KR20100077052A - Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours - Google Patents
Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours Download PDFInfo
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- KR20100077052A KR20100077052A KR1020107013008A KR20107013008A KR20100077052A KR 20100077052 A KR20100077052 A KR 20100077052A KR 1020107013008 A KR1020107013008 A KR 1020107013008A KR 20107013008 A KR20107013008 A KR 20107013008A KR 20100077052 A KR20100077052 A KR 20100077052A
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Abstract
Description
본 발명은 방사성핵종에 결합하는 신규한 펩티드를 사용한 종양의 진단 및 치료를 위한 새로운 방법에 관한 것이다.The present invention relates to a novel method for diagnosis and treatment of tumors using novel peptides that bind to radionuclides.
종양은 새로운 혈관의 생성 (혈관신생) 없이 소정의 중량 보다 무거워질 수 없고, 미세관 밀도 및 종양 침투성 사이의 관련성이 다수의 종양에 대해 보고되어 있다 (Folkman (1995), Nature Med., 1, 27-31). 더욱이, 혈관신생은 대다수의 안과 질환과 관련되어 시력의 손실을 초래한다 (Lee et al., Surv. Ophthalmol. 43, 245-269 (1998); Friedlander, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9764-9769 (1996)). 혈관신생의 표지를 선택적으로 표적할 수 있는 분자는 종양 및 혈관 증식으로 특징되는 다른 질병, 예를 들면 당뇨병성 망막증 및 노화-관련 황반변성의 진단 및 치료를 위한 임상적 기회를 제공할 것이다. 혈관신생의 표지는 종양 혈관과 관련된 대다수의 공격적 고형 종양에 발현되고 따라서 정맥 내로 주사되는 특이한 결합체에 쉽게 접근될 것이다 (Pasqualini et al., (1997), Nature Biotechnol., 15, 542-546; Neri et al. (1997), Nature Biotechnol., 15, 1271-1275). 신생혈관구조의 표적 폐색은 종양 경색 및 붕괴를 초래할 수 있다 (O'Reilly et al. (1996), Nature Med., 2, 689-692; Huang et al. (1997), Science, 275, 547-550).Tumors cannot be heavier than a given weight without the creation of new blood vessels (angiogenesis), and the association between microtubule density and tumor permeability has been reported for a number of tumors (Folkman (1995), Nature Med., 1, 27-31). Moreover, angiogenesis is associated with the majority of eye diseases, leading to loss of vision (Lee et al., Surv. Ophthalmol. 43, 245-269 (1998); Friedlander, M. et al., Proc. Natl. Acad. Sci. USA 93, 9764-9769 (1996)). Molecules capable of selectively targeting markers of angiogenesis will provide clinical opportunities for diagnosis and treatment of tumors and other diseases characterized by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration. Markers of angiogenesis are expressed in the majority of aggressive solid tumors associated with tumor vessels and will therefore be readily accessible to specific conjugates that are injected intravenously (Pasqualini et al., (1997), Nature Biotechnol., 15, 542-546; Neri et al. (1997), Nature Biotechnol., 15, 1271-1275). Target occlusion of neovascular structures can lead to tumor infarction and disruption (O'Reilly et al. (1996), Nature Med., 2, 689-692; Huang et al. (1997), Science, 275, 547- 550).
피브로넥틴 분자로 별법적 스프라이싱에 의해 삽입되는, 생쥐, 쥐 및 인간에서 동일한 91개 아미노산 서열인 피브로넥틴의 ED-B 도메인은 신생혈관구조 주위에 특이적으로 축적되고 (Castellani et al. (1994), Int. J. Cancer 59, 612-618) 분자적 중재를 위한 표적이 될 수 있다. 실로, 항-ED-B 단쇄 Fv 항체 단편 (scFv)이 종양 가진 생쥐의 종양 혈관 주위에 선택적으로 축적되고, 항체 친화도가 표적 성능을 아마도 결정한다는 것을 형광 기술로 최근 밝혔다 (Neri et al. (1997), Nature Biotechnol., 15, 1271-1275; WO 97/45544).The ED-B domain of fibronectin, a 91 amino acid sequence identical in mice, rats and humans, inserted by alternative splicing into fibronectin molecules, accumulates specifically around the neovascular structure (Castellani et al. (1994), Int. J. Cancer 59, 612-618) may be a target for molecular intervention. Indeed, it was recently revealed by fluorescence techniques that anti-ED-B single chain Fv antibody fragment (scFv) selectively accumulates around tumor vessels in tumor-bearing mice, and that antibody affinity presumably determines target performance (Neri et al. (Neri et al. 1997), Nature Biotechnol., 15, 1271-1275; WO 97/45544).
추가로, 나노몰 이하의 해리 상수를 가진 피브로넥틴의 ED-B 도메인에 특이적으로 결합하는 항체 및 항체 단편 뿐만 아니라 그의 방사성동위원소 표지된 유도체는 WO 99/58570에 기재되어 있다. 고-친화도 인간 항체 단편 중의 하나인 L19로 불리는 125I로 표지된 항체 단편의 생체분포는 종양 가진 생쥐에서 이미 연구되었다 (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192-198). L19-항체를 포함하는 방사성동위원소 표지된 콘쥬게이트 및 혈관신생의 감지 및 치료를 위한 그의 용도는 WO 01/62800에 개시되어 있다. In addition, antibodies and antibody fragments that specifically bind to the ED-B domain of fibronectin with sub-nanomolar dissociation constants, as well as radioisotope-labeled derivatives thereof, are described in WO 99/58570. The biodistribution of an antibody fragment labeled with 125 I called L19, one of the high-affinity human antibody fragments, has already been studied in tumor-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192-198). Radioisotope labeled conjugates comprising L19-antibodies and their use for the detection and treatment of angiogenesis are disclosed in WO 01/62800.
피치아 파스토리스 (Pichia pastoris)의 피브로넥틴의 B-동종체의 ED-B 도메인에 결합하는 기능화된 단쇄 Fv 항체 단편의 재조합 제조는 이미 기술되어 있다 (Marty et al., Protein Expression and Purification 21, 156-164 (2001)). The recombinant preparation of functionalized single chain Fv antibody fragments that bind to the ED-B domain of the B- homogen of fibronectin from Pichia pastoris has already been described (Marty et al., Protein Expression and Purification 21, 156). -164 (2001)).
추가로, scFv 항체 단편과 99mTc와의 C-말단 시스테인 펩티드를 통한 방사성동위원소 표지는 문헌 [George et al., Proc. Natl. Acad. Sci. USA, Vol. 92 pp. 8358-8362, 1995, 및 Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868-872, 1996]에 기재되어 있다.In addition, radioisotope labeling via a C-terminal cysteine peptide with scFv antibody fragment and 99m Tc is described in George et al., Proc. Natl. Acad. Sci. USA, Vol. 92 pp. 8358-8362, 1995, and Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868-872, 1996].
그러나, 방사성핵종이 방사성의 약품에 특히 적절하기 때문에, 향상된 약동학 성질을 가지고, 방사성동위원소, 예를 들면 테크네튬 또는 레늄으로 쉽게 표지될 수 있는 항체 단편의 제공에 대한 임상적 필요성은 아직 존재한다.However, since radionuclides are particularly suitable for radiopharmaceuticals, there is still a clinical need for the provision of antibody fragments that have improved pharmacokinetic properties and can be easily labeled with radioisotopes such as technetium or rhenium.
그러므로, 향상된 약동학 성질, 특히 표적 특이성 및(또는) 생체내 안정도를 가지고, 테크네튬 또는 레늄 같은 방사성동위원소에 쉽게 결합할 수 있는 항체 단편을 제공하는 것이 본 발명의 목적이다.Therefore, it is an object of the present invention to provide an antibody fragment that has improved pharmacokinetic properties, in particular target specificity and/or stability in vivo, and can readily bind to radioisotopes such as technetium or rhenium.
<발명의 요약><Summary of the invention>
본 발명은 The present invention
aa) 표 1에 보여진 상보성-결정 지역 HCDR3 및(또는) LCDR3을 포함하는 피브로넥틴의 세포외 도메인 B (ED-B)에 대한 항원-결합 부위의 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 HCDR3 지역의 5개 이하의 아미노산 및 LCDR3 지역의 6개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 그의 변이체;aa) the sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and/or LCDR3 shown in Table 1 or having the same function as the peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions and/or substitutions of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region;
ab) 표 1에 보여진 상보성-결정 지역 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 및 LCDR3을 포함하는 피브로넥틴의 세포외 도메인 B (ED-B)에 대한 항원-결합 부위의 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 HCDR1 지역의 3개 이하의 아미노산, HCDR2 지역의 8개 이하의 아미노산, HCDR3 지역의 5개 이하의 아미노산, LCDR1 지역의 6개 이하의 아미노산, LCDR2 지역의 4개 이하의 아미노산 및 LCDR3 지역의 6개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 그의 변이체;ab) The sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin including the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or a peptide according to SEQ ID NO: 1 3 or less amino acids in the HCDR1 region, 8 or less amino acids in the HCDR2 region, 5 or less amino acids in the HCDR3 region, 6 or less amino acids in the LCDR1 region, 4 or less amino acids in the LCDR2 region, and Variants thereof that are deletions, insertions and/or substitutions of up to 6 amino acids in the LCDR3 region;
ac) 서열번호 1 (L19)에 따른 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 30개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 서열번호 1의 변이체, 및ac) a variant of SEQ ID NO: 1, which is a deletion, insertion and/or substitution of 30 or less amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and
ba) 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys (서열번호 2) (여기서, Xaa1, Xaa2, 및 Xaa3은 각각 임의의 자연 발생의 아미노산을 독립적으로 나타냄) 또는ba) amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys (SEQ ID NO: 2) (wherein Xaa 1 , Xaa 2 , and Xaa 3 each independently represent any naturally occurring amino acid) or
bb) 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys-Xaa4 (서열번호 3) (여기서, Xaa1, Xaa2, Xaa3, 및 Xaa4는 각각 임의의 자연 발생의 아미노산을 독립적으로 나타냄) 또는bb) amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 4 (SEQ ID NO: 3) (wherein Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 each independently represents any naturally occurring amino acid) or
bc) 아미노산 서열 (His)n (서열번호 4) (여기서 n은 4 내지 6의 정수를 나타냄)bc) amino acid sequence (His) n (SEQ ID NO: 4) (where n represents an integer from 4 to 6)
을 포함하고, 여기서 aa), ab) 또는 ac)의 C-말단이 펩티드 결합을 통해 서열번호 2, 서열번호 3, 또는 서열번호 4의 서열들 중의 하나의 N-말단에 결합되는 펩티드를 포함하는 화합물을 기술한다.Including, wherein the C-terminus of aa), ab) or ac) comprises a peptide bonded to the N-terminus of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 through peptide bonds. Describe the compound.
