JP6449272B2 - タンパク質の血清半減期を増加する組成物及び方法 - Google Patents
タンパク質の血清半減期を増加する組成物及び方法 Download PDFInfo
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Description
(a)GSGFGINGVI(SEQ ID NO: 1)、GSGFGVNGVI(SEQ ID NO: 2)、GNVFTIAAMG(SEQ ID NO: 3)、GNVFTIAAMA(SEQID NO: 4)、GNVFTIDAMG(SEQ ID NO: 5)、GSVFSIDAMG(SEQ ID NO: 6)、GNVFGIDAVG(SEQ ID NO: 7)、GSIFSIKVMG(SEQ ID NO: 8)及びGSIFPLNDMG(SEQ ID NO: 9)から成る群から選ばれる1つのアミノ酸配列を含む相補性決定領域(CDR)1と、
(b)LIKSDGYTNYRESVKG(SEQID NO: 10)、LIKSDGYTNYRESVRG(SEQ ID NO: 11)、GITTGGSTNYADSVKG(SEQ ID NO: 12)、GMTNGGKTNYADSVKG(SEQ ID NO: 13)、AMTNAGSTNYADSVKG(SEQ ID NO: 14)、ATTTSGSSTNYADSVKG(SEQ ID NO: 15)、DITSGGSTDYSDSVKG(SEQ ID NO: 16)及びTITRGGTTNYADSVKG(SEQ ID NO: 17)から成る群から選ばれる1つのアミノ酸配列を含む相補性決定領域(CDR)2と、
(c)PGVP(SEQ ID NO: 18)、VTKWAARVGGSAEYE(SEQ ID NO: 19)、RSKLIATINNPYDY(SEQ ID NO: 20)、RSKLIARINNPYEY(SEQ ID NO: 21)、RPKQATLIRDDY(SEQ ID NO: 22)、DLGCSGAGSCPDY(SEQ ID NO: 23)及びDNRVGGSY(SEQID NO: 24)から成る群から選ばれる1つのアミノ酸配列を含む相補性決定領域(CDR)3と、から選ばれた配列の1種又は複数種を含むトランスフェリンと特異的に結合する単離抗体又はそのフラグメントに関する。
(1)トランスフェリンと特異的に結合する抗体又はそのフラグメント、前記標的タンパク質、及び選択的なリンカを含む融合タンパク質であって、前記抗体又はそのフラグメントは標的タンパク質のカルボキシル末端又はアミノ末端に融合され、且つ選択的なリンカが抗体と標的タンパク質のカルボキシル末端又はアミノ末端を分離する融合タンパク質を取得するステップと、
(2)融合タンパク質をトランスフェリンと結合することにより、単独な標的タンパク質に対して、標的タンパク質の半減期を増加するステップと、を含む。
(a)融合タンパク質遺伝子をコードする発現ベクターを取得するステップと、
(b)発現ベクターを宿主細胞に導入して組換え細胞を取得するステップと、
(c)組換え細胞を培養して、融合タンパク質の発現を許可するステップと、
(d)組換え細胞から前記融合タンパク質を取得するステップとを含む方法により取得される。
(1)一体的に接続されている、トランスフェリンと特異的に結合する抗体又はそのフラグメントをコードする第1のヌクレオチド配列、及び選択的なリンカをコードする第2のヌクレオチド配列を含む発現ベクターと、
(2)宿主細胞と、
(3)トランスフェリンと、を含む標的タンパク質の半減期を増加するためのシステムに関する。
(a)GSGFGINGVI(SEQ ID NO: 1)、GSGFGVNGVI(SEQ ID NO: 2)、GNVFTIAAMG(SEQ ID NO: 3)、GNVFTIAAMA(SEQID NO: 4)、GNVFTIDAMG(SEQ ID NO: 5)、GSVFSIDAMG(SEQ ID NO: 6)、GNVFGIDAVG(SEQ ID NO: 7)、GSIFSIKVMG(SEQ ID NO: 8)及びGSIFPLNDMG(SEQ ID NO: 9)から成る群から選ばれたアミノ酸配列を含む相補性決定領域(CDR)1と、
(b)LIKSDGYTNYRESVKG(SEQID NO: 10)、LIKSDGYTNYRESVRG(SEQ ID NO: 11)、GITTGGSTNYADSVKG(SEQ ID NO: 12)、GMTNGGKTNYADSVKG(SEQ ID NO: 13)、AMTNAGSTNYADSVKG(SEQ ID NO: 14)、ATTTSGSSTNYADSVKG(SEQ ID NO: 15)、DITSGGSTDYSDSVKG(SEQ ID NO: 16)及びTITRGGTTNYADSVKG(SEQ ID NO: 17)から成る群から選ばれたアミノ酸配列を含む相補性決定領域CDR2と、
(c)PGVP(SEQ ID NO: 18)、VTKWAARVGGSAEYE(SEQ ID NO: 19)、RSKLIATINNPYDY(SEQ ID NO: 20)、RSKLIARINNPYEY(SEQ ID NO: 21)、RPKQATLIRDDY(SEQ ID NO: 22)、DLGCSGAGSCPDY(SEQ ID NO: 23)及びDNRVGGSY(SEQID NO: 24)から選ばれたアミノ酸配列を含む相補性決定領域CDR3と、から成る群から選ばれた1種又は複数種の配列を含むトランスフェリンと特異的に結合する抗体又はそのフラグメントを提供する。
