterial agents, antioxidants, reference agents, inert gases, etc.) are also included.
[0101]
“Adjuvant” refers to a compound or mixture that enhances the immune response to an antigen. Adjuvants can function as tissue depots that slowly release antigen or as lymphoid activators that non-specifically enhance immune responses (Hood et al., Immunology, Second Ed., Benjamin / Cummings : Menlo Park, California, p.384 (1984)). Initial challenge with antigen alone without adjuvants often fails to elicit a humoral or cellular immune response. Adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gel (eg, aluminum hydroxide), surfactant (eg, lysolecithin), pluronic polyol, polyanion, peptide, oil or hydrocarbon emulsion, keyhole limpet It includes, but is not limited to, hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (baccile Galmette-Guerin) and Corynebacterium parvum. Preferably the adjuvant is pharmaceutically acceptable.
[0102]
Controlled or sustained release compositions include formulations as lipophilic depots (eg fatty acids, waxes, oils). Also included in the present invention is a particulate composition coated with a polymer (eg, poloxamer or poloxamine) and a compound, which is conjugated to an antibody directed against a tissue specific receptor, ligand or antigen. Or conjugated with a ligand for a tissue-specific receptor. Other embodiments of the composition of the present invention include particulate forms with protective coatings, protease inhibitors or penetration enhancers for various administration routes (including parenteral, nasal and oral routes).
[0103]
When administered, compounds are often rapidly cleared from mucosal surfaces or the circulatory system, and thus pharmacological activity is relatively short. As a result, in order to maintain a therapeutic effect, it is necessary to administer a relatively large dose of the bioactive substance many times. Modification of the compound by covalent attachment of a water-soluble polymer (eg polyethylene glycol, copolymers of polyethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline) is more effective after intravenous injection than the corresponding unmodified compound. It is known to exhibit a longer blood half-life. (Abuchowski et al., (1981); Newmark et al., (1982); Katre et al., (1987)). Such modifications also increase the solubility of the compound in aqueous solution, prevent aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity can be achieved by administration of the polymeric compound adduct with fewer doses or less doses than with the unmodified compound.
[0104]
Dosage: A sufficient amount includes from about 1 μg / kg to about 1000 mg / kg, but is not limited to this amount. This amount will be 10 mg / kg. A composition in pharmaceutically acceptable form contains a pharmaceutically acceptable carrier. As described above, the present invention includes pharmaceutical compositions comprising vectors, vaccines, polypeptides, nucleic acids and antibodies, anti-antibodies, and agents that compete with pneumococci for pathogenic activity (eg, attachment to host cells). A therapeutic composition is provided.
[0105]
The manufacture of therapeutic compositions containing active ingredients is well known in the art. Typically, such compositions can be prepared so that they can be injected as an aerosol of the polypeptide administered to the nasopharynx, or as either a liquid solution or suspension, but the solution, suspension prior to injection. Solid preparations suitable for suspensions and liquids can also be produced. The preparation may be emulsified. The active ingredient for treatment is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which increase the effectiveness of the active ingredient.
[0106]
The active ingredient can be formulated into the therapeutic composition as neutral pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and inorganic acids (eg, hydrochloric acid, phosphoric acid) or organic acids (eg, acetic acid, oxalic acid, tartaric acid, mandelic acid) Etc.). Salts formed from free carboxyl groups are also from, for example, inorganic bases such as sodium hydroxide, potassium, ammonium, calcium, or iron, and from organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine. Can be guided.
[0107]
When at least about 75% by weight of the protein, DNA, or vector of the composition is “A” (“A” is just one protein, DNA molecule, vector, etc.) (depending on the species classification to which A and B belong) ), Compositions containing “A” are substantially free of “B” (where “B” is one or more contaminating proteins, DNA molecules, vectors, etc.). Preferably "A" comprises at least about 90% by weight of the A + B material in the composition, most preferably at least about 99% by weight.
[0108]
As used herein, the phrase “therapeutically effective amount” refers to clinically in at least about 15%, preferably up to 50%, more preferably up to at least 90%, most preferably in host activity, function and response. Prevent significant lack. Alternatively, a therapeutically effective amount is sufficient to provide a significant improvement in the host's clinical condition. In the present invention, the lack of host response is manifested by persistent or spreading bacterial infections. Significant improvements in the host's clinical status include a reduction in bacterial load, removal of bacteria from colonized host cells, a reduction in fever or inflammation associated with infection, or a decrease in symptoms associated with infection. .
[0109]
In accordance with the present invention, the components of the therapeutic composition of the present invention can be introduced parenterally, transmucosally (eg, oral, nasal, pulmonary or enteral) or transdermally. Preferably, administration includes but is not limited to parenteral, such as intravenous injection, intraarterial, intramuscular endothelium, subcutaneous, intraperitoneal, intraventricular, and intracranial. Oral or pulmonary administration is preferred to activate mucosal immunity. Because pneumococci generally colonize the nasopharynx and lung mucosa, mucosal immunity is a particularly effective protective treatment. As used in connection with the therapeutic compositions of the present invention, the term “unit dose” refers to a physically separate unit as a single human dose, each unit producing the desired therapeutic effect. Contains a predetermined amount of active substance calculated in conjunction with the required diluent (ie carrier or excipient).
[0110]
In another embodiment, the active compounds are delivered as excipients, in particular liposomes (see literature: Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of infectious Diesease and Cancer, Lopez-Berestein & Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., Pp.317-327; see generally )
[0111]
In yet another embodiment, the therapeutic compound is delivered in a controlled release system. For example, the polypeptide can be administered using intravenous infusions, implanted osmotic pumps, transdermal patches, liposomes, or other modes of administration. In some embodiments, pumps can be used (see the following references: Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1978); Buchwald et al., Surgery 88 : 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (see for example: Medical Applications of Controlled Release, Langer & Wise, eds., CRC Press, Boca Raton, Florida (1974); Controlled Drug Bioavailability , Drug Product Design and Performance, Smolen & Ball, eds., Wiley, New York (1984); Ranger & Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25: 351 (1989); Howard et al., Neurosurg. 71: 105 (1989)).
[0112]
In yet another embodiment, the controlled release system is placed in the vicinity of the therapeutic target (eg, the brain) and thus only requires a portion of the systemic dose (see, eg, Goodson, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)). Preferably, the controlled release device is introduced at a site or in the vicinity of the tumor where the subject's immune activation is inappropriate. Other control systems have been reviewed by Langer (Science 249: 1527-1533 (1990)).