화합물은 바람직하게 단쇄 항체 단편, 특히 scFv 단편이다. 추가로, 화합물은 방사성동위원소, 예를 들면 테크네튬, 예를 들면 94mTc, 99mTc, 레늄, 예를 들면 186Re, 188Re, 또는 다른 동위원소, 예를 들면 203Pb, 67Ga, 68Ga, 43Sc, 44Sc, 47Sc, 110mIn, 111In, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 90Y, 121Sn, 161Tb, 153Sm, 166Ho, 105Rh, 177Lu, 72As 및 18F의 방사성동위원소로 바람직하게 콘쥬게이션된다.The compound is preferably a single chain antibody fragment, in particular an scFv fragment. Additionally, the compound may be a radioisotope, such as technetium, such as 94m Tc, 99m Tc, rhenium, such as 186 Re, 188 Re, or other isotopes, such as 203 Pb, 67 Ga, 68 Ga. , 43 Sc, 44 Sc, 47 Sc, 110m In, 111 In, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 90 Y, 121 Sn, 161 Tb, 153 Sm, 166 Ho, 105 Rh, 177 Lu, 72 As and 18 F radioisotopes are preferably conjugated.
본 발명은 또한 활성 약제로서 상기 화합물과 함께 생리적으로 허용가능한 보조제, 희석제 및(또는) 담체를 포함하는 제약 조성물을 기술한다.The present invention also describes pharmaceutical compositions comprising a physiologically acceptable adjuvant, diluent and/or carrier together with the compound as an active agent.
본 발명은 또한 The present invention also
aa) 표 1에 보여진 상보성-결정 지역 HCDR3 및(또는) LCDR3을 포함하는 피브로넥틴의 세포외 도메인 B (ED-B)에 대한 항원-결합 부위의 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 HCDR3 지역의 5개 이하의 아미노산 및 LCDR3 지역의 6개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 그의 변이체;aa) the sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and/or LCDR3 shown in Table 1 or having the same function as the peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions and/or substitutions of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region;
ab) 표 1에 보여진 상보성-결정 지역 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 및 LCDR3을 포함하는 피브로넥틴의 세포외 도메인 B (ED-B)에 대한 항원-결합 부위의 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 HCDR1 지역의 3개 이하의 아미노산, HCDR2 지역의 8개 이하의 아미노산, HCDR3 지역의 5개 이하의 아미노산, LCDR1 지역의 6개 이하의 아미노산, LCDR2 지역의 4개 이하의 아미노산 및 LCDR3 지역의 6개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 그의 변이체;ab) The sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin including the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or a peptide according to SEQ ID NO: 1 3 or less amino acids in the HCDR1 region, 8 or less amino acids in the HCDR2 region, 5 or less amino acids in the HCDR3 region, 6 or less amino acids in the LCDR1 region, 4 or less amino acids in the LCDR2 region, and Variants thereof that are deletions, insertions and/or substitutions of up to 6 amino acids in the LCDR3 region;
ac) 서열번호 1 (L19)에 따른 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 30개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 서열번호 1의 변이체, 및ac) a variant of SEQ ID NO: 1, which is a deletion, insertion and/or substitution of 30 or less amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and
ba) 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys (서열번호 2) (여기서, Xaa1, Xaa2, 및 Xaa3은 각각 임의의 자연 발생의 아미노산을 독립적으로 나타냄) 또는ba) amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys (SEQ ID NO: 2) (wherein Xaa 1 , Xaa 2 , and Xaa 3 each independently represent any naturally occurring amino acid) or
bb) 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys-Xaa4 (서열번호 3) (여기서, Xaa1, Xaa2, Xaa3, 및 Xaa4는 각각 임의의 자연 발생의 아미노산을 독립적으로 나타냄) 또는bb) amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 4 (SEQ ID NO: 3) (wherein Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 each independently represents any naturally occurring amino acid) or
bc) 아미노산 서열 (His)n (서열번호 4) (여기서 n은 4 내지 6의 정수를 나타냄)bc) amino acid sequence (His) n (SEQ ID NO: 4) (where n represents an integer from 4 to 6)
을 포함하고, 여기서 aa), ab) 또는 ac)의 C-말단이 펩티드 결합을 통해 서열번호 2, 서열번호 3, 또는 서열번호 4의 서열들 중의 하나의 N-말단에 결합되는 펩티드의 방사성동위원소, 예를 들면 테크네튬 또는 레늄의 방사성동위원소를 결합하기 위한 용도를 기술한다. Including, wherein the C-terminus of aa), ab) or ac) is a radioactive isotope of the peptide bonded to the N-terminus of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 through peptide bond Describes the use of an element, such as technetium or rhenium, for binding radioactive isotopes.
항체 단편 L19는 하기의 서열에 의해 정의된다 (서열번호 1):Antibody fragment L19 is defined by the following sequence (SEQ ID NO: 1):
(VH) (VH)
EVQLLESGGG LVQPGGSLRL SCAASGFTFSEVQLLESGGG LVQPGGSLRL SCAASGFTFS
SFSMSWVRQA PGKGLEWVSS ISGSSGTTYY SFSMSWVRQA PGKGLEWVSS ISGSSGTTYY
ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF PYFDYWGQGT LVTVSS TAVYYCAKPF PYFDYWGQGT LVTVSS
(링커) (Linker)
GDGSSGGSGG AS GDGSSGGSGG AS
(VL)(VL)
EIVLTQSPGT LSLSPGERAT LSCRASQSVS EIVLTQSPGT LSLSPGERAT LSCRASQSVS
SSFLAWYQQK PGQAPRLLIY YASSRATGIP SSFLAWYQQK PGQAPRLLIY YASSRATGIP
DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ
QTGRIPPTFG QGTKVEIKQTGRIPPTFG QGTKVEIK
30개 이하의 아미노산의 결실, 삽입 및(또는) 치환은 서열번호 1의 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 또는 30개 아미노산의 결실, 삽입 및(또는) 치환이다. 그러나, 상보성-결정 지역 (CDR) 내에서 청구된 펩티드, 예를 들면 서열번호 1의 펩티드, 아미노산 (aa)의 결실, 삽입 및(또는) 치환인 변이체는 하기 표 1에 정의된 최대 변이를 초과해서는 안된다 (HCDR: 중쇄의 CDR; LCDR: 경쇄의 CDR).Deletion, insertion and/or substitution of 30 or less amino acids are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 of SEQ ID NO: 1. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids deletion, insertion and/or substitution. However, within the complementarity-determining region (CDR) the claimed peptide, e.g., a peptide of SEQ ID NO: 1, a variant that is a deletion, insertion and/or substitution of an amino acid (aa) exceeds the maximum variation defined in Table 1 below. Should not (HCDR: CDR of heavy chain; LCDR: CDR of light chain).
TYYADSV
KGSISGSSGT
TYYADSV
KG
(7,6,5,4,3,2,1)8
(7,6,5,4,3,2,1)
SSFLARASQSVS
SSFLA
PTCQQTGRIP
PT
CDR들은 문헌 [E. A. Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, National Institutes for Health, Bethesda, MD, 5th Edition, 1991]에 따라 정의되었다.CDRs are described in E. A. Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, National Institutes for Health, Bethesda, MD, 5th Edition, 1991].
바람직한 것은 서열번호 1 (L19)에 따른 서열 또는 20개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 서열번호 1의 변이체를 포함하는 펩티드이다. Preferred are peptides comprising a sequence according to SEQ ID NO: 1 (L19) or a variant of SEQ ID NO: 1 which is a deletion, insertion and/or substitution of 20 or fewer amino acids.
표 1에 보여진 CDR 서열의 변이 및 특히 서열번호 1에 따른 펩티드와 동일한 기능을 가진 결실, 삽입 및(또는) 치환인 서열번호 1의 변이를 포함하는 펩티드는 비아코아 (BIAcore)에 의해 측정된 나노몰 이하 범위의 (즉, 10-9보다 낮은) 해리 상수 Kd로 피브로넥틴의 ED-B 도메인에 결합하는 펩티드로서 정의된다 (W099/58570, 실시예 2 및 표 2 참조).The mutations in the CDR sequences shown in Table 1 and particularly the peptides comprising the mutations in SEQ ID NO: 1, which are deletions, insertions, and/or substitutions having the same function as the peptides according to SEQ ID NO: 1, were measured by BIAcore. It is defined as a peptide that binds to the ED-B domain of fibronectin with a dissociation constant K d in the sub-molar range (i.e., less than 10 -9) (see WO99/58570, Example 2 and Table 2).
바람직한 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys (서열번호 2)은 서열 Gly-Gly-Gly-Cys (서열번호 5) 및 Gly-Cys-Gly-Cys (서열번호 6)이다. 가장 바람직한 것은 서열 Gly-Gly-Gly-Cys (서열번호 5)이다.Preferred amino acid sequences Xaa 1 -Xaa 2 -Xaa 3 -Cys (SEQ ID NO: 2) are the sequences Gly-Gly-Gly-Cys (SEQ ID NO: 5) and Gly-Cys-Gly-Cys (SEQ ID NO: 6). Most preferred is the sequence Gly-Gly-Gly-Cys (SEQ ID NO: 5).
바람직한 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys-Xaa4 (서열번호 3)은 서열 Gly-Gly-Gly-Cys-Ala (서열번호 7) 및 Gly-Cys-Gly-Cys-Ala (서열번호 8)이다. 가장 바람직한 것은 서열 Gly-Gly-Gly-Cys-Ala (서열번호 7)이다.The preferred amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 4 (SEQ ID NO: 3) is the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO: 7) and Gly-Cys-Gly-Cys-Ala (SEQ ID NO: 8). Most preferred is the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO: 7).
아미노산 서열 (His)n (서열번호 4)을 포함하는 화합물에서, n이 정수 6인 화합물이 바람직하다.In the compound containing the amino acid sequence (His) n (SEQ ID NO: 4), a compound in which n is an integer 6 is preferred.
테크네튬 또는 레늄의 바람직한 방사성동위원소는 동위원소 94mTc,99mTc, 186Re 및 188Re이다. 가장 바람직한 것은 방사성동위원소 99mTc이다.Preferred radioisotopes of technetium or rhenium are the isotopes 94m Tc, 99m Tc, 186 Re and 188 Re. Most preferred is the radioisotope 99m Tc.
<발명의 상세한 설명><Detailed description of the invention>
단쇄 항체 단편 L19 (서열번호 1)는 이전에 종양 가진 생쥐에서 이 화합물의 생체분포를 조사하기 위해 125I으로 표지되었다 (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192-198). 결과는 생체내에서의 종양 혈관의 선택적 표적이 달성될 수 있음을 보여준다. 그러나, 놀랍게도 단쇄 항체 단편 L19의 약동학적 성질은 펩티드 ba), bb) 또는 bc)로 콘쥬게이션되고 테크네튬 또는 레늄의 방사성동위원소로 표지될 때 상당히 향상될 수 있음이 발견되었다. 동위원소 99mTc가 방사화학적 성질 (99Mo/99mTc 생성기를 통해 쉽게 얻을 수 있고, 140KeV의 단일 감마-광자를 방출하며, 고도의 광자 선속을 가지며, 6시간의 반감기로 붕괴됨) 및 비용-효용 때문에 일상의 임상적 SPECT를 위해 선택되는 방사성동위원소 표지이다. 치료적 용도를 위해, 화학적으로 동종인 동위원소 186Re 및 188Re로의 표지가 특히 바람직하다 (Hsieh, B.T., et al., Nucl. Med. Biol., 1999, 26(8), 967-972; 973-976, Zamora, P.O., et al., Anticancer Res., 1997, 17(3B), 1803-1838). The single chain antibody fragment L19 (SEQ ID NO: 1) was previously labeled with 125 I to investigate the biodistribution of this compound in tumor-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192-198). The results show that selective targeting of tumor vessels in vivo can be achieved. However, it was surprisingly found that the pharmacokinetic properties of the single chain antibody fragment L19 can be significantly improved when conjugated with peptides ba), bb) or bc) and labeled with a radioisotope of technetium or rhenium. The isotope 99m Tc has radiochemical properties ( easily obtainable through the 99 Mo/ 99m Tc generator, emits a single gamma-photon of 140KeV, has a high photon flux, decays with a half-life of 6 hours) and cost- It is the radioisotope label of choice for routine clinical SPECT because of its utility. For therapeutic use, labeling with the chemically homogeneous isotopes 186 Re and 188 Re is particularly preferred (Hsieh, BT, et al., Nucl. Med. Biol., 1999, 26(8), 967-972; 973 -976, Zamora, PO, et al., Anticancer Res., 1997, 17(3B), 1803-1838).