(a)トランスフェリンと特異的に結合する抗体又はそのフラグメント、前記標的タンパク質、及び選択的なリンカをコードする融合タンパク質を取得し、前記抗体又はそのフラグメントが標的タンパク質のカルボキシル末端又はアミノ末端に融合され、選択的なリンカが抗体と標的タンパク質のカルボキシル末端又はアミノ末端を分離するステップと、
(b)ステップ(a)の発現ベクターを細胞に導入して組換え細胞を取得するステップ、
(c)組換え細胞を成長させ、かつ融合タンパク質の発現を許可する条件、
(d)組換え細胞から前記融合タンパク質を取得するステップを含む。
(1)トランスフェリンと特異的に結合する抗体又はそのフラグメントをコードする第1ヌクレオチド配列、及び選択的な第2ヌクレオチド配列をコードするリンカヘッドを含み、第1と第2ヌクレオチド配列は操作可能に接続される発現ベクター、
(2)宿主細胞、及び
(3)トランスフェリンを含む標的タンパク質の半減期を増加するシステムに関する。
1.Sleep, D., J. Cameron, and L.R. Evans, Albumin as a versatile platform for drug half-life extension. Biochim Biophys Acta.
2.Kontermann, R.E., Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol. 22(6): p. 868-76.
3.Lee, L.S., et al., Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds. Bioconjug Chem, 1999. 10(6): p. 973-81.
4.Tuettenberg, J., et al., Pharmacokinetics, pharmacodynamics, safety and tolerability of APG101, a CD95-Fc fusion protein, in healthy volunteers and two glioma patients. Int Immunopharmacol. 13(1): p. 93-100.
5.Nolte, M.W., et al., Improved kinetics of rIX-FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs. J Thromb Haemost. 10(8): p. 1591-9.
6.Holt, L.J., et al., Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng Des Sel, 2008. 21(5): p. 283-8.
7.Walker, A., et al., Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon. Protein Eng Des Sel. 23(4): p. 271-8.
8.Stork, R., D. Muller, and R.E. Kontermann, A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G. Protein Eng Des Sel, 2007. 20(11): p. 569-76.
9.Matsubara, M., et al., Single dose GLP-1-Tf ameliorates myocardial ischemia/reperfusion injury. J Surg Res. 165(1): p. 38-45.
10.Keefe, D., et al., In vitro characterization of an acetylcholine receptor-transferrin fusion protein for the treatment of myasthenia gravis. Autoimmunity. 43(8): p. 628-39.
11.Arbabi Ghahroudi, M., et al., Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett, 1997. 414(3): p. 521-6.
12.Tanha, J., A. Muruganandam, and D. Stanimirovic, Phage display technology for identifying specific antigens on brain endothelial cells. Methods Mol Med, 2003. 89: p. 435-49.
13.Zhang, J., et al., A pentavalent single-domain antibody approach to tumor antigen discovery and the development of novel proteomics reagents. J Mol Biol, 2004. 341(1): p. 161-9.
14.Cortez-Retamozo, V., et al., Efficient cancer therapy with a nanobody-based conjugate. Cancer Res, 2004. 64(8): p. 2853-7.