[0113]
The subject for whom the administration of the active ingredient described above is an effective treatment for bacterial infection is a human, but the subject may be any animal. Thus, as will be readily appreciated by those skilled in the art, the methods and pharmaceutical compositions of the invention are administered for veterinary use to any animal, particularly mammals including but not limited to: Suitable for: livestock animals (eg cats or dogs, but not limited to), livestock animals (eg but not limited to cattle, horses, goats, sheep and pigs), wild animals (wild or zoos) Research animals (eg, mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc.).
[0114]
The therapeutic methods and compositions of the present invention provide a therapeutically effective amount of the active ingredient. The therapeutically effective dose is based on the characteristics of the patient (age, weight, sex, condition, complications, other diseases, etc.) as known to those skilled in the art. Can be easily determined. Furthermore, while conducting routine examinations, more specific information can be obtained regarding the appropriate dosage level for treatment of various symptoms in various patients, and the person skilled in the art can determine the content of the treatment, age Appropriate dosages can be ascertained taking into account the patient's general condition.
[0115]
In general, for intravenous injection or infusion, the dosage will be less than intraperitoneal, intramuscular, or other routes of administration. The dosage regimen will vary according to the circulation half-life and the dosage form used. The composition will be administered in a therapeutically effective amount in a manner compatible with the dosage form. The exact amount of active ingredient required for administration will be unique to each person according to the judgment of the clinician. However, suitable dosages are about 0.1 to 20 mg, preferably about 0.5 to about 10 mg, more preferably 1 to several mg of active ingredient / kg body weight / person / day, depending on the route of administration. Appropriate schedules for initiating and boosting dosages will also vary, but are typically followed by repeated dosing at 1 hour or more intervals, or other dosings following the starting dosing . Alternatively, continuous intravenous infusion sufficient to maintain 10 nanomolar to 10 micromolar concentrations in the blood is also contemplated.
[0116]
Administration with other compounds: for the treatment of bacterial infections, in combination with one or more pharmaceutical compositions used in the treatment of bacterial infections, including but not limited to the following: Active ingredients can be administered: (1) antibiotics; (2) soluble carbohydrates that inhibit bacterial adhesion; (3) other small molecules that inhibit bacterial adhesion; (4) bacterial metabolism, transport, A substance that suppresses transformation; (5) a bacterial lysis promoting substance; or (6) a vaccine against an antibacterial antibody or other bacterial antigen. Other possible active ingredients include anti-inflammatory agents such as steroids and non-steroidal anti-inflammatory agents. Administration can be simultaneous (eg, administration of a mixture of the active ingredient and antibiotics of the invention) or sequential.
[0117]
Thus, in certain embodiments, the therapeutic composition can further comprise an effective amount of the active ingredient and one or more of the following active ingredients: antibiotics, steroids, and the like. A typical formulation is shown below:
Intravenous formulation I:
Ingredient mg / ml
Cefotaxime 250.0
Polypeptide 10.0
Dextrose USP 45.0
Sodium Bisulfite USP 3.2
Editate disodium USP 0.1
Adjust by adding an appropriate amount of water for injection 1.0ml
[0118]
Intravenous formulation II
Ingredient mg / ml
Ampicillin 250.0
Polypeptide 10.0
Sodium Bisulfite USP 3.2
Disodium Edit USP 0.1
Adjust by adding an appropriate amount of water for injection 1.0ml
[0119]
Intravenous formulation III
Ingredient mg / ml
Gentamicin (as sulfate) 40.0
Polypeptide 10.0
Sodium Bisulfite USP 3.2
Disodium Edit USP 0.1
Adjust by adding an appropriate amount of water for injection 1.0ml
[0120]
Intravenous formulation IV
Ingredient mg / ml
Polypeptide 10.0
Dextrose USP 45.0
Sodium Bisulfite USP 3.2
Editate disodium USP 0.1
Adjust by adding an appropriate amount of water for injection 1.0ml
[0121]
Intravenous formulation V
Ingredient mg / ml
Polypeptide antagonist 5.0
Sodium Bisulfite USP 3.2
Disodium Edit USP 0.1
Adjust by adding an appropriate amount of water for injection 1.0ml
[0122]
Thus, in certain instances where it is desired to reduce or suppress infection caused by bacterial-mediated binding to a bacterial host cell, or where an antibody against the bacteria or a ligand thereof or an antibody against the ligand is desired, the polypeptide comprises: Introduced to inhibit the interaction between host cells and bacteria.
[0123]
Further contemplated herein is pulmonary delivery of a polypeptide of the invention or derivative thereof having lectin activity as an adhesion factor inhibitor. Adhesion factor inhibitors (or derivatives thereof) are transported into the mammalian lung where they interfere with the binding of bacteria (ie streptococci, preferably pneumococci) to host cells. Other reports on the preparation of proteins for pulmonary delivery are found in the art (Adjei et al., Pharmaceutical Research, 7: 565-569 (1990); Adjei et al., International Journal of Pharmaceutics, 63: 135 -144 (1990) (leuprolide acetate); Braquet et al., J. Cardiovascular Pharmacology, 13 (suppl. 5): 143-146 (1989) (endothelin-1); Hubbard et al., Annals Int. Med Vol. III, pp. 206-212 (1989) (α1-antitrypsin); Smith et al., J. Clin. Invest. 84: 1145-1146 (1989) (α1-proteinase); Oswein et al., "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (1990) (recombinant human growth factor); Debs et al., J. Immunol. 140: 3482-3488 (1988) (interferon) -Γ and tumor necrosis factor α); Platz et al., US Pat. No. 5,284,656 (granulocyte colony stimulating factor)). Methods and compositions for pulmonary delivery of drugs are described in US Pat. No. 5,451,569 (September 19, 1995, Wong et al.).
[0124]
All such devices require the use of a formulation suitable for dispersion of the adhesion factor inhibitor (or derivative thereof). Typically, each formulation is specific to the type of device used and requires the use of a suitable propellant other than the usual diluents, adjuvants, and / or carriers useful for therapy. Furthermore, liposomes, microcapsules or microspheres, inclusion complexes or other types of carriers are also contemplated. Chemically modified adhesion factor inhibitors can also be produced in various dosage forms depending on the type of chemical modification or the type of equipment used.