본 발명의 펩티드는 피브로넥틴의 세포외 ED-B 도메인에 대한 재조합 scFv 항체 L19 (서열번호 1)의 유도체이고, 도 1에 따른 유전 공학을 통해 제조되었다. 하기의 펩티드들이 제조되었다:The peptide of the present invention is a derivative of the recombinant scFv antibody L19 (SEQ ID NO: 1) against the extracellular ED-B domain of fibronectin, and was prepared through genetic engineering according to FIG. 1. The following peptides were prepared:
L19 (서열번호 1)L19 (SEQ ID NO: 1)
L19His:L19His:
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA
PGKGLEWVSSPGKGLEWVSS
51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED 51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF TAVYYCAKPF
101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS 101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS
LSPGERATLS LSPGERATLS
151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR 151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR
FSGSGSGTDF FSGSGSGTDF
201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKAAAL201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKAAAL
EHHHHHHEHHHHHH
(서열번호 9) (SEQ ID NO: 9)
AP38: AP38:
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA
PGKGLEWVSSPGKGLEWVSS
51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED 51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF TAVYYCAKPF
101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS 101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS
LSPGERATLS LSPGERATLS
151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR 151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR
FSGSGSGTDF FSGSGSGTDF
201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC 201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC
(서열번호 10) (SEQ ID NO: 10)
AP39: AP39:
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA
PGKGLEWVSSPGKGLEWVSS
51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED 51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF TAVYYCAKPF
101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS 101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS
LSPGERATLS LSPGERATLS
151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR 151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR
FSGSGSGTDF FSGSGSGTDF
201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC A201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC A
(서열번호 11) (SEQ ID NO: 11)
L19-GlyCysGlyCys: L19-GlyCysGlyCys:
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA
PGKGLEWVSSPGKGLEWVSS
51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED 51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF TAVYYCAKPF
101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS 101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS
LSPGERATLS LSPGERATLS
151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR 151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR
FSGSGSGTDF FSGSGSGTDF
201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC 201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC
(서열번호 12)(SEQ ID NO: 12)
L19-GlyCysGlyCysAla:L19-GlyCysGlyCysAla:
1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA 1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA
PGKGLEWVSS PGKGLEWVSS
51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED 51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF TAVYYCAKPF
101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS 101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS
LSPGERATLS LSPGERATLS
151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR 151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR
FSGSGSGTDF FSGSGSGTDF
201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC A 201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC A
(서열번호 13) (SEQ ID NO: 13)
펩티드의 제조는 하기의 실시예에 자세히 기술한다 ("실시예" 참조).The preparation of the peptide is described in detail in the following examples (see "Examples").
항체 단편 L19는 이. 콜라이에서의 발현으로 원래 제조되었다 (WO 99/58570 참조). 그러나, scFv 항체 단편의 대규모 제조의 경우, 이 발현계는 불만족스러운 것으로 발견되었다. 또다른 발현계인 효모 발현계, 특히 피치아 파스토리스 (Pichia pastoris) 발현계가 시험되었다. 본 출원인들은 효모, 예를 들면 피치아 파스토리스 (Pichia pastoris)가 고도로 생활성인 항체 단편, 예를 들면 단편 AP39의 발현을 일반적으로 할 수 있으나, 생체약물의 경제적 제조를 위해 필요한 리터 배양 당 250mg 항체 단편까지의 고수율 발현은 항상 발현 벡터 (예를 들면, pGAP)에 의해서만 얻을 수 있고, 메탄올 유도성 벡터 (예를 들면, pPIC9K)에 의해서는 얻을 수 없음을 발견하였다. 이 항상 발현계의 추가 이점은 유도성 효모 발현과 비교하여 단순화되고 강한 발효 과정이다. 예상과 달리, 본 출원인들은 단편의 N-말단이 알파-신호 서열의 Kex2-절단 부위에 직접 융합된 발현 카세트가 사용될 때만 항체 단편, 예를 들면 단편 AP39의 적절한 신호 서열 프로세싱이 관찰됨을 발견하였다.Antibody fragment L19 is E. Originally prepared for expression in E. coli (see WO 99/58570). However, for large-scale production of scFv antibody fragments, this expression system was found to be unsatisfactory. Another expression system, the yeast expression system, in particular the Pichia pastoris expression system, was tested. Applicants believe that yeast, such as Pichia pastoris , is generally capable of expressing highly bioactive antibody fragments, such as fragment AP39, but 250 mg antibody per liter culture required for economical production of biopharmaceuticals. It has been found that high-yield expression up to fragments can always be obtained only with expression vectors (eg pGAP) and not with methanol inducible vectors (eg pPIC9K). An additional advantage of this always expression system is a simplified and robust fermentation process compared to inducible yeast expression. Contrary to expectations, Applicants have found that proper signal sequence processing of antibody fragments, such as fragment AP39, is observed only when an expression cassette in which the N-terminus of the fragment is fused directly to the Kex2-cleavage site of the alpha-signal sequence is used.
펩티드들은 진단 및 치료 용도, 특히 침투성 종양 및 종양 전이의 진단 및 치료에 적절하다. 바람직한 진단 용도는 SPECT (단일 광자 방출 전산화 단층촬영, Single Photon Emission Computed Tomography) 및 PET (양전자 방출 단층촬영, Positron Emission Tomography)이다.The peptides are suitable for diagnostic and therapeutic uses, in particular for diagnosis and treatment of invasive tumors and tumor metastases. Preferred diagnostic applications are SPECT (Single Photon Emission Computed Tomography) and PET (Positron Emission Tomography).
상기의 펩티드들은 상기의 방사성동위원소, 예를 들면 테크네튬 및 레늄의 방사성동위원소, 바람직하게는 방사성핵종 94mTc, 99mTc, 186Re, 및 188Re의 표지에 특히 적절하다. 펩티드의 표지를 위해, 펩티드는 적절한 환원제, 예를 들면 염화 주석 또는 트리스(2-카르복시에틸)포스핀 (TCEP)으로 먼저 환원된다. 결과의 환원된 펩티드는 99mTc 생성기 용리액 또는 188Re 생성기 용리액 및 염화 주석과 반응하여 본 발명의 화합물로 될 수 있는 SH-기를 제시한다 (자세한 것은 하기의 실시예 참조). 간접적 표지는 킬레이팅 리간드의 예비-콘쥬게이션 및 후속하는 인듐, 이트륨, 란탄족 등과 같은 방사성동위원소의 복합화에 의해 실시된다. 킬레이팅 리간드는 바람직하게 에틸렌 디아민 테트라아세트산 (EDTA), 디에틸렌 트리아민 펜타아세트산 (DTPA), 시클로헥실 1,2-디아민테트라아세트산 (CDTA), 에틸렌글리콜-O,0'-비스(2-아미노에틸)-N,N,N',N'-디아세트산 (HBED), 트리에틸렌 테트라아민 헥사아세트산 (TTHA), 1,4,7,10-테트라아자시클로도데칸-N,N',N"'-테트라아세트산 (DOTA), 1,4,7-트리아자시클로노난-N,N',N"-트리아세트산 (NOTA), 및 1,4,8,11-테트라아자시클로테트라데칸-N,N',N",N"'-테트라아세트산 (TETA)으로부터 펩티드 화합물의 아민 또는 티올기로 유도된다. 킬레이팅 리간드는 적절한 커플링기, 예를 들면 활성 에스테르, 말레이미드, 티오카르바메이트 또는 α-할로겐화 아세트아미드 잔기를 보유한다. 킬레이팅 리간드를 아민기, 예를 들면 라이신 잔기의 ε-NH2-기에 콘쥬게이션하기 위해, 펩티드 화합물의 사전 환원은 필요로 하지 않는다. 방사성동위원소 표지된 펩티드는 방사선-진단 및 방사선-치료 용도에 적절하다.The above peptides are particularly suitable for labeling the above radioisotopes, such as those of technetium and rhenium, preferably the radionuclides 94m Tc, 99m Tc, 186 Re, and 188 Re. For labeling of the peptide, the peptide is first reduced with a suitable reducing agent such as tin chloride or tris(2-carboxyethyl)phosphine (TCEP). The resulting reduced peptide reacts with a 99m Tc generator eluent or 188 Re generator eluent and tin chloride to reveal an SH-group that can be a compound of the present invention (see Examples below for details). Indirect labeling is carried out by pre-conjugation of chelating ligands and subsequent complexation of radioisotopes such as indium, yttrium, lanthanides, and the like. The chelating ligand is preferably ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), cyclohexyl 1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,0'-bis(2-amino Ethyl)-N,N,N',N'-diacetic acid (HBED), triethylene tetraamine hexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N"'-Tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), and 1,4,8,11-tetraazacyclotetradecane-N, It is derived from the amine or thiol group of the peptide compound from N',N",N"'-tetraacetic acid (TETA). The chelating ligand carries a suitable coupling group, such as an active ester, maleimide, thiocarbamate or α-halogenated acetamide moiety. In order to conjugate the chelating ligand to the amine group, for example the ε-NH 2 -group of the lysine residue, prior reduction of the peptide compound is not required. Radioisotope labeled peptides are suitable for radiation-diagnostic and radiation-therapeutic applications.
결과의 방사성동위원소 표지된 펩티드는 동물 실험에서 예상치 못한 이점을 보인다. 예를 들면, 누드 생쥐에서의 표지된 펩티드, 예를 들면 99mTc-표지된 AP39 (서열번호 11)의 배출은 신장을 통해 24시간내 70% 이상, 예를 들면 80.63%가 일어나는 반면, 125I으로 표지된 L19 (서열번호 1)는 누드 생쥐에서의 배출이 신장을 통해 24시간내 67.79%만이 일어났다. 표지된 펩티드, 예를 들면 99mTc-표지된 AP39의 종양 대 혈액 비가 5시간 후 5:1 이상, 바람직하게 8:1 이상, 예를 들면 약 10:1인 반면, 125I으로 표지된 L19에서는 이 비가 단지 약 3:1이다. 이는 또한 종종 덜 선호적인 생체분포 특징을 보이는 다른 99mTc-표지된 scFv 항체와 비교해 예상치 못한 반응이다. 예를 들면, 문헌 [Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868-872, 1996]은 본 발명에 기재된 펩티드의 값, 예를 들면 99mTc-표지된 AP39의 경우 1.3%와 비교해 매우 높은, 24시간 후 단지 4:1의 종양 대 혈액 비, 및 24시간 후 9%의 신장 축적을 보이는 99mTc-표지된 scFv 항체를 보고한다 (하기 실시예 13 참조).The resulting radioisotope labeled peptides show unexpected advantages in animal experiments. For example, in nude mice, excretion of a labeled peptide, e.g. 99m Tc-labeled AP39 (SEQ ID NO: 11), occurs over 70%, e.g. 80.63%, within 24 hours through the kidney, while 125 I Labeled L19 (SEQ ID NO: 1) was excreted in nude mice only 67.79% within 24 hours through the kidney. The tumor to blood ratio of the labeled peptide, e.g. 99m Tc-labeled AP39, after 5 hours is at least 5:1, preferably at least 8:1, e.g. about 10:1, whereas in L19 labeled with 125 I This ratio is only about 3:1. This is also an unexpected response compared to other 99m Tc-labeled scFv antibodies, which often exhibit less favorable biodistribution characteristics. See, eg, Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868-872, 1996] is very high compared to the values of the peptides described in the present invention, for example 1.3% for 99m Tc-labeled AP39, a tumor to blood ratio of only 4:1 after 24 hours, and after 24 hours. A 99m Tc-labeled scFv antibody showing 9% renal accumulation is reported (see Example 13 below).