15.Luo, F.R., et al., Correlation of pharmacokinetics with the antitumor activity of Cetuximab in nude mice bearing the GEO human colon carcinoma xenograft. Cancer Chemother Pharmacol, 2005. 56(5): p. 455-64.
Claims (29)
- トランスフェリンと、特異的に結合する、単離された単一ドメイン抗体(sdAb)又はその抗原結合性(antigen−binding)フラグメントであって、その単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメントは、それぞれ、以下のアミノ酸配列を有する相補性決定領域(CDR)1、CDR2およびCDR3を含む。
(a)SEQ ID NO:1、SEQ ID NO:10および、SEQ ID NO:18;
(b)SEQ ID NO:3、SEQ ID NO:12および、SEQ ID NO:19;
(c)SEQ ID NO:6、SEQ ID NO:14および、SEQ ID NO:21;
(d)SEQ ID NO:7、SEQ ID NO:15および、SEQ ID NO:22;または
(e)SEQ ID NO:9、SEQ ID NO:17および、SEQ ID NO:24。 - SEQ ID NO:1、SEQ ID NO:10、及びSEQ ID NO:18のアミノ酸配列を含む、請求項1に記載の単離された単一ドメイン抗体又はその抗原結合性フラグメント。
- SEQ ID NO:3、SEQ ID NO:12及びSEQ ID NO:19のアミノ酸配列を含む、請求項1に記載の単離された単一ドメイン抗体又はその抗原結合性フラグメント。
- SEQ ID NO:6、SEQ ID NO:14及びSEQ ID NO:21のアミノ酸配列を含む、請求項1に記載の単離された単一ドメイン抗体又はその抗原結合性フラグメント。
- SEQ ID NO:7、SEQ ID NO:15及びSEQ ID NO:22のアミノ酸配列を含む、請求項1に記載の単離された単一ドメイン抗体又はその抗原結合性フラグメント。
- SEQ ID NO:9、SEQ ID NO:17及びSEQ ID NO:24のアミノ酸配列を含む、請求項1に記載の単離された単一ドメイン抗体又はその抗原結合性フラグメント。
- 以下からなる群から選択される、アミノ酸配列を含む、請求項1乃至6のいずれか1項に記載の、単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメント。
A60219:QVQLVESGGGLVQAGGSLRLSCVASGSGFGINGVIWYRQAPGKQRELVALIKSDGYTNYRESVKGRFTISRDDAKNTVWLQMNALEPEDTGVYYCKTPGVPFGQGTQVTVSS(SEQ ID NO:26)、
A60401:QVKLEESGGGLVQAGGSLRLSCEASGNVFTIAAMGWFRQAPGKERELVAGITTGGSTNYADSVKGRFTISRDNAQNTMYLQMNSLRPEDTAAYSCNAVTKWAARVGGSAEYEYWGQGTQVTVSS(SEQ ID NO:28)、
A69449:QVQLVESGGGVVQAGGSLRLSCVASGSVFSIDAMGWYRQAPGNQRELVAAMTNAGSTNYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNARSKLIARINNPYEYWGQGTQVTVSS(SEQ ID NO:56)、
A69433:QVKLEESGGGLVQAGGSLRLSCVASGNVFGIDAVGWYRQAPGKQRELVAATTTSGSSTNYADSVKGRFTISRDIAKNTVYLQMDSLKPEDTAVYYCYARPKQATLIRDDYWGQGTQVTVSS(SEQ ID NO:58)、および
A69447:QVKLEESGGGSVQAGGSLRLSCTGSGSIFPLNDMGWYRQAPGKQRELVATITRGGTTNYADSVKGRFTISRDSNAKNTVYLQMNSLKVEDTAVYYCNMDNRVGGSYWGQGTQVTVSS(SEQ ID NO:62)。 - SEQ ID NO:26またはSEQ ID NO:28のアミノ酸配列を含む、請求項1または請求項7に記載の単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメント。
- トランスフェリンに対して、10−9M以下の解離定数(Kd)を有する、請求項1乃至8のいずれか一項に記載の、単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメント。
- 前記トランスフェリンが、ヒトトランスフェリン又はサルトランスフェリンである、請求項1乃至9のいずれか一項に記載の単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメント。
- 組換えにより生成される、請求項1乃至10のいずれか一項に記載の、単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメント。