[0125]
Formulations suitable for use with nebulizers (either jet or ultrasonic) typically include an adhesion factor inhibitor (and derivatives thereof). The adhesion factor is dissolved in water at a concentration of about 0.1 to 25 mg of biologically active adhesion factor inhibitor per ml of solution. The formulation can also include a buffer and a simple sugar (eg, for adhesion factor inhibitor stabilization and regulation of osmotic pressure). The nebulizer formulation can also include a surfactant to reduce or prevent face-induced aggregation of the adhesion factor inhibitor caused by atomization of the solution as it forms the aerosol.
[0126]
Formulations using dose metered inhalation devices generally comprise a finely divided powder comprising an adhesion factor inhibitor (or a derivative thereof) suspended in a propellant together with a surfactant. The propellant can be any common material used for this purpose. For example, chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon or hydrocarbon (including trichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoromethane) or combinations thereof. Suitable surfactants are sorbitan trioleate and soy lecithin. Oleic acid is also useful as a surfactant.
[0127]
Liquid aerosol formulations include an adhesion factor inhibitor and a dispersant in a pharmaceutically acceptable diluent. The dry powder aerosol formulation of the present invention comprises a finely divided solid of an adhesion factor inhibitor and a dispersant. The formulation must be nebulized using either a liquid or dry powder aerosol formulation. That is, the formulation must be subdivided into liquid or solid particles to ensure that the nebulized dose actually reaches the nasal cavity or lung mucosa. The term “aerosol particles” is used herein to refer to liquid or solid particles that are suitable for nasal or pulmonary administration (ie reach the mucosa). Other considerations are also important, such as construction of the delivery device, additional ingredients of the formulation, and particle properties. The characteristics of pulmonary administration of drugs are well known in the art, but the formulation process, nebulization means and delivery device construction require very routine experimentation by those skilled in the art. In certain embodiments, the average dynamic diameter of the substance will be 5 micrometers or less to ensure that the drug particles reach the alveoli (LL Wearley, Crit. Rev. in Ther. Drug Carrier Systems 8: 333 (1991)).
[0128]
Aerosol delivery systems, such as pressurized dose metered inhalers and dry powder inhalers, are disclosed in the literature (SP Newman, Aerosols and the Lung, SW Clarke & D. Davia, eds., Pp.197-22). Can be used.
In yet another embodiment, as discussed in detail below, the aerosol formulations of the present invention comprise other therapeutically or pharmacologically active ingredients (eg, antibiotics, steroids, non-adjuvants) in addition to adhesion factor inhibitors. Steroidal anti-inflammatory agents and the like (but not limited to).
[0129]
Liquid aerosol formulations: The present invention provides aerosol formulations and dosage forms for use in the treatment of subjects suffering from bacterial infections (eg streptococci, especially pneumococci). In general, such dosage forms comprise an adhesion factor inhibitor in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include, but are not limited to, sterile water, saline, buffered saline, dextrose solution, and the like. In a particular embodiment, the diluent that can be used in the present invention or the pharmaceutical formulation of the present invention is phosphate buffered saline or buffered saline solution (generally pH is 7.0 to 8.0), or water.
[0130]
The liquid aerosol formulation of the present invention can include pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers, surfactants and excipients as optional ingredients. The formulation can include a carrier. A carrier is a macromolecule that can be dissolved in the circulatory system and is physiologically acceptable, where physiologically acceptable means that one of skill in the art allows the patient to inject the carrier as part of a treatment plan. To do. Preferably, the carrier is relatively stable in the circulatory system with an acceptable blood half-life for clearance. Such macromolecules include but are not limited to soybean lecithin, oleic acid and sorbitan trioleate. Sorbitan trioleate is preferred.
[0131]
The formulations of the present invention can also include other agents useful for maintaining pH, stabilizing the solution, or adjusting the osmotic pressure. For example, such agents include, but are not limited to, salts (eg, sodium chloride, potassium chloride), carbohydrates (eg, glucose, galactose or mannose) and the like.
The present invention further contemplates liquid aerosol formulations comprising an adhesion factor inhibitor and another therapeutically effective agent (eg, antibiotics, steroids, non-steroidal anti-inflammatory agents, etc.).
[0132]
Aerosol dry powder formulation: The aerosol formulation is also intended to be manufactured as a dry powder formulation comprising a finely divided powder form of an adhesion factor inhibitor and a dispersant.
Formulations dispensed from powder inhalation devices include finely divided dry powders containing adhesion factor inhibitors (or derivatives), and in addition, swelling agents such as lactose, sorbitol, sorbitol, etc. to facilitate dispersion of the powder from the device. Sugar or mannitol may be included, for example, in an amount of 50 to 90% of the formulation weight. Adhesion factor inhibitors (or derivatives) are particles with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, most advantageous for the most effective delivery to the terminal lung. Should be manufactured in a chemical form. In another embodiment, the dry powder formulation can comprise a finely divided dry powder, wherein the finely divided dry powder comprises an adhesion factor inhibitor, a dispersant, and also a swelling agent. Swelling agents useful in combination with the present formulations include agents such as lactose, sorbitol, sucrose, or mannitol. The amount of swelling agent is an amount that facilitates the distribution of powder from the device.
The present invention further contemplates dry powder formulations comprising an adhesion factor inhibitor and another therapeutically effective agent (eg, antibiotics, steroids, non-steroidal anti-inflammatory agents, etc.).
[0133]
What is intended to be used herein is an oral dosage form, which is generally described in the literature (Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA 18042), Chapter 89. : This document is incorporated herein by reference). Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Furthermore, liposomal vesicles or proteinaceous vesicles can also be used to formulate the present compositions (eg, proteinaceous microspheres are reported in US Pat. No. 4,925,637). Liposome encapsulation may be used, and liposomes can be derivatized with various polymers (eg, US Pat. No. 5,013,556). Possible therapeutic solid dosage forms are described in the literature (K. Marshall, in: Modern Pharmaceutics, GS Banker & CT Rhodes, eds., Chapter 10, 1979: this document is incorporated herein by reference) ) In general, the formulation comprises the component (or a chemically modified version thereof) and an inert component that protects the gastric environment and releases the biologically active substance into the intestine.