더욱이, 본 발명의 표지된 펩티드, 예를 들면 99mTc-표지된 AP39의 생체내 안정도는 125I으로 표지된 L19의 생체내 안정도에 비해 매우 높다. 본 발명자들은 펩티드, 예를 들면 99mTc-표지된 AP39의 주사 2시간 후 혈청 내 단지 10% 이하, 예를 들면 3%의 방사능이 대사물에 기인한 반면, 125I으로 표지된 L19의 주사 2시간 후 혈청 내 49%의 방사능이 유리 요오드일 수 있는 대사물에 기인했음을 발견했다. 펩티드, 예를 들면 99mTc-표지된 AP39의 향상된 생체내 안정도는 또한 표적 ED-B에의 결합 능력의 계속된 보존을 반영한다. 본 발명자들은 펩티드, 예를 들면 99mTc-표지된 AP39의 주사 2시간 후 혈청내 50% 이상, 예를 들면 74%의 방사능이 ED-B에 결합할 수 있는 반면, 125I으로 표지된 L19의 주사 2시간 후 혈청 내 단지 27%의 방사능이 ED-B에 결합할 수 있음을 발견하였다. 본 발명의 화합물은 또한 고도의 종양 축적을 보인다. 예를 들면, Tc-99m-AP39 및 In-111-MX-DTPA-ε-HN(Lys)-AP39는 주사후 (post injection, p.i.) 1시간에 10.7 (Tc-99m) 또는 12.9 (In-111) % 그람당 주사량 (ID/g)의 고도 종양 축적을 보였다. 그러므로, 종양 흡수는 다른 공지의 In-111 또는 Tc-99m 표지된 항체 단편에 비해 상당히 높다 (예를 들면, Kobayashi et al., J. Nuc. Med., Vol. 41(4), pp. 755-762, 2000; Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868-872, 1996). Moreover, the in vivo stability of the labeled peptide of the present invention, such as 99m Tc-labeled AP39, is very high compared to the in vivo stability of L19 labeled with 125 I. We found that only 10% or less, e.g. 3% of radioactivity in the serum 2 hours after injection of a peptide, e.g. 99m Tc-labeled AP39, is due to metabolites, whereas injection 2 of L19 labeled with 125 I After hours, it was found that 49% of the radioactivity in the serum was due to a metabolite that could be free iodine. The improved in vivo stability of the peptide, eg 99m Tc-labeled AP39, also reflects the continued preservation of the ability to bind to the target ED-B. We found that at least 50%, e.g., 74% of radioactivity in serum 2 hours after injection of a peptide, e.g. 99m Tc-labeled AP39, is capable of binding to ED-B, while that of L19 labeled with 125 I. It was found that only 27% of the radioactivity in the serum could bind to ED-B 2 hours after injection. The compounds of the present invention also show a high degree of tumor accumulation. For example, Tc-99m-AP39 and In-111-MX-DTPA-ε-HN(Lys)-AP39 are 10.7 (Tc-99m) or 12.9 (In-111 )% Injection per gram (ID/g) showed high tumor accumulation. Therefore, tumor uptake is significantly higher compared to other known In-111 or Tc-99m labeled antibody fragments (e.g., Kobayashi et al., J. Nuc. Med., Vol. 41(4), pp. 755 -762, 2000; Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868-872, 1996).
화합물들은 진단 및 치료의 용도에 적절하다. 이들은 바람직하게는 비경구 투여, 더 바람직하게는 정맥내 주사에 의해 환자에 적용된다. 인간 투여량은 바람직하게는 방사선진단 용도를 위해서는 환자 당 0.1 내지 1mg의 범위, 및 방사선치료 용도를 위해서는 환자 당 0.1 내지 100mg의 범위이다. The compounds are suitable for diagnostic and therapeutic uses. They are preferably applied to the patient by parenteral administration, more preferably by intravenous injection. The human dosage is preferably in the range of 0.1 to 1 mg per patient for radiodiagnostic use, and in the range of 0.1 to 100 mg per patient for radiotherapy use.
본 발명의 화합물의 제조 및 표지 방법은 하기 실시예에서 더 완전히 예시된다. 이들 실시예들은 예시를 위해 보여진 것이지 한정을 위해서가 아니다.The preparation and labeling methods of the compounds of the present invention are more fully exemplified in the following examples. These embodiments have been shown for purposes of illustration and not limitation.
도 1은 피브로넥틴의 세포외 ED-B 도메인에 대한 재조합 scFv 항체 L19 (서열번호 1)의 유도체를 보여준다.
도 2는 F9 (기형암)-가진 누드 생쥐의 Tc-99m-AP39의 종양 대 혈액 비 (평균±SD, n=3)를 보여준다.
도 3은 물질 주사 5시간 후의 F9 (기형암)-가진 누드 생쥐에서의 Tc-99m-AP39의 신티그램을 보여준다.
도 4는 물질 주사 24시간 후의 F9 (기형암)-가진 누드 생쥐에서의 Tc-99m-AP39의 신티그램을 보여준다.1 shows a derivative of the recombinant scFv antibody L19 (SEQ ID NO: 1) against the extracellular ED-B domain of fibronectin.
Figure 2 shows the tumor-to-blood ratio (mean±SD, n=3) of Tc-99m-AP39 in nude mice with F9 (teratocarcinoma)-.
Figure 3 shows the scintigram of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing
Figure 4 shows the scintigram of Tc-99m-AP39 in nude mice with F9 (teratocarcinoma)- 24 hours after injection of the substance.
실시예 1: L19 유도체의 제조Example 1: Preparation of L19 derivative
피브로넥틴의 스플라이스 변이체의 세포외 도메인 B (ED-B)에 대한 재조합 항체 (scFv L19, 약칭 L19)가 출발 물질을 형성했다. scFv L19를 합성 인간 항체 레퍼토리로부터 파지 디스플레이 선별법에 의해 단리하였다 (Neri et al., 1997, Nature Biotechnol. 15: 1271; Pini et al., 1998, J. Biol. Chem. 273: 21769). 이 재조합 항체 단편은 소위 단쇄 항체 단편 (scFv)의 형태이고 링커 서열에 의해 연결된 VH 및 VL 지역으로 구성되어 있다 (서열번호 1 참조). scFv L19는 ED B에 대해 극히 고도의 친화도를 가진다 (Kd: 5.4x10-11M).A recombinant antibody (scFv L19, abbreviated L19) against the extracellular domain B (ED-B) of the splice variant of fibronectin formed the starting material. scFv L19 was isolated from a synthetic human antibody repertoire by phage display selection (Neri et al., 1997, Nature Biotechnol. 15: 1271; Pini et al., 1998, J. Biol. Chem. 273: 21769). This recombinant antibody fragment is in the form of a so-called single chain antibody fragment (scFv) and is composed of VH and VL regions linked by linker sequences (see SEQ ID NO: 1). scFv L19 has extremely high affinity for ED B (K d : 5.4x10 -11 M).
다양한 L19의 유도체를 유전 조작에 의해 제조하였다 (도 1 참조). L19를 변형시키기 위해, scFv 코딩 DNA를 추가 서열을 코딩하는 프라이머들을 이용해 PCR (중합효소 연쇄 반응, polymerase chain reaction)에 의해 증폭하고, 발현 벡터 내로 클로닝하였다.Various derivatives of L19 were prepared by genetic engineering (see Fig. 1). To modify L19, scFv-encoding DNA was amplified by PCR (polymerase chain reaction) using primers encoding additional sequences, and cloned into an expression vector.
L19의 유도체:Derivatives of L19:
L19: 추가의 말단 변형 없음 L19: no further terminal modifications
L19 His: Ni 킬레이트 크로마토그래피 및 방사성동위원소 결합을 위한 C-말단 His6 도메인 (His 태그)L19 His: C-terminal His 6 domain for Ni chelate chromatography and radioisotope binding (His tag)
AP38: 치료 및 진단에 사용될 수 있는 물질 (예를 들면, 방사성 동위원소)의 결합 (Cys를 통해)을 위한 C-말단 GlyGlyGlyCys 도메인 AP38: C-terminal GlyGlyGlyCys domain for binding (via Cys) of substances (e.g., radioactive isotopes) that can be used for treatment and diagnosis
AP39: 치료 및 진단에 사용될 수 있는 물질 (예를 들면, 방사성 동위원소)의 결합 (Cys를 통해)을 위한 C-말단 GlyGlyGlyCysAla 도메인 AP39: C-terminal GlyGlyGlyCysAla domain for binding (via Cys) of substances (e.g., radioactive isotopes) that can be used for treatment and diagnosis
L19-GlyCysGlyCys: 치료 및 진단에 사용될 수 있는 물질 (예를 들면, 방사성 동위원소)의 결합 (Cys를 통해)을 위한 C-말단 GlyCysGlyCys 도메인 L19-GlyCysGlyCys: C-terminal GlyCysGlyCys domain for binding (via Cys) of substances (e.g., radioactive isotopes) that can be used for treatment and diagnosis
L19-GlyCysGlyCysAla: 치료 및 진단에 사용될 수 있는 물질 (예를 들면, 방사성 동위원소)의 결합 (Cys를 통해)을 위한 C-말단 GlyCysGlyCysAla 도메인 L19-GlyCysGlyCysAla: C-terminal GlyCysGlyCysAla domain for binding (via Cys) of substances (e.g., radioactive isotopes) that can be used for treatment and diagnosis.