- 請求項1−11のいずれか一項に記載の、単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメントをコードするcDNA又は合成DNAを含む核酸。
- 請求項12に記載の核酸を含む発現ベクター。
- 請求項13に記載の発現ベクターを含む、組換え宿主細胞。
- 単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメント、標的タンパク質、および所望により、リンカーを含む、融合タンパク質であって、
上記単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメントは、トランスフェリンと特異的に結合し、そして、標的タンパク質のカルボキシル末端又はアミノ末端に融合され、そして、
上記リンカーは、所望により、前記抗体と、前記標的タンパク質のカルボキシル末端又はアミノ末端を分離(separates)し、そして、
上記単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメントは、それぞれ、以下のアミノ酸配列を有する相補性決定領域(CDR)1、CDR2およびCDR3を含む、融合タンパク質。
(a)SEQ ID NO:1、SEQ ID NO:10および、SEQ ID NO:18;
(b)SEQ ID NO:3、SEQ ID NO:12および、SEQ ID NO:19;
(c)SEQ ID NO:6、SEQ ID NO:14および、SEQ ID NO:21;
(d)SEQ ID NO:7、SEQ ID NO:15および、SEQ ID NO:22;または
(e)SEQ ID NO:9、SEQ ID NO:17および、SEQ ID NO:24。 - 請求項15に記載の融合タンパク質であって、前記単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメントが、
A60219:QVQLVESGGGLVQAGGSLRLSCVASGSGFGINGVIWYRQAPGKQRELVALIKSDGYTNYRESVKGRFTISRDDAKNTVWLQMNALEPEDTGVYYCKTPGVPFGQGTQVTVSS(SEQ ID NO:26)、
A60401:QVKLEESGGGLVQAGGSLRLSCEASGNVFTIAAMGWFRQAPGKERELVAGITTGGSTNYADSVKGRFTISRDNAQNTMYLQMNSLRPEDTAAYSCNAVTKWAARVGGSAEYEYWGQGTQVTVSS(SEQ ID NO:28)、
A69449:QVQLVESGGGVVQAGGSLRLSCVASGSVFSIDAMGWYRQAPGNQRELVAAMTNAGSTNYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNARSKLIARINNPYEYWGQGTQVTVSS(SEQ ID NO:56)、
A69433:QVKLEESGGGLVQAGGSLRLSCVASGNVFGIDAVGWYRQAPGKQRELVAATTTSGSSTNYADSVKGRFTISRDIAKNTVYLQMDSLKPEDTAVYYCYARPKQATLIRDDYWGQGTQVTVSS(SEQ ID NO:58)及び
A69447:QVKLEESGGGSVQAGGSLRLSCTGSGSIFPLNDMGWYRQAPGKQRELVATITRGGTTNYADSVKGRFTISRDSNAKNTVYLQMNSLKVEDTAVYYCNMDNRVGGSYWGQGTQVTVSS(SEQ ID NO:62)
からなる群から選ばれたアミノ酸配列を含む、融合タンパク質。 - 単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメントが、SEQ ID NO:26またはSEQ ID NO:28のアミノ酸配列を含む、請求項15または請求項16に記載の融合タンパク質。
- 請求項15〜17のいずれか1項に記載の融合タンパク質をコードする、核酸配列を含む核酸。
- 請求項18に記載の核酸を含む発現ベクター。
- 請求項19に記載の発現ベクターを含む組換え宿主細胞。
- 標的タンパク質の半減期を増加するための方法であって、その方法が、
(1)融合タンパク質を製造すること。ここで、この融合タンパク質は、単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメント、標的タンパク質、および所望により、リンカーを含み、
上記単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメントは、トランスフェリンと特異的に結合し、そして、標的タンパク質のカルボキシル末端又はアミノ末端に融合され、そして、
上記リンカーは、所望により、前記単一ドメイン抗体と前記標的タンパク質のカルボキシル末端又はアミノ末端を分離し、そして、
(2)トランスフェリンに融合タンパク質を、体外において、暴露し、それにより、標的タンパク質単独と比較して、融合タンパク質における標的タンパク質の半減期を増加すること、