[0134]
Also specifically contemplated are oral dosage forms of the above derived components. The component can be chemically modified so that oral delivery of the derivative is effective. In general, an intended chemical modification is one that attaches at least one molecular moiety to the component molecule itself. In this case, the molecular moiety allows (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. What is further desired is an increase in the overall stability of the ingredients and an increase in systemic circulation time. Such molecular moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline (Abuchowski & Davis, 1981, "Soluble Polymer-Enzyme Adducts", In: Enzymes as Drugs, Hocenberg & Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383; Newmark, et al., J. Appl. Biochem. 4: 185-189 (1982)). Other polymers that can be used are poly-1,3-dioxolene and poly-1,3,6-thioxoken. Preferred for pharmaceutical use is a polyethylene glycol molecular moiety as indicated above.
[0135]
In the case of the component (derivative), the location of release will be the stomach, small intestine (duodenum, jejunum, ileum) or large intestine. Those skilled in the art can use formulations that do not dissolve in the stomach and are released elsewhere in the duodenum or small intestine. Preferably, detrimental effects of the gastric environment due to release will be avoided by protection of the protein (or derivative) or by releasing the biologically active substance prior to the gastric environment (eg, the small intestine).
[0136]
In order to ensure complete resistance to gastric acid, an impervious coating is necessary at least at pH 5.0. Examples of more common inert ingredients used as enteric skin are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings can be used as mixed films.
[0137]
The coating or coating mixture can also be used for tablets, but this is not intended for protection from the stomach. This includes sugar coatings or coatings that make tablets easier to swallow. Capsules can use a hard shell (eg, gelatin) for dry therapeutic (ie, powder) delivery and a soft gelatin shell for a liquid dosage form. Cashew shell material may be thick starch or other food paper. In the case of pills, lozenges, die-cut tablets or powder tablets, wet integration techniques can be used.
[0138]
The peptide therapeutic can be included in the granule or pellet form as fine multiparticulates with a particle size of about 1 mm. Formulation of the substance for capsule administration may be carried out as a powder, a light compression plug or a tablet. The therapeutic agent may be manufactured by compression.
Coloring and flavoring agents can be included. For example, the protein (or derivative) can be formulated (eg, liposomes or microsphere vesicles) and subsequently mixed into an edible product (eg, a cold beverage containing colorants and flavors).
[0139]
The therapeutic agent can be diluted or increased in volume with an inert substance. These diluents include carbohydrates, particularly mannitol, a-lactose, lactose anhydride, cellulose, sucrose, modified dextran and starch. Certain inorganic salts can also be used as fillers. These fillers include calcium triphosphate, magnesium carbonate and sodium chloride. Some commercial diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress, and Avicell.
[0140]
The disintegrant can be included in the solid dosage form of the therapeutic agent formulation. Substances used as disintegrants include starch (including the commercially available disintegrant Explotab based on starch). Sodium starch glycolate, amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite can all be used.
[0141]
Another form of disintegrant is an insoluble cation exchange resin. Powdered rubber can be used as a disintegrant and binder. In addition, these binders include powdered rubbers such as agar, karaya, and tragacanth gum. Alginic acid and its sodium salt are also useful as disintegrants. The binder can hold the therapeutic agent together to form a hard tablet. Binders include materials derived from natural products such as gum arabic, gum tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Both polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can be used in alcoholic solutions to granulate the therapeutic agent.
[0142]
An anti-friction agent can be included in the therapeutic agent formulation to prevent sticking during the formulation process. Lubricants can also be used as a layer between the therapeutic agent and the mold wall. These lubricants include stearic acid (including magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, carbowax 4000 and 6000 can also be used.
A slip agent may be added that improves the flow characteristics of the drug during formulation and promotes recombination during compression. Sliding agents include starch, talc, pyrogenic silica, and hydrated silicoaluminate.
[0143]
A surfactant can be added as a wetting agent to facilitate dissolution of the therapeutic agent in an aqueous environment. Surfactants include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. A cationic detergent may be used. This includes benzalkonium chloride or benzethonium chloride. Possible nonionic detergents that can be included in the formulation as surfactants are Lauromacrogol 400, polyoxy 40 stearate, polyoxyethylene hydrated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. These surfactants will be included in the protein or derivative preparation alone or as a mixture in various proportions.
Additives that can enhance the uptake of the polypeptide (or derivative) are, for example, the fatty acids oleic acid, linoleic acid, linolenic acid.
[0144]
Pulmonary delivery: Also contemplated herein is pulmonary delivery of the polypeptide (or derivative thereof). The polypeptide (or derivative) is transported to the mammalian lung upon inhalation and coats the mucosal surface of the alveoli. Other reports on this include: Adjei et al., Pharmaceutical Research, 7: 565-569 (1990); Adjei et al., International J. Pharmaceutics, 6: 135-144 (1990) (Lupro Liquetacetate); Braquet et al., J. Cardiovascular Pharmacology 13 (suppl. 5): 143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine, vol. III, pp. 206- 212 (1989) (a1-antitrypsin); Smith et al., J. Clin. Invest. 84: 1145-1146 (1989) (a1-proteinase); Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., J. Immunol. 140: 3482-3488 (1988) (interferon-γ and tumor necrosis factor α) and Platz et al., US Pat. No. 5,284,656 (granulocyte colony stimulating factor). Methods and compositions for pulmonary delivery of drugs for systemic effects are described in US Pat. No. 5,451,569 (September 19, 1995, Wong et al.).
[0145]
Contemplated for use in the practice of the present invention are a wide range of mechanical devices for pulmonary delivery of therapeutic products, including nebulizers, dose metered inhalers, and powder inhalers ( All of these are well known to those skilled in the art, but are not limited thereto.
Formulations suitable for use with a nebulizer (either jet or ultrasonic) typically contain biologically active protein in water at a concentration of about 0.1 to 25 mg / ml of solution. It will contain the dissolved polypeptide (or derivative). The formulation can also include buffers and simple sugars (eg, for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also include a surfactant to reduce or prevent surface-induced aggregation caused by atomization of the solution during aerosol formation.
[0146]
A formulation generally used with a dose metered inhalation device will include a finely divided powder containing the polypeptide (or derivative) suspended in the propellant with the aid of a surfactant. The propellant can be any of the common materials used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, And 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be useful as a surfactant.