L19 유도체의 재조합 제조:Recombinant production of L19 derivatives:
기술된 L19 유도체를 원핵 및 진핵 발현계에서 제조하였다.The described L19 derivatives were prepared in prokaryotic and eukaryotic expression systems.
a) 이. 콜라이에서의 L19 제조a) this. L19 production in coli
다양한 L19 유도체 (AP38, AP39, L19-GlyCysGlyCys, L19-GlyCysGlyCysAla, L19, L19His)를 코딩하는 DNA 서열을 IPTG-유도성 프로모터 및 엠피실린 내성 표지와 함께 원핵 발현 벡터 (pDN5, Pini et al., 1997, J. Immunol. Methods 206: 171, Pini et al., 1998, J. Biol. Chem. 273: 21769; pET, 노바젠 (Novagen)) 내로 클로닝했다. 재조합 단백질의 주변세포질로의 분비를 가능하게 하기 위해, scFv의 N-말단에 Pel B 신호 서열을 융합한 발현 카세트를 제조하는데 이 벡터를 사용했다. 이. 콜라이 (TG1, BL21DE3 및 HB2151)를 이 발현 벡터로 형질전환 후 엠피실린 선별로 안정한 생산 균주를 얻을 수 있었다. scFv를 제조하기 위해, 프로모터를 억제하기 위해 성장기 (37℃)에서 1%의 포도당의 존재하에 이 균주들을 배양하였다. 배양액에서의 scFv 발현을 IPTG 첨가 및 30℃에서 16시간까지의 인큐베이팅에 의해 유도했다. 가용성 및 항원-결합 scFv 물질을 이. 콜라이 균주의 완전한 추출액으로부터, 주변세포질 분획으로부터 또는 정제 및 수율면에서 특히 효율적인 것으로 증명된 배양 상층액으로부터 단리할 수 있었다. 제조를 10리터까지의 배양 부피로 진탕 플라스크 및 발효기에서 실시했다.DNA sequences encoding various L19 derivatives (AP38, AP39, L19-GlyCysGlyCys, L19-GlyCysGlyCysAla, L19, L19His) were prepared in prokaryotic expression vectors (pDN5, Pini et al., 1997 , J. Immunol.Methods 206:171, Pini et al., 1998, J. Biol. Chem. 273: 21769; pET, Novagen). In order to enable secretion of the recombinant protein into the periplasm, this vector was used to prepare an expression cassette in which the Pel B signal sequence was fused to the N-terminus of the scFv. this. E. coli (TG1, BL21DE3, and HB2151) were transformed with this expression vector, and then a stable production strain was obtained by selection of mpicillin. To prepare scFv, these strains were cultured in the presence of 1% glucose in the growing phase (37° C.) to inhibit the promoter. The expression of scFv in the culture was induced by addition of IPTG and incubation at 30°C for up to 16 hours. Soluble and antigen-binding scFv substances are used in E. E. coli strains could be isolated from complete extracts, from periplasmic fractions or from culture supernatants that proved to be particularly efficient in terms of purification and yield. Preparation was carried out in shake flasks and fermentors with culture volumes up to 10 liters.
b) 피치아 파스토리스 (b) Pichia Pastoris ( Pichia pastorisPichia pastoris )에서의 L19 유도체 제조) Of L19 derivatives
효모 피치아 파스토리스 (Pichia pastoris)에서의 제조를 위해 L19His, AP38, AP39, L19-GlyCysGlyCys 및 L19-GlyCysGlyCysAla-코딩 DNA 서열을 PCR로 증폭하고 이. 콜라이 및 발현 벡터 pPIC9K 및 pGAP (인비트로젠 (Invitrogen)) 내로 클로닝하였다. 이종 유전자의 발현을 위해, pPIC9K는 메탄올-유도성 프로모터 (AOX1)를 함유하고, pGAP는 GAPDH 효소의 항상성 프로모터를 함유한다. 추가로, 이들 벡터들은 각각 외래 유전자의 선별/증폭을 위한 제네티신 내성 유전자 및 제오신 내성 유전자 및 재조합 산물의 발현 및 분비를 위한 신호 서열 (효모 α인자에서)을 함유한다. 사용된 AP39 발현 카세트는 신호 서열 제거를 위해 자연 α인자 프로세싱의 다른 절단 부위가 아닌 단지 Kex2 절단 부위만을 함유하는 융합 단백질 (α인자 신호+ L19 유도체)을 코딩한다. 안정한 트랜스펙션된 PP 클론들은 피치아 파스토리스 (Pichia pastoris) 균주로의 선형화된 벡터의 전기천공 (예를 들면, 균주 GS115내로의 pPIC9K-AP39, 균주 X33내로의 pGAP-AP39) 및 후속하는 제네티신 또는 제오신 선별로 얻어졌다. 상기 L19 유도체를 가용성 분비 단백질로서 제조하기 위해 이들 클론을 사용하는 것이 가능했다. 클론들을 BMGY 배지 또는 기초 미네랄 배지에서 30℃에서 배양했다. pPIC 기재 클론들에 발현기 동안 프로모터 유도를 위해 메탄올을 첨가했다. 재조합 산물은 정확히 프로세싱된 말단 및 고도 항원-결합 활성을 가졌다. 얻을 수 있는 수율 (비정제된 생활성 산물/배양 상층액 리터)은 배양 조건 및 방법 조절에 따라 하기와 같았다: 예를 들면, pPIC9K-AP39/GS115 (진탕 플라스크 5mg/l, 발효기 10-15mg/l); pGAP-AP39/X33 (진탕 플라스크 30-40mg/l, 발효기 100-250mg/1). For preparation in the yeast Pichia pastoris , L19His, AP38, AP39, L19-GlyCysGlyCys and L19-GlyCysGlyCysAla-encoding DNA sequences were amplified by PCR and E. E. coli and expression vectors pPIC9K and pGAP (Invitrogen) were cloned. For the expression of heterologous genes, pPIC9K contains the methanol-inducible promoter (AOX1), and pGAP contains the homeostatic promoter of the GAPDH enzyme. In addition, these vectors each contain a geneticin resistance gene for selection/amplification of foreign genes and a signal sequence for expression and secretion of a zeocin resistance gene and a recombinant product (in the yeast α factor). The AP39 expression cassette used encodes a fusion protein (a factor signal + L19 derivative) containing only the Kex2 cleavage site and not other cleavage sites of natural α factor processing for signal sequence removal. Stable transfected PP clones were electroporated of a linearized vector into the Pichia pastoris strain (e.g., pPIC9K-AP39 into strain GS115, pGAP-AP39 into strain X33) and subsequent agents. It was obtained by screening either neticin or zeocin. It was possible to use these clones to prepare the L19 derivative as a soluble secreted protein. Clones were cultured at 30° C. in BMGY medium or basal mineral medium. Methanol was added to the pPIC based clones for promoter induction during the expression phase. Recombinant products had precisely processed terminal and high antigen-binding activity. The yield achievable (unpurified bioactive product/liter of culture supernatant) was as follows depending on the culture conditions and method control: for example, pPIC9K-AP39/GS115 (shaking
L19 유도체를 친화성 크로마토그래피 (r단백질 A, 스트림라인 파마시아 (Streamline Pharmacia) 또는 ED B 항원 칼럼) 및 후속하는 크기 배제 크로마토그래피를 사용해 피치아 파스토리스 (Pichia pastoris) 또는 이. 콜라이 배양 상층액으로부터 정제했다. 추가 프로세싱에 사용된 정제된 AP39 분획은 동종이량체 구조 (서브유니트의 거의 모든 부분이 공유적으로 연결된) 및 고도 항원-결합 활성을 가졌다.The L19 derivative was subjected to affinity chromatography (rprotein A, Streamline Pharmacia or ED B antigen column) and subsequent size exclusion chromatography to Pichia pastoris or E. It was purified from the coli culture supernatant. The purified AP39 fraction used for further processing had a homodimeric structure (almost all parts of the subunit are covalently linked) and high antigen-binding activity.
실시예 2aExample 2a
환원된 AP38의 합성 [환원된 L19-(Gly)Synthesis of Reduced AP38 [Reduced L19-(Gly) 33 -Cys-OH] -Cys-OH]
156㎕ PBS (인산염 완충 식염수)/10% 글리세린 내의 240㎍ (4.29nmol) S-S-이량체 AP38 용액에 50㎕ TCEP-용액 (14.34mg TCEPxHCl/5ml 수성 Na2HPO4, 0.1M, pH=7.4)을 첨가했다. 반응 혼합물을 실온에서 1시간 동안 서서히 진탕하였다. SH-단량체 AP38을 NAP-5 칼럼 (아메르샴 (Amersham), 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다. 단리된 산물의 SDS-PAGE 분석은 S-S-이량체 AP38에서 SH-단량체 AP38로의 정량적 전환을 증명했다. 50 μl TCEP-solution (14.34mg TCEPxHCl/5ml aqueous Na 2 HPO 4 , 0.1M, pH=7.4) in 240 μg (4.29 nmol) SS-dimer AP38 solution in 156 μl PBS (phosphate buffered saline)/10% glycerin Was added. The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS). SDS-PAGE analysis of the isolated product demonstrated a quantitative conversion from SS-dimer AP38 to SH-monomer AP38.
수율: 79.4㎍/220㎕ PBS (33.1%). Yield: 79.4 μg/220 μl PBS (33.1%).
실시예 2bExample 2b
Tc-99m-AP38 [Tc-99m-L19-(Gly)Tc-99m-AP38 (Tc-99m-L19-(Gly) 33 -Cys-OH]의 합성 -Cys-OH] synthesis
2.37mg 디소듐-L-타르트레이트를 작은 병에 넣은 후 220㎕ PBS 내의 79.4㎍ 환원된 AP38을 첨가하고, 용액을 100㎕ 수성 Na2HPO4-완충제 (1M, pH=10.5)로 희석했다. 50㎕ Tc-99m 생성기 용리액 (24h) 및 10㎕ SnCl2-용액 (5mg SnCl2/1ml 0.1M HCl)을 첨가했다. 반응 혼합물을 37℃에서 반시간 동안 진탕하였다. Tc-99m-표지된 AP38을 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.2.37 mg disodium-L-tartrate was placed in a small bottle, 79.4 μg reduced AP38 in 220 μl PBS was added, and the solution was diluted with 100 μl aqueous Na 2 HPO 4 -buffer (1M, pH=10.5). 50 μl Tc-99m generator eluent (24h) and 10 μl SnCl 2 -solution (5 mg SnCl 2 /1 ml 0.1M HCl) were added. The reaction mixture was shaken at 37° C. for half an hour. Tc-99m-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 39.7%. Radiochemical yield: 39.7%.
방사화학적 순도: 92.5% (SDS-PAGE). Radiochemical purity: 92.5% (SDS-PAGE).
비활성도 (Specific activity): 17.7MBq/nmol. Specific activity: 17.7MBq/nmol.
면역반응성: 88.7% Immune Reactivity: 88.7%
실시예 3aExample 3a
환원된 AP39 [환원된 L19-(Gly)Reduced AP39 [Reduced L19-(Gly) 33 -Cys-Ala-OH]의 합성-Cys-Ala-OH] synthesis
135㎕ PBS/10% 글리세린 내의 240㎍ (4.29nmol) S-S-이량체 AP39 용액에 50㎕ TCEP-용액 (14.34mg TCEPxHCl/5ml 수성 Na2HPO4, 0.1M, pH=7.4)을 첨가했다. 반응 혼합물을 실온에서 1시간 동안 서서히 진탕하였다. SH-단량체 AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다. 단리된 산물의 SDS-PAGE 분석은 S-S-이량체 AP39에서 SH-단량체 AP39로의 정량적 전환을 증명했다.To a solution of 240 μg (4.29 nmol) SS-dimer AP39 in 135 μl PBS/10% glycerin was added 50 μl TCEP-solution (14.34 mg TCEPxHCl/5 ml aqueous Na 2 HPO 4 , 0.1 M, pH=7.4). The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS). SDS-PAGE analysis of the isolated product demonstrated a quantitative conversion from SS-dimer AP39 to SH-monomer AP39.