ここで、単一ドメイン抗体またはそのフラグメントが、それぞれ、以下のアミノ酸配列を有する相補性決定領域(CDR)1、CDR2およびCDR3を含む、方法。
(a)SEQ ID NO:1、SEQ ID NO:10および、SEQ ID NO:18;
(b)SEQ ID NO:3、SEQ ID NO:12および、SEQ ID NO:19;
(c)SEQ ID NO:6、SEQ ID NO:14および、SEQ ID NO:21;
(d)SEQ ID NO:7、SEQ ID NO:15および、SEQ ID NO:22;または
(e)SEQ ID NO:9、SEQ ID NO:17および、SEQ ID NO:24。 - 前記暴露ステップが、トランスフェリンを含む血清に、体外において、融合タンパク質を投与することを含む、請求項21に記載の方法。
- 前記単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメントが、
A60219:QVQLVESGGGLVQAGGSLRLSCVASGSGFGINGVIWYRQAPGKQRELVALIKSDGYTNYRESVKGRFTISRDDAKNTVWLQMNALEPEDTGVYYCKTPGVPFGQGTQVTVSS(SEQ ID NO:26)、
A60401:QVKLEESGGGLVQAGGSLRLSCEASGNVFTIAAMGWFRQAPGKERELVAGITTGGSTNYADSVKGRFTISRDNAQNTMYLQMNSLRPEDTAAYSCNAVTKWAARVGGSAEYEYWGQGTQVTVSS(SEQ ID NO:28)、
A69449:QVQLVESGGGVVQAGGSLRLSCVASGSVFSIDAMGWYRQAPGNQRELVAAMTNAGSTNYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNARSKLIARINNPYEYWGQGTQVTVSS(SEQ ID NO:56)、
A69433:QVKLEESGGGLVQAGGSLRLSCVASGNVFGIDAVGWYRQAPGKQRELVAATTTSGSSTNYADSVKGRFTISRDIAKNTVYLQMDSLKPEDTAVYYCYARPKQATLIRDDYWGQGTQVTVSS(SEQ ID NO:58)、又は
A69447:QVKLEESGGGSVQAGGSLRLSCTGSGSIFPLNDMGWYRQAPGKQRELVATITRGGTTNYADSVKGRFTISRDSNAKNTVYLQMNSLKVEDTAVYYCNMDNRVGGSYWGQGTQVTVSS(SEQ ID NO:62)
のアミノ酸配列を含む、請求項21または請求項22に記載の方法。 - 単離された単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメントが、SEQ ID NO:26またはSEQ ID NO:28のアミノ酸配列を含む、請求項21〜23のいずれか1項に記載の方法。
- 前記融合タンパク質が、
(a)融合タンパク質をコードする発現ベクターを取得するステップ;
(b)発現ベクターを細胞に導入して組換え細胞を取得するステップ;
(c)融合タンパク質の発現を許可する条件で、組換え細胞を生長するステップ;および
(d)組換え細胞又はその上澄み液から、融合タンパク質を取得するステップ、
を含む方法により生成される、請求項21乃至24のいずれか1項に記載の方法。 - 請求項15〜17のいずれか1項に記載の融合タンパク質の有効量を含む、組成物。
- トランスフェリンを更に含む、請求項26に記載の組成物。
- 標的タンパク質の半減期を増加する方法であって、その方法は、請求項26または請求項27に記載の組成物を、体外において、トランスフェリンに暴露することにより、融合タンパク質における標的タンパク質の半減期を増加する方法。
- 標的タンパク質の半減期を増加するためのシステムであって、そのシステムは、
(1)トランスフェリンと特異的に結合する、単一ドメイン抗体又はその抗原結合性(antigen−binding)フラグメントをコードする第1のヌクレオチド配列と、
所望により、リンカーをコードする第2のヌクレオチド配列とを含む発現ベクター;ここで、第1および、第2のヌクレオチド配列は、作動可能に連結しており(operably linked);
(2)宿主細胞;および
(3)トランスフェリン、を含み、
ここで、単一ドメイン抗体またはその抗原結合性(antigen−binding)フラグメントは、それぞれ、以下のアミノ酸配列を有する相補性決定領域(CDR)1、CDR2およびCDR3を含む。
(a)SEQ ID NO:1、SEQ ID NO:10および、SEQ ID NO:18;
(b)SEQ ID NO:3、SEQ ID NO:12および、SEQ ID NO:19;
(c)SEQ ID NO:6、SEQ ID NO:14および、SEQ ID NO:21;
(d)SEQ ID NO:7、SEQ ID NO:15および、SEQ ID NO:22;または
(e)SEQ ID NO:9、SEQ ID NO:17および、SEQ ID NO:24。
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