[0147]
The formulation to be administered from a powder inhalation device will comprise a finely divided dry powder containing the polypeptide (or derivative). The formulation can also include a bulking agent such as lactose, sorbitol, sucrose or mannitol, to facilitate dispersion of the powder from the device, from about 50 to 90% by weight of the formulation. The protein (or derivative) is most advantageously produced in granular form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for the most effective delivery to the terminal lung. It should be.
[0148]
Nasal delivery:
Nasal or nasopharyngeal delivery of the polypeptide (or derivative) is also contemplated. Nasal delivery allows the polypeptide to be delivered directly to the upper airway mucosa after the therapeutic agent is administered to the nose, without the need to administer the product directly to the lungs. Nasal delivery formulations include formulations containing dextran or cyclodextran.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention, but should not be construed as limiting the scope of the invention.
[0149]
Example
Detailed description of the experiment
Example 1: Peptide truncated form of choline-binding protein A (CbpA)
A polypeptide containing a truncated N-terminal fragment of CbpA (serotype 4) was generated. PCR primers SJ533 and SJ537 were used to amplify full-length CbpA, and primers were designed based on the derived N-terminal amino acid sequence of CbpA. The 5 ′ forward primer SJ533 was 5′GGCGGATCCATGGGA (A, G) AA (C, T) GA (A, G) GG3 ′. This degenerate primer designed from the amino acid sequence of XENEG incorporated both the BamHI and NcoI restriction sites and the ATG start codon. The 3 ′ reverse primer SJ537 was 5 ′ GCCGTCGAACTTAGTTTACCCATTCACCATTGGC3 ′. This primer incorporated a cloning SalI restriction site and a natural stop codon from CbpA. This primer is based on both type 4 and R6x sequences.
[0150]
The PCR product was prepared from genomic DNA as a template by using primers SJ533 and SJ537 and amplifying for 30 cycles at an annealing temperature of 50 ° C. using a high fidelity enzyme (Boehringer Mannheim). The resulting PCR product was purified using the QIA Quick PCR Purification Kit (Qiagen, Inc), subsequently digested with BamHI and SalI restriction enzymes, and further digested with BamHI, XbaI and SmaI restriction enzymes (Qiagen, Inc).
[0151]
Polypeptide R2:
Using the naturally occurring PvuII site at the end of the second repeat region, ie, the C region shown in FIG. 1 (type 4 sequence nucleic acid 1228), a truncated CbpA gene containing only the 5 ′ portion of the gene Produced. To create truncated clones, full-length clones PMI580 (type 4) or PMI581 (R6x) were designed using PvuII and XbaI, the resulting fragment was ligated into the expression vector pQE30, and the appropriate host was transformed. . The protein was expressed and purified. In this case, the stop codon utilized by the expression vector is downstream of the insert, so the expressed protein is larger than the expected size of the insert due to the additional nucleic acid at the 5 ′ end of the cloning site. The amino acid sequence of polypeptide R2 is shown in SEQ ID NO: 1.
[0152]
Polypeptide R1:
A similar method was used to express only the first repeat region within the N-terminal region of CbpA (ie, the A region of polypeptide R1). A naturally occurring XmnI site between two amino repeats (type 4 sequence nucleic acid 856) was used. CbpA full length clone PMI580 was digested with XmnI and AatII. Vector pQE30 was digested with AatII and SmaI. Once again, the two size fragments were ligated to transform E. coli and clones were screened for inserts. One positive clone was selected and the recombinant protein was purified from the strain.
All polypeptides were expressed and purified with the Qia expression system (Qiagen) using E. coli and pQE30 vectors. The amino terminus of the His patch protein is detected by host and western analysis using both anti-histidine and protein specific antibodies.
[0153]
Purification of R1 and R2:
To induce and purify recombinant protein from E. coli, single colonies were picked from plated bacteria containing the recombinant plasmid and in 6 ml LB buffer containing 50 μg / ml kanamycin and 100 μg / ml ampicillin. Grow overnight at 37 ° C. This 6.0 ml culture was added to 1 L of LB containing antibiotics at the above concentrations. Incubate at 37 ° C for A₆₀₀Shake to = −0.400. 1M 1PTG was added to 1 L culture at a final concentration of 1 mM. The culture was then shaken at 37 ° C. for 3-4 hours. A 1 L culture was spun at 4000 rpm for 15 minutes in a model J-6B centrifuge. The supernatant was discarded and the pellet was stored at -20 ° C.
[0154]
1 L of pellet is added to 25 ml of 50 mM NaH₂PO_FourResuspended in 10 mM Tris, 6MGuCl, 300 mM NaCl, pH 8.0 (Buffer A). This mixture is centrifuged at room temperature for 30 minutes and output with a 50% Cuty Cycle for 30 seconds twice using a microchip in a VibraCell Sonicator (Sonics and Materials, Inc., Danbury, Conn.). And sonication at setting 7. The mixture was centrifuged with JA20 rotor at 10K for 5 minutes, the supernatant was removed and discarded. The supernatant was loaded onto a 10 ml Talon (Clonetech, Palo Alto, Calif.) Resin column coupled to a GradiFrac system (Pharmacia Biotech, Upsala, Sweden). The column was equilibrated with 100 ml of buffer A and further washed with 200 ml of this buffer. 100% 50 mM NaH as final target buffer₂PO_Four, 8M urea, 20 mM MES, pH 6.0 (buffer), and a volume-based pH gradient was run at a total volume of 100 ml. The protein eluted at about 30% buffer B elution peak was collected and combined.
[0155]
For refolding, dialysis was performed with 2 L PBS at room temperature for about 3 hours using a 14000 molecular weight cut-off dialysis tube. The sample was subsequently dialyzed overnight at 4 ° C. with 2 L of PBS. Additional buffer exchange was performed by adding PBS during spin down and re-rotation during protein concentration on a Centriprep-30 spin column. The protein concentration was determined by BCA protein assay and the purity was further visualized using Coomassie stained 4-20% SDS-PAGE gel (FIG. 3).