수율: 135.9㎍/180㎕ PBS (56.2%). Yield: 135.9 μg/180 μl PBS (56.2%).
실시예 3bExample 3b
Tc-99m-AP39 [Tc-99m-L19-(Gly)Tc-99m-AP39 (Tc-99m-L19-(Gly) 33 -Cys-Ala-OH]의 합성-Cys-Ala-OH] synthesis
4.2mg 디소듐-L-타르트레이트를 작은 병에 넣은 후 180㎕ PBS 내의 135.9㎍ 환원된 AP39를 첨가하고, 용액을 100㎕ 수성 Na2HPO4-완충제 (1M, pH=10.5)로 희석했다. 100㎕ Tc-99m 생성기 용리액 (24h) 및 10㎕ SnCl2-용액 (5mg SnCl2/1ml 0.1M HCl)을 첨가했다. 반응 혼합물을 37℃에서 반시간 동안 진탕하였다. Tc-99m-표지된 AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.4.2 mg disodium-L-tartrate was placed in a small bottle, 135.9 μg reduced AP39 in 180 μl PBS was added, and the solution was diluted with 100 μl aqueous Na 2 HPO 4 -buffer (1M, pH=10.5). 100 μl Tc-99m generator eluent (24h) and 10 μl SnCl 2 -solution (5 mg SnCl 2 /1 ml 0.1M HCl) were added. The reaction mixture was shaken at 37° C. for half an hour. Tc-99m-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 50.1%. Radiochemical yield: 50.1%.
방사화학적 순도: 91.5% (SDS-PAGE). Radiochemical purity: 91.5% (SDS-PAGE).
비활성도: 21.4MBq/nmol. Specific activity: 21.4 MBq/nmol.
면역반응성: 96.4% Immune Reactivity: 96.4%
실시예 4Example 4
Re-188-AP38 [Re-188-L19-(Gly)Re-188-AP38 [Re-188-L19-(Gly) 33 -Cys-OH]의 합성-Cys-OH] synthesis
2.37mg 디소듐-L-타르트레이트를 작은 병에 넣은 후 310㎕ PBS 내의 112㎍ 환원된 AP38을 첨가하고, 용액을 100㎕ 수성 Na2HPO4-완충제 (1M, pH=10.5)로 희석했다. 100㎕ Re-188 생성기 용리액 및 50㎕ SnCl2-용액 (5mg SnCl2/1ml 0.1M HCl)을 첨가했다. 반응 혼합물을 37℃에서 한시간반 동안 진탕하였다. Re-188-표지된 AP38을 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.2.37 mg disodium-L-tartrate was placed in a small bottle, 112 µg reduced AP38 in 310 µl PBS was added, and the solution was diluted with 100 µl aqueous Na 2 HPO 4 -buffer (1M, pH=10.5). 100 μl Re-188 generator eluent and 50 μl SnCl 2 -solution (5 mg SnCl 2 /1 ml 0.1M HCl) were added. The reaction mixture was shaken at 37° C. for an hour and a half. Re-188-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 28.3%. Radiochemical yield: 28.3%.
방사화학적 순도: 91.1% (SDS-PAGE). Radiochemical purity: 91.1% (SDS-PAGE).
비활성도: 15.3MBq/nmol. Specific specificity: 15.3 MBq/nmol.
면역반응성: 89.9% Immune Reactivity: 89.9%
실시예 5Example 5
Re-188-AP39 [Re-188-L19-(Gly)Re-188-AP39 (Re-188-L19-(Gly) 33 -Cys-Ala-OH]의 합성 -Cys-Ala-OH] synthesis
2.37mg 디소듐-L-타르트레이트를 작은 병에 넣은 후 303㎕ PBS 내의 112㎍ 환원된 AP39를 첨가하고, 용액을 100㎕ 수성 Na2HPO4-완충제 (1M, pH=10.5)로 희석했다. 100㎕ Re-188 생성기 용리액 및 50㎕ SnCl2-용액 (5mg SnCl2/1ml 0.1M HCl)을 첨가했다. 반응 혼합물을 37℃에서 한시간반 동안 진탕하였다. Re-188-표지된 AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.2.37 mg disodium-L-tartrate was placed in a small bottle, 112 μg reduced AP39 in 303 μl PBS was added, and the solution was diluted with 100 μl aqueous Na 2 HPO 4 -buffer (1M, pH=10.5). 100 μl Re-188 generator eluent and 50 μl SnCl 2 -solution (5 mg SnCl 2 /1 ml 0.1M HCl) were added. The reaction mixture was shaken at 37° C. for an hour and a half. Re-188-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 33.5%. Radiochemical yield: 33.5%.
방사화학적 순도: 92.3% (SDS-PAGE). Radiochemical purity: 92.3% (SDS-PAGE).
비활성도: 18.5MBq/nmol. Specific specificity: 18.5 MBq/nmol.
면역반응성: 92.5% Immune Reactivity: 92.5%
실시예 6aExample 6a
환원된 L19-Gly-Cys-Gly-Cys-OH의 합성Synthesis of reduced L19-Gly-Cys-Gly-Cys-OH
160㎕ PBS/10% 글리세린 내의 240㎍ (4.29nmol) S-S-이량체 L19-Gly-Cys-Gly-Cys-OH 용액에 75㎕ TCEP-용액 (14.34mg TCEPxHCl/5ml 수성 Na2HPO4, 0.1M, pH=7.4)을 첨가했다. 반응 혼합물을 실온에서 1시간 동안 서서히 진탕하였다. SH-단량체 L19-Gly-Cys-Gly-Cys-OH를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다. 단리된 산물의 SDS-PAGE 분석은 S-S-이량체 L19-Gly-Cys-Gly-Cys-OH에서 SH-단량체 L19-Gly-Cys-Gly-Cys-OH로의 정량적 전환을 증명했다.In 160 μl PBS/10% glycerin 240 μg (4.29 nmol) SS-dimer L19-Gly-Cys-Gly-Cys-OH in 75 μl TCEP-solution (14.34 mg TCEPxHCl/5 ml aqueous Na 2 HPO 4 , 0.1 M , pH=7.4) was added. The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer L19-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS). SDS-PAGE analysis of the isolated product demonstrated the quantitative conversion of SS-dimer L19-Gly-Cys-Gly-Cys-OH to SH-monomer L19-Gly-Cys-Gly-Cys-OH.
수율: 80.4㎍/210㎕ PBS (33.5%). Yield: 80.4 μg/210 μl PBS (33.5%).
실시예 6bExample 6b
Tc-99m-L19-Gly-Cys-Gly-Cys-OH의 합성Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-OH
2.37mg 디소듐-L-타르트레이트를 작은 병에 넣은 후 210㎕ PBS 내의 80.4㎍ 환원된 L19-Gly-Cys-Gly-Cys-OH를 첨가하고, 용액을 100㎕ 수성 Na2HPO4-완충제 (1M, pH=10.5)로 희석했다. 50㎕ Tc-99m 생성기 용리액 (24h) 및 10㎕ SnCl2-용액 (5mg SnCl2/1ml 0.1M HCl)을 첨가했다. 반응 혼합물을 37℃에서 반시간 동안 진탕하였다. Tc-99m-표지된 L19-Gly-Cys-Gly-Cys-OH를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.2.37mg disodium-L-tartrate was put in a small bottle, then 80.4 µg reduced L19-Gly-Cys-Gly-Cys-OH in 210 µl PBS was added, and the solution was added to 100 µl aqueous Na 2 HPO 4 -buffer ( 1M, pH=10.5). 50 μl Tc-99m generator eluent (24h) and 10 μl SnCl 2 -solution (5 mg SnCl 2 /1 ml 0.1M HCl) were added. The reaction mixture was shaken at 37° C. for half an hour. Tc-99m-labeled L19-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 37.7%. Radiochemical yield: 37.7%.
방사화학적 순도: 91.5% (SDS-PAGE). Radiochemical purity: 91.5% (SDS-PAGE).
비활성도: 19.7MBq/nmol. Specific activity: 19.7 MBq/nmol.
면역반응성: 89.7%Immune Reactivity: 89.7%
실시예 7aExample 7a
환원된 L19-Gly-Cys-Gly-Cys-Ala-OH의 합성Synthesis of reduced L19-Gly-Cys-Gly-Cys-Ala-OH
155㎕ PBS/10% 글리세린 내의 240㎍ (4.29nmol) S-S-이량체 L19-Gly-Cys-Gly-Cys-Ala-OH 용액에 75㎕ TCEP-용액 (14.34mg TCEPxHCl/5ml 수성 Na2HPO4, 0.1M, pH=7.4)을 첨가했다. 반응 혼합물을 실온에서 1시간 동안 서서히 진탕하였다. SH-단량체 L19-Gly-Cys-Gly-Cys-Ala-OH를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다. 단리된 산물의 SDS-PAGE 분석은 S-S-이량체 L19-Gly-Cys-Gly-Cys-Ala-OH에서 SH-단량체 L19-Gly-Cys-Gly-Cys-Ala-OH로의 정량적 전환을 증명했다. 75 μl TCEP-solution (14.34 mg TCEPxHCl/5 ml aqueous Na 2 HPO 4 , in 240 μg (4.29 nmol) SS-dimer L19-Gly-Cys-Gly-Cys-Ala-OH solution in 155 μl PBS/10% glycerin 0.1M, pH=7.4) was added. The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer L19-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS). SDS-PAGE analysis of the isolated product demonstrated a quantitative conversion from SS-dimer L19-Gly-Cys-Gly-Cys-Ala-OH to SH-monomer L19-Gly-Cys-Gly-Cys-Ala-OH.
수율: 81.2㎍/215㎕ PBS (33.8%). Yield: 81.2 μg/215 μl PBS (33.8%).
실시예 7bExample 7b
Tc-99m-L19-Gly-Cys-Gly-Cys-Ala-OH의 합성Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-Ala-OH
2.37mg 디소듐-L-타르트레이트를 작은 병에 넣은 후 215㎕ PBS 내의 81.2㎍ 환원된 L19-Gly-Cys-Gly-Cys-Ala-OH를 첨가하고, 용액을 100㎕ 수성 Na2HPO4-완충제 (1M, pH=10.5)로 희석했다. 50㎕ Tc-99m 생성기 용리액 (24h) 및 10㎕ SnCl2-용액 (5mg SnCl2/1ml 0.1M HCl)을 첨가했다. 반응 혼합물을 37℃에서 반시간 동안 진탕하였다. Tc-99m-표지된 L19-Gly-Cys-Gly-Cys-Ala-OH를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.2.37mg disodium-L-tartrate was put in a small bottle, and then 81.2 µg reduced L19-Gly-Cys-Gly-Cys-Ala-OH in 215 µl PBS was added, and the solution was added to 100 µl aqueous Na 2 HPO 4- It was diluted with buffer (1M, pH=10.5). 50 μl Tc-99m generator eluent (24h) and 10 μl SnCl 2 -solution (5 mg SnCl 2 /1 ml 0.1M HCl) were added. The reaction mixture was shaken at 37° C. for half an hour. Tc-99m-labeled L19-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 35.6%. Radiochemical yield: 35.6%.
방사화학적 순도: 93.5% (SDS-PAGE). Radiochemical purity: 93.5% (SDS-PAGE).
비활성도: 19.1MBq/nmol. Specific specificity: 19.1 MBq/nmol.
면역반응성: 88.7%Immune Reactivity: 88.7%
실시예 8aExample 8a
시스테인-SH 기에의 EDTA, CDTA, TETA, DTPA, TTHA, HBED, DOTA, NOTA, 및 D03A형 킬레이트제의 특이적 콘쥬게이션을 위한 환원된 AP39의 합성Synthesis of reduced AP39 for specific conjugation of EDTA, CDTA, TETA, DTPA, TTHA, HBED, DOTA, NOTA, and D03A type chelating agents to cysteine-SH groups
50㎕ TCEP-용액 (14.34mg TCEPxHCl/5ml 수성 Na2HPO4, 0.1M, pH=7.4)을 450㎕ PBS 내의 400㎍ (7.1nmol) AP39 용액에 첨가했다. 반응 혼합물을 37℃에서 1시간 동안 서서히 진탕하였다. 환원된 AP39를 NAP-5 칼럼 (아메르샴, 용리액: 아세트산 나트륨 완충제, 0.1M, pH 5.0)을 이용해 젤-크로마토그래피로 정제했다. 단리된 산물의 SDS-PAGE 분석은 AP39에서 환원된 AP39로의 완전한 전환을 증명했다.50 μl TCEP-solution (14.34 mg TCEPxHCl/5 ml aqueous Na 2 HPO 4 , 0.1 M, pH=7.4) was added to a 400 μg (7.1 nmol) AP39 solution in 450 μl PBS. The reaction mixture was shaken slowly at 37° C. for 1 hour. The reduced AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: sodium acetate buffer, 0.1M, pH 5.0). SDS-PAGE analysis of the isolated product demonstrated complete conversion from AP39 to reduced AP39.
수율: 140㎍/200㎕ (35%). Yield: 140 μg/200 μl (35%).
실시예 8bExample 8b
MX-DTPA-말레이미드 (1,4,7-트리아자-2-(N-말레이미도 에틸렌 MX-DTPA-maleimide (1,4,7-triaza-2-(N-maleimido ethylene pp -아미노)벤질-1,7-비스(카르복시메틸)-4-카르복시메틸 6-메틸 헵탄)의 합성Synthesis of -amino)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl 6-methyl heptane)
512mg (1mmol) {[3-(4-아미노-페닐)-2-(비스-카르복시메틸-아미노)-프로필]-[2-(비스-카르복시메틸-아미노)-프로필]-아미노}-아세트산 (마크로시클릭스 인크. (Macrocyclics Inc.), 미국 텍사스주 달라스 소재) 및 707mg (7mmol) 트리에틸아민을 3ml 건조 DMF에 용해했다. 1ml 건조 DMF 내의 400mg (1.5mmol) 3-(2,5-디옥소-2,5-디히드로-피롤-1-일)-프로피온산 2,5-디옥소-피롤리딘-1-일 에스테르 (알드리치 (Aldrich))를 적가했다. 용액을 50℃에서 5시간 교반했다. 30ml 디에틸에테르를 천천히 첨가했다. 반응 혼합물을 추가로 30분간 교반했다. 침전물을 여과에 의해 수집했다. 조생성물을 RP-HPLC (아세토니트릴-:물-:트리플루오르아세트산/3:96.9:0.1→99.9:0:0.1)에 의해 정제했다. 수율: 61% (405mg, 0.61mmol). MS-ESI: 664=M++1. 512mg (1mmol) {[3-(4-amino-phenyl)-2-(bis-carboxymethyl-amino)-propyl]-[2-(bis-carboxymethyl-amino)-propyl]-amino}-acetic acid ( Macrocyclics Inc., Dallas, TX) and 707 mg (7 mmol) triethylamine were dissolved in 3 ml dry DMF. 400mg (1.5mmol) 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester in 1 ml dry DMF ( Aldrich) was added dropwise. The solution was stirred at 50°C for 5 hours. 30 ml diethyl ether was slowly added. The reaction mixture was stirred for an additional 30 minutes. The precipitate was collected by filtration. The crude product was purified by RP-HPLC (acetonitrile-:water-:trifluoroacetic acid/3:96.9:0.1→99.9:0:0.1). Yield: 61% (405mg, 0.61mmol). MS-ESI: 664=M + +1.
실시예 8cExample 8c
In-111-MX-DTPA-말레이미드-S(Cys)-AP39-R의 합성 Synthesis of In-111-MX-DTPA-maleimide-S(Cys)-AP39-R
(R = 환원된)(R = reduced)
200㎕ 아세트산 나트륨 완충제 (0.1M, pH 5) 내의 140㎍ (5nmol) AP39-R을 50㎕ 용해된 1,4,7-트리아자-2-(N-말레이미도 에틸렌 p-아미노)벤질-1,7-비스(카르복시메틸)-4-카르복시메틸 6-메틸헵탄 (500㎕ 아세트산 나트륨 완충제 0.1M pH 5 내의 0.25mg DTPA-말레이미드)과 37℃에서 3시간 반응시켰다. 반응 혼합물을 200ml 아세트산 나트륨 완충제 (0.1M, pH 6)로 2x1h로 슬라이드-어-라이저 (Slide-A-Lyzer) 10,000 MWCO (피어스 인크. (Pierce Inc.), 미국 일리노이주 락포드 소재)를 사용해 투석했다. 1,4,7-triaza-2-(N-maleimido ethylene p -amino)benzyl-1 dissolved in 50 μl of 140 μg (5 nmol) AP39-R in 200 μl sodium acetate buffer (0.1 M, pH 5) ,7-bis(carboxymethyl)-4-carboxymethyl 6-methylheptane (0.25 mg DTPA-maleimide in 500 μl sodium acetate buffer 0.1M pH 5) was reacted at 37°C for 3 hours. The reaction mixture was 200 ml sodium acetate buffer (0.1 M, pH 6) at 2×1 h using a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, Ill.). Dialysis.
80㎕ [In-111]InCl3 용액 (HCl, 1N, 40MBq, 아메르샴 인크.)을 첨가하고 반응 혼합물을 37℃에서 30분간 가열했다. In-111 표지된 DTPA-말레이미드-S(Cys)-AP39-R을 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.80 [mu]l [In-111]InCl 3 solution (HCl, 1N, 40MBq, Amersham Inc.) was added and the reaction mixture was heated at 37[deg.] C. for 30 minutes. In-111 labeled DTPA-maleimide-S(Cys)-AP39-R was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 54%. Radiochemical yield: 54%.
방사화학적 순도: 94% (SDS-PAGE). Radiochemical purity: 94% (SDS-PAGE).
비활성도: 6.2MBq/nmol. Specific specificity: 6.2 MBq/nmol.
면역반응성: 86%Immune Reactivity: 86%
실시예 9 Example 9
In-111-MX-DTPA-ε-HN(Lys)-AP39의 합성 Synthesis of In-111-MX-DTPA-ε-HN(Lys)-AP39
111㎕ PBS 내의 200㎍ (3.6nmol) 비환원된 AP39를 300㎕ 붕산 나트륨 완충제 (0.1M, pH 8.5)로 희석하고 200ml 붕산 나트륨 완충제 (0.1M, pH 8.5)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 2x1h로 투석했다. 50㎕ 1,4,7-트리아자-2-(p-이소티오시아나토)벤질-1,7-비스(카르복시메틸)-4-카르복시메틸-6-메틸헵탄 (MX-DTPA) 용액 (500㎕ 붕산 나트륨 완충제 0.1M pH 8.5 내 용해된 0.33mg MX-DTPA)을 첨가하고 반응 혼합물을 37℃에서 3시간 가열했다. 반응 혼합물을 200ml 아세트산 나트륨 완충제 (0.1M, pH 6.0)로 각각 2x1h 및 1x17h (밤샘)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 투석했다.200 μg (3.6 nmol) non-reduced AP39 in 111 μl PBS was diluted with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO ( Pierce Inc., Rockford, Illinois) and dialyzed at 2x1h. 50 μl 1,4,7-triaza-2-( p -isothiocyanato)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl-6-methylheptane (MX-DTPA) solution (500 0.33 mg MX-DTPA) dissolved in [mu]l sodium borate buffer 0.1M pH 8.5) was added and the reaction mixture was heated at 37[deg.] C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) at 2x1h and 1x17h (overnight) respectively using Slide-A-Riser 10,000 MWCO (Pierce Inc., Rockford, IL).
80㎕ [In-111]InCl3 용액 (HCl, 1N, 40MBq, 아메르샴 인크.)을 첨가하고 반응 혼합물을 37℃에서 30분간 가열했다. In-111 표지된 MX-DTPA-ε-HN(Lys)-AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.80 [mu]l [In-111]InCl 3 solution (HCl, 1N, 40MBq, Amersham Inc.) was added and the reaction mixture was heated at 37[deg.] C. for 30 minutes. In-111 labeled MX-DTPA-ε-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 70%. Radiochemical yield: 70%.
방사화학적 순도: 85% (SDS-PAGE). Radiochemical purity: 85% (SDS-PAGE).
비활성도: 7.6MBq/nmol. Specific activity: 7.6 MBq/nmol.
면역반응성: 74%Immune Reactivity: 74%
실시예 10Example 10
In-111-DOTA-C-벤질-p-NCS-ε-HN(Lys)-AP39의 합성Synthesis of In-111-DOTA-C-benzyl-p-NCS-ε-HN(Lys)-AP39
114㎕ PBS 내의 200㎍ (3.6nmol) 비환원된 AP39를 300㎕ 붕산 나트륨 완충제 (0.1M, pH 8.5)로 희석하고 200ml 붕산 나트륨 완충제 (0.1M, pH 8.5)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 2x1h로 투석했다. 50㎕ 1,4,7,10-테트라아자-2-(p-이소티오시아나토)벤질 시클로도데칸-1,4,7,10-테트라아세트산 (벤질-p-SCN-DOTA, 마크로시클릭스 인크., 미국 텍사스주 달라스 소재) 용액 (5ml 붕산 나트륨 완충제 0.1M pH 8.5 내 용해된 1.5mg 벤질-p-SCN-DOTA)을 용액에 첨가하고 반응 혼합물을 37℃에서 3시간 가열했다. 반응 혼합물을 200ml 아세트산 나트륨 완충제 (0.1M, pH 6.0)로 각각 2x1h 및 1x17h (밤샘)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 투석했다.Dilute 200 μg (3.6 nmol) non-reduced AP39 in 114 μl PBS with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO ( Pierce Inc., Rockford, Illinois) and dialyzed at 2x1h. 50 μl 1,4,7,10-tetraaza-2-( p -isothiocyanato)benzyl cyclododecane-1,4,7,10-tetraacetic acid (benzyl-p-SCN-DOTA, macrocyclics Inc., Dallas, TX) solution (1.5 mg benzyl-p-SCN-DOTA dissolved in 5 ml sodium borate buffer 0.1 M pH 8.5) was added to the solution and the reaction mixture was heated at 37° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) at 2x1h and 1x17h (overnight) respectively using Slide-A-Riser 10,000 MWCO (Pierce Inc., Rockford, IL).
80㎕ [In-111]InCl3 용액 (HCl, 1N, 40MBq, 아메르샴 인크.)을 첨가하고 반응 혼합물을 37℃에서 30분간 가열했다. In-111 표지된 DOTA-C-벤질-p-NCS-ε-HN(Lys)-AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.80 [mu]l [In-111]InCl 3 solution (HCl, 1N, 40MBq, Amersham Inc.) was added and the reaction mixture was heated at 37[deg.] C. for 30 minutes. In-111 labeled DOTA-C-benzyl-p-NCS-ε-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 74%. Radiochemical yield: 74%.
방사화학적 순도: 94% (SDS-PAGE). Radiochemical purity: 94% (SDS-PAGE).
비활성도: 12.3MBq/nmol. Specific activity: 12.3 MBq/nmol.
면역반응성: 73%Immune Reactivity: 73%
실시예 11Example 11
Y-88-MX-DTPA-ε-HN(Lys)-AP39의 합성Synthesis of Y-88-MX-DTPA-ε-HN(Lys)-AP39
115㎕ PBS 내의 200㎍ (3.6nmol) 비환원된 AP39를 300㎕ 붕산 나트륨 완충제 (0.1M, pH 8.5)로 희석하고 200ml 붕산 나트륨 완충제 (0.1M, pH 8.5)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 2x1h로 투석했다. 50㎕ MX-DTPA 용액 (500㎕ 붕산 나트륨 완충제 0.1M pH 8.5 내 용해된 0.33mg MX-DTPA)을 첨가하고 반응 혼합물을 37℃에서 3시간 가열했다. 반응 혼합물을 200ml 아세트산 나트륨 완충제 (0.1M, pH 6.0)로 각각 2x1h 및 1x17h (밤샘)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 투석했다.Dilute 200 μg (3.6 nmol) non-reduced AP39 in 115 μl PBS with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO ( Pierce Inc., Rockford, Illinois) and dialyzed at 2x1h. 50 μl MX-DTPA solution (0.33 mg MX-DTPA dissolved in 500 μl sodium borate buffer 0.1 M pH 8.5) was added and the reaction mixture was heated at 37° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) at 2x1h and 1x17h (overnight), respectively, using Slide-A-Riser 10,000 MWCO (Pierce Inc., Rockford, IL).
100㎕ [Y-88]YCl3 용액 (HCl, 1N, 75MBq, 오크 리찌 내셔널 랩. (Oak Ridge National Lab.))을 첨가하고 반응 혼합물을 37℃에서 30분간 가열했다. Y-88 표지된 MX-DTPA-ε-HN(Lys)-AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.100 μl [Y-88]YCl 3 solution (HCl, 1N, 75MBq, Oak Ridge National Lab.) was added and the reaction mixture was heated at 37° C. for 30 minutes. Y-88 labeled MX-DTPA-ε-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 65%. Radiochemical yield: 65%.
방사화학적 순도: 93% (SDS-PAGE). Radiochemical purity: 93% (SDS-PAGE).
비활성도: 10.2MBq/nmol. Specific specificity: 10.2 MBq/nmol.
면역반응성: 72%Immune Reactivity: 72%
실시예 12Example 12
Lu-177-DOTA-C-벤질-p-NCS-ε-HN(Lys)-AP3의 합성Synthesis of Lu-177-DOTA-C-benzyl-p-NCS-ε-HN(Lys)-AP3
110㎕ PBS 내의 200㎍ (3.6nmol) 비환원된 AP39를 300㎕ 붕산 나트륨 완충제 (0.1M, pH 8.5)로 희석하고 200ml 붕산 나트륨 완충제 (0.1M, pH 8.5)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 2 x 1h로 투석했다. 50㎕ 벤질-p-SCN-DOTA 용액 (5ml 붕산 나트륨 완충제 0.1M pH 8.5 내 용해된 1.5mg)을 첨가하고 반응 혼합물을 37℃에서 3시간 가열했다. 반응 혼합물을 200ml 아세트산 나트륨 완충제 (0.1M, pH 6.0)로 각각 2x1h 및 1x17h (밤샘)로 슬라이드-어-라이저 10,000 MWCO (피어스 인크., 미국 일리노이주 락포드 소재)를 사용해 투석했다.200 μg (3.6 nmol) non-reduced AP39 in 110 μl PBS was diluted with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO ( Pierce Inc., Rockford, Illinois) and dialyzed at 2 x 1h. 50 μl benzyl-p-SCN-DOTA solution (1.5 mg dissolved in 5 ml sodium borate buffer 0.1 M pH 8.5) was added and the reaction mixture was heated at 37° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) at 2x1h and 1x17h (overnight), respectively, using Slide-A-Riser 10,000 MWCO (Pierce Inc., Rockford, IL).
200㎕ [Lu-177]LuCl3 용액 (HCl, 1N, 80MBq, NRH-페텐 (NRH-Petten, 네덜란드))을 첨가하고 반응 혼합물을 37℃에서 30분간 가열했다. Lu-177 표지된 DOTA-C-벤질-p-NCS-ε-HN(Lys)-AP39를 NAP-5 칼럼 (아메르샴, 용리액: PBS)을 이용해 젤-크로마토그래피로 정제했다.200 μl [Lu-177]LuCl 3 solution (HCl, 1N, 80MBq, NRH-petene (NRH-Petten, Netherlands)) was added and the reaction mixture was heated at 37° C. for 30 minutes. Lu-177 labeled DOTA-C-benzyl-p-NCS-ε-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).
방사화학적 수율: 74%. Radiochemical yield: 74%.
방사화학적 순도: 95% (SDS-PAGE). Radiochemical purity: 95% (SDS-PAGE).
비활성도: 19MBq/nmol. Specific specificity: 19 MBq/nmol.
면역반응성: 71%Immune Reactivity: 71%
실시예 13Example 13
종양 가진 누드 생쥐 내로 1회 정맥 주사 후 피치아 파스토리스 (Pichia pastoris)에서 발현된 Tc-99m-AP39의 기관 분포 및 배출Organ distribution and excretion of Tc-99m-AP39 expressed in Pichia pastoris after one intravenous injection into tumor-bearing nude mice
본 발명의 물질을 F9 (기형암)-가진 동물 (체중 약 25g) 내로 약 74kBq의 투여량으로 정맥 내 주사하였다. 다양한 기관에서의 방사능 농도 및 분뇨의 방사능을 물질의 투여 후 다양한 시간에 γ계수기를 이용해 측정했다. 추가로, 종양 대 혈액 비를 종양 및 혈액에서의 본 발명의 물질의 농도를 기초로 다양한 시간에 측정했다.The substance of the present invention was injected intravenously into an F9 (teratocarcinoma)-bearing animal (weight about 25 g) at a dose of about 74 kBq. The concentration of radioactivity in various organs and radioactivity of manure were measured using a γ counter at various times after administration of the substance. Additionally, the tumor to blood ratio was measured at various times based on the concentration of the substance of the invention in the tumor and blood.
F9 (기형암)-가진 누드 생쥐의 Tc-99m-AP39의 생체분포 (평균±SD, n=3)는 표 2에 보여진다:The biodistribution (mean±SD, n=3) of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice is shown in Table 2:
F9 (기형암)-가진 누드 생쥐의 Tc-99m-AP39의 배출 (평균±SD, n=3)은 표 3에 보여진다:The excretion (mean±SD, n=3) of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice is shown in Table 3:
F9 (기형암)-가진 누드 생쥐의 Tc-99m-AP39의 종양 대 혈액 비 (평균±SD, n=3)는 도 2에 보여진다. The tumor to blood ratio (mean±SD, n=3) of Tc-99m-AP39 in nude mice with F9 (teratocarcinoma)- is shown in FIG. 2.
본 발명의 결과는 고형 종양에서의 축적에 대한 본 발명의 물질의 우수한 잠재성과 함께 동시에 우수한 배출을 보여준다.The results of the present invention show good excretion at the same time with the good potential of the substances of the present invention for accumulation in solid tumors.
실시예 14Example 14
종양 가진 누드 생쥐 내로 1회 정맥 주사 후 In-111-MX-DTPA-ε-HN(Lys)-AP39의 기관 분포Organ distribution of In-111-MX-DTPA-ε-HN(Lys)-AP39 after one intravenous injection into tumor-bearing nude mice
본 발명의 물질을 F9 (기형암)-가진 동물 (체중 약 25g) 내로 약 48kBq의 투여량으로 정맥 내 주사하였다. 다양한 기관에서의 방사능 농도 및 분뇨의 방사능을 물질의 투여 후 다양한 시간에 γ계수기를 사용해 측정했다. The substance of the present invention was injected intravenously into an F9 (teratocarcinoma)-bearing animal (weight about 25 g) at a dose of about 48 kBq. The concentration of radioactivity in various organs and the radioactivity of manure were measured using a γ counter at various times after administration of the substance.
F9 (기형암)-가진 누드 생쥐의 In-111-MX-DTPA-ε-HN(Lys)-AP39의 생체분포 (평균±SD, n=3)는 표 4에 보여진다:The biodistribution (mean±SD, n=3) of In-111-MX-DTPA-ε-HN(Lys)-AP39 in F9 (teratocarcinoma)-bearing nude mice is shown in Table 4:
F9 (기형암)-가진 누드 생쥐의 In-111-MX-DTPA-ε-HN(Lys)-AP39의 종양 대 혈액 비 (평균±SD, n=3)는 표 5에 보여진다.The tumor-to-blood ratio (mean±SD, n=3) of In-111-MX-DTPA-ε-HN(Lys)-AP39 in nude mice with F9 (teratocarcinoma)- is shown in Table 5.
본 발명의 결과는 고형 종양에서의 축적에 대한 본 발명의 물질의 우수한 잠재성과 함께 우수한 생체분포 및 종양 대 혈액 비를 보여준다.The results of the present invention show excellent biodistribution and tumor to blood ratio with the excellent potential of the substances of the present invention for accumulation in solid tumors.
실시예 15Example 15
종양 가진 누드 생쥐 내로 1회 정맥 주사 후 피치아 파스토리스에서 발현된 Tc-99m-AP39의 영상 촬영Imaging of Tc-99m-AP39 Expressed in Pichia Pastoris After One Intravenous Injection into Tumor-bearing Nude Mice
본 발명의 물질을 F9 (기형암)-가진 동물 (체중 약 25g) 내로 약 9.25MBq의 투여량으로 정맥 내 주사하였다. 물질의 투여 후 다양한 시간에 감마-카메라 영상 촬영을 실시했다. The substance of the present invention was injected intravenously into an F9 (teratocarcinoma)-bearing animal (weight about 25 g) at a dose of about 9.25 MBq. Gamma-camera imaging was performed at various times after administration of the substance.
F9 (기형암)-가진 누드 생쥐에서의 Tc-99m-AP39의 섬광조영술은 도 3 및 도 4에 보여진다. 도 3은 물질 주사 5시간 후의 신티그램을 보여주고 도 4는 물질 주사 24시간 후의 신티그램을 보여준다. Scintography of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice is shown in FIGS. 3 and 4. Figure 3 shows the
본 발명의 결과는 고형 종양의 영상 촬영에 대한 본 발명의 물질의 우수한 잠재성을 보여준다.
The results of the present invention show the excellent potential of the substances of the present invention for imaging solid tumors.
SEQUENCE LISTING <110> Schering AG <120> New methods for diagnosis and treatment of tumours <130> 27