[0156]
Example 2: Lectin activity of polypeptides R1 and R2
LNnt is a carbohydrate analog of the receptor for pneumococci present on eukaryotic cells. CbpA-deficient Streptococcus pneumoniae mutants were unable to attach to either eukaryotic cells or immobilized sugars, indicating that CbpA is an attachment ligand. CbpA is a modular protein that is divided into two regions: an N-terminal functional domain and a C-terminal choline binding domain (FIG. 1). In order to determine whether the complete CbpA activity is localized at the unique N-terminus (modeled by R2) or its fragments (modeled by R1), the polypeptides R1 and R2 are biologically active. Was analyzed. It was investigated whether only the N-terminal domain (R2) has lectin-binding biological activity in the absence of the choline binding domain (CBD). This was investigated using full length CbpA and polypeptide R2 (a truncated form that lost the CBD region beyond the PvuII site of the proline-rich region).
[0157]
In the assay, tissue culture wells were coated with the following glycoconjugates known to be recognized by CbpA: LNnt-albumin, 3 ′ sialyl lactose-albumin and negative control albumin. The plate is subsequently blocked with albumin and washed, and either full-length CbpA polypeptide R2 or polypeptide R1 is added for 15 minutes (0.8 μg / ml) followed by 30 minutes of fluorescein-labeled R6 pneumococci without washing. In addition, the bacteria were washed and counted visually.
[0158]
Binding of R6 to carbohydrate in the absence of added peptide was a positive control, which was taken as 100% (Table 1). In three separate experiments, CbpA full length or polypeptide R2 competitively inhibited binding of pneumococci to surface coated LNnt. CbpA full length suppressed 71.64% and 63% of the control, and polypeptide R2 suppressed 65%, 53% and 74% of the control. The equivalent activities of CbpA and R2 indicate that the choline binding domain is not required for CbpA's LNnt lectin activity and that R2 is a candidate for LNnt lectin.
[0159]
In contrast to binding to LNnt, pneumococcal binding to 3 ′ sialyl lactose was not inhibited by R2 (79% and 101%) compared to full length CbpA (74% and 66%). This indicates that sialic acid recognition activity is lost when CBD is removed. In contrast, R1 appears to be active in sialic acid recognition and this property is shared with CbpA but is clearly masked by R2. This indicates that the folding of the polypeptide into the functional domain is affected by the composition and length of the polypeptide. A slight sequence change was found in other strains (see Figure 2). Given the high homology between R1 and R2, both R1 and R2 are required for lectin activity, or they are both lectins (± sialic acid) with slightly different properties It is further possible.
[0160]
Table 1. Inhibition of purified glycoconjugate binding to R6 pneumococci by CbpA soluble form

N = 3 LNnt experiments, 3 wells each
N = 2 sialyl lactose experiment, 3 wells each
[0161]
Lectin activity correlates with cell binding activity:
Human cells have surface molecules that contain carbohydrates (glycoproteins and glycolipids), and bacteria bind to these glycoconjugates with this carbohydrate, regardless of the very different protein or lipid framework. Thus, bacteria with polypeptides having lectin activity in vitro can adhere to human cell surfaces. A direct proportional relationship between in vitro lectin activity and cell binding activity is known for pneumococci. For example, LNnt competitively inhibits pneumococcal binding to TNF-activated A549 human lung cells and blocks the progression of pneumonia in vivo. To confirm that the CbpA truncated lectin activity reflects cell binding activity, CbpA and truncated were tested for inhibition of pneumococcal and lung cell binding (Table 2). Full-length CbpA and polypeptide R2 inhibited pneumococcal adherence to lung cells by 58% and 63%, respectively, relative to the control. Polypeptide R1 is not effective, suggesting that R2's LnNT binding activity is required, and explains the binding of Streptococcus pneumoniae to lung cells.
[0162]
Table 2

N = 2 2 or 3 well experiments each
[0163]
LNnt lectin activity depends on R2:
The N-terminal region of CbpA contains two repeats of about 110 amino acids each (see FIG. 1, regions A and C within polypeptide 2). To examine the relative contribution of these two domains to biological activity, R1 containing only domain A was compared to R2 and full-length CbpA. Polypeptide R1 did not inhibit LNnt attachment at all (91, 92 and 112% of the wild type) when tested in an adhesion assay. However, polypeptide R1 showed some inhibition with respect to binding to sialyl lactose (68 and 40% of controls). This indicates that polypeptide R2 is required for LNnt lectin activity and R2 is a candidate substance for LNnt lectin domain. In contrast, R1 appears to have activity in sialic acid recognition.
[0164]
Antibodies to the N-terminal domain of CbpA block cell binding:
If the N-terminal domain of CbpA binds to cells, interference with N-terminal domain activity will prevent or reverse the binding of cells or purified sugar conjugates to bacteria. One such interference mechanism is an antibody.
[0165]
Table 3. Inhibition of R6 Streptococcus pneumoniae binding to LNnT coated surface by anti-CbpA R2 antibody

Undiluted 5 μl rabbit antibody + 5 μl 2 × 10⁷  Pre-incubate R6x at room temperature for 30 minutes, then add to LNnt coated wells and attach assay. Two separate experiments are shown.
[0166]
Antisera raised against the recombinant N-terminal domain of CbpA (R2) were tested for the ability to block pneumococcal adherence to LNnt. Rabbit polyclonal anti-CbpA antiserum (5 μl) +5 μl 2 × 10⁷The labeled bacteria were incubated at room temperature for 30 minutes. This mixture was layered on immobilized LNnt for 30 minutes, followed by 3 washes with PBS to remove unbound bacteria. Bacteria bound to the plate are counted under a microscope and the results are shown as mean + standard deviation of 6 wells. The results shown in Table 3 demonstrate that antisera raised against R2 block pneumococcal binding to LNnt. FIG. 5 shows quantitative curves of pre-immunization and anti-CbpAR2 antibodies for the inhibition of pneumococcal R6x binding to LNnt model receptors. Over 70% pneumococcal adherence was blocked by anti-R2 at dilutions of 1: 100 and 1: 200. Furthermore, the loss of activity at a dilution of 1: 400 indicates the specificity of this action.
[0167]
The CbpA used to produce the antisera shown in Table 3 and FIG. 5 was serotype 4 CbpA. The pneumococcal R6x strain used in the adhesion inhibition assay is derived from serotype 2. The ability of antibodies to block the attachment of heterologous serotypes of bacteria indicates cross-protective activity that exceeds the active form. Such activity is highly desirable for effective vaccine immunization.
[0168]
Antibody activity against the native structure of the N-terminus of CbpA:
CbpA can be purified on its choline affinity column from its natural host, pneumococci, as described by Rosenow et al. Alternatively, a polyhistidine tag can be added by manipulation of the end of the gene so that the transcribed protein is extended by several histidine residues. These residues facilitate the nickel affinity matrix purification of the full length polypeptide, which, unlike the shorter truncated form, is advantageous for retaining the natural three-dimensional structure. In particular, CbpA purified by these biochemical means from E. coli or other host bacteria as well as pneumococci retains its natural three-dimensional structure. When used as an immunogen, CbpA, folded in nature, produces antibodies that are potentially different from those induced by immunization with a truncated shape that may be folded differently. Similarly, CbpA used as a therapeutic agent will have a three-dimensional structure that differs from a truncated shape with its improved ability to block adhesion. Given these, it would be advantageous to produce CbpA as a full-length protein that allows it to fold in its native three-dimensional structure, followed by biochemical cleavage of the C-terminus (CBD). . For example, treatment with hydroxylamine would cleave CbpA at choline-binding protein A serotype R6x and amino acid 475 of serotype 4 and separate the N-terminus and C-terminus. As a result, the N-terminal fragment is suitable as a therapeutic or immunogen.
[0169]
Alternatively, natural CbpA can be used as an immunogen, resulting in antisera against active structures. The biologically active anti-N-terminal antibody in this mixture can be concentrated by removing the antibody against BD by absorption. Such an antibody can contain 200 μl of serum at 1 × 10⁸Of CbpA-deficient bacteria and incubated at room temperature for 1 hour. Other choline binding proteins of this mutant were removed by absorption with anti-CBD antibody and removed from the antiserum by centrifugation to remove the bacteria.
To demonstrate the biological activity of the absorbed anti-CbpA antibody, the ability of the absorbed antiserum to block pneumococcal adherence to the LNnt model receptor was examined. R6x pneumococci were incubated with 1: 600 dilution of antiserum and added to wells coated with LNnt albumin.
[0170]
Table 4 Absorbed anti-CbpA antiserum inhibits adhesion

[0171]
These results indicate that the antibody against the N-terminal domain of Cbp / A with its native structure potently blocks attachment. This activity is much higher than the truncated activity of FIG. 5 (the latter showed no activity at a dilution of 1: 600). This activity of the absorbed anti-CbpA antiserum was also clearly shown in the titration experiment of FIG. Baseline adherence to Streptococcus pneumoniae type 4 LNnt coated wells is indicated by a triangle symbol. Pre-incubation with various dilutions of pneumococci with unabsorbed (square symbol) or absorbed (diamond symbol) antisera has been shown to reduce the resulting adhesion. The fact that both antisera show similar reduction in adhesion indicates that most of the blocking activity of the antibody against CbpA is at the N-terminus (ie, removal of the antibody against the choline binding domain by absorption is biological activity Does not decrease).
[0172]
Example 3: Passive protection with anti-R2 antiserum:
Production of rabbit immune serum:
Rabbit immune sera against polypeptides R2 (CbpA truncated) and CbpA were generated in Covance (Denver, PA). After collecting preimmune sera, New Zealand white rabbits were immunized with 250 μg R2 (preparation 483: 58 above, containing both amino-terminal repeats) in Freund's complete adjuvant. On day 21, rabbits were boosted with 125 μg R2 in incomplete Freund's adjuvant and blood was collected on day 31. A second rabbit was similarly immunized with purified CbpA.
[0173]
Mouse passive defense:
Passive immunization was performed by administering 100 μl of 1: 2 diluted rabbit anti-R2 serum or preimmune serum in sterile PBS (preimmune and day 31 immune serum) to the peritoneal cavity of C3H / HeJ mice (5 / group). . One hour after serum administration, mice were challenged with 1600 CFU of Streptococcus pneumoniae serotype 6B (SP317 strain). Mice were monitored for survival for 14 days. Eighty percent of mice immunized with rabbit immune serum raised against polypeptide R2 survived the challenge (FIG. 4). All mice immunized with preimmune rabbit serum died by day 7.
This result indicates that an antibody specific for CbpA has a protective effect against systemic pneumococcal infection. This result further indicates that the choline binding region is not required for protection. This is because, for protection, an antibody specific for the truncated protein polypeptide R2 (which lacks a conserved choline binding repeat) is sufficient. Furthermore, sera induced against CbpA serotype 4 showed protection against attack by serotype 6B.
[0174]
Example 4: Active protection with anti-R1 antiserum:
CbpH truncated protein R1 (15 μg in 50 μl PBS and 50 μl Freund's complete adjuvant) was administered to the abdominal cavity of C3H / HeJ mice (10 mice / group) for passive immunization. A group of 10 sham immunized mice received PBS and adjuvant. The second immunization was performed 4 weeks later with 15 μg of protein administered intraperitoneally with Freund's incomplete adjuvant (the Sham immunized group received PBS and Freund's incomplete adjuvant (IFA)). Blood was collected at 3, 6, and 9 weeks for analysis of the immune response (retroorbital bleed). Serum collected from 10 CbpA immunized mice at week 9 had an anti-CbpA truncated ELISA endpoint titer of 4096000. No antibodies were detected in sera obtained from sham immunized mice. Mice were challenged at 10 weeks with 560 CFU of Streptococcus pneumoniae serotype 6B (SPSJ2p strain, provided by P. Flynn (St. Jude Children's Research Hospital, Memphis, TN)). Mice were monitored for survival for 14 days. 80% of mice immunized with CbpA truncated protein R1 survived challenge. All Siamese immunized mice died by day 8 (Figure 7).
[0175]
This result indicates that the recombinant fragment of CbpA induces the production of specific antibodies that can protect against systemic pneumococcal infection and lethality. Furthermore, this result indicates that the choline binding region is unnecessary for protection because the immunogen is the truncated protein R1. This result further suggests that a single amino terminal repeat is sufficient to elicit a protective response. Cross-protection is also evident as recombinant pneumococcal protein was made based on the serotype 4 DNA sequence and protection was observed following challenge with the serotype 6B isolate.
[0176]
Example 5: Prevention of nasopharyngeal colonization in cub rats:
The N-terminal domain of CbpA competitively suppressed pneumococcal adherence in vitro. To elucidate the therapeutic utility of peptides with this activity, larval rats were administered truncated peptides and subsequently challenged with pneumococci to examine nasopharyngeal colonization.
Rats were administered nasally with 10 μl PBS containing 0.8 μg polypeptide R2 or R1, or 10 μl PBS without protein. After 15 minutes, type 3 Streptococcus pneumoniae (SIII strain) (1 × 10^Five10 μl) containing cfer was introduced nasally. To determine the ability of the polypeptide to competitively inhibit pneumococcal adherence and colonization, nasal washes were performed at 72 hours and the number of recovered pneumococci quantified for each of four animals per group. Rats given SIII only showed 2200, 6500, 6900 and 8700 (average 6075) colonies per 10 μl. Animals treated with the truncated R2 showed the greatest reduction (3600, 3500, 2500, 2100), with an average of 2925 bacteria per 10 μl (48% of control). Animals treated with the truncated R1 also showed reduced colony formation (5000, 4800, 3500, 1600) with an average of 3725 (61% of control).
This experiment showed that administration of the peptides of the present invention to animals in an animal treatment experimental design can protect the animals from subsequent pneumococcal challenge.
[0177]
Discussion:
As these experiments show, polypeptide R2 is: 1) elicits protective antibodies when administered as a vaccine antigen and is a preferred composition as a vaccine formulation; 2) transports to the airway and / or nasopharyngeal receptor as a peptide When used, it is a preferred composition as a prophylactic and therapeutic agent for competitively preventing pneumococcal adherence and for colonization and invasive symptoms. Furthermore, the CbpA truncated form functions as a lectin without CBD. Two carbohydrates are recognized: LNnt by the peptide containing both N-terminal repeats (A and C) in FIG. 1 and also only the most N-terminal N-terminal repeat (A) Sialic acid is recognized by the peptide. Truncated forms containing N-terminal repeat polypeptides R1 and R2 show lectin activity in cell culture assays as well.
[0178]
Important properties of polypeptide R2 activity include: 1) Complete correlation of biological activity of polypeptide R2 and full-length CbpA for recognition of purified glycoconjugate receptor analogs, lung cells and animal models. Correlations are also apparent for antibodies against them. 2) Cross protection between bacteria from in vitro assays with type 4 derived bacteria and other serotypes (eg 6B and 2). This is important for useful vaccines, prophylactics and therapeutics.
[0179]
Although the present invention has been described in detail and further described in detail with reference to various specific materials, methods and examples, the present invention is directed to specific materials, combinations of materials, and methods selected for that purpose. It will be understood that this is not a limitation. As will be appreciated by those skilled in the art, many variations are implied. Similarly, any references cited herein with respect to the disclosure of the present invention are incorporated herein by reference.
[0180]
[Brief description of the drawings]
FIG. 1 shows choline-binding protein A (CbpA), recombinant truncated R1 (from about amino acid 16 to amino acid 321 at the N-terminus of CbpA shown in FIG. 2), and R2 (N-terminal of CbpA shown in FIG. 2). The terminal amino acids 16 to 444) are schematically shown. Domain A is from about amino acid 153 to amino acid 321 at the N-terminus of the CbpA amino acid sequence shown in FIG. Domain B is from about amino acid 270 to amino acid 326 at the N-terminus of the CbpA amino acid sequence shown in FIG. C is from about amino acid 327 to amino acid 433 at the N-terminus of the CbpA amino acid sequence shown in FIG.
FIGS. 2A-B are comparisons of homology among various serotypes of nucleic acid and amino acid sequences of the N-terminal region of CbpA.
FIG. 3 shows the expression and purification of recombinants R1 and R2.
FIG. 4 is the result of passive protection in mice. Immune sera against recombinant R2 protected mice from lethal attack by S. pneumoniae.
FIG. 5 shows titration of anti-R2 antibody against R6x attachment to LNnT-HSA coated plates.
FIG. 6 shows the titers of anti-CbpA antibody and absorbed anti-CbpA antibody measured for the activity of blocking pneumococcal adsorption to LNnT-HSA-coated plates.
FIG. 7 shows the results of active protection in mice. Immune sera against recombinant R1 protected mice from lethal attack by Streptococcus pneumoniae (560 cfu for serotype 6B).
[Sequence Listing]

Claims (10)

A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 3, pharmaceutical compositions comprising a polypeptide that does not bind to choline and a pharmaceutically acceptable carrier or diluent. Use of a pharmaceutical composition for the manufacture of a medicament for eliciting an immune response in a subject exposed to or infected with Streptococcus pneumoniae, wherein the pharmaceutical composition does not bind choline. comprises a polypeptide having the amino acid sequence of protein a truncated form, said polypeptide, characterized by comprising the amino acid sequence shown in SEQ ID NO: 1 or 3 used. It said medicament Use according to claim 2 is to induce a protective immune response. Use of a pharmaceutical composition for the manufacture of a medicament for preventing infection by Streptococcus pneumoniae in a subject, wherein said pharmaceutical composition does not bind choline to an N-terminal choline-binding protein A truncated amino acid sequence Use wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 or 3 . Use according to claim 4 , wherein the pharmaceutical composition is for delivery to the respiratory tract or nasopharynx.   A vaccine comprising a polypeptide and a pharmaceutically acceptable adjuvant or carrier, wherein the polypeptide consists of the amino acid sequence defined by SEQ ID NO: 1 or 3, and does not bind to choline.   A vaccine comprising an isolated nucleic acid sequence molecule encoding a polypeptide and a pharmaceutically acceptable adjuvant or carrier, wherein the polypeptide comprises the amino acid sequence defined by SEQ ID NO: 1 or 3, Vaccine characterized by not binding to. Use of a vaccine according to any one of claims 6 or 7 for the manufacture of a medicament for treating a subject exposed to or infected with S. pneumoniae.   Use of a pharmaceutical composition for the manufacture of a medicament for inducing an immune response in a subject exposed to or infected with S. pneumoniae, said pharmaceutical composition being defined by SEQ ID NO: 1 or 3. A use characterized in that it comprises a polypeptide that comprises an amino acid sequence that does not bind to choline.   Use of a pharmaceutical composition for the manufacture of a medicament for preventing infection by Streptococcus pneumoniae in a subject comprising the amino acid sequence defined by SEQ ID NO: 1 or 3 and binding to choline Use characterized in that it comprises a polypeptide that does not.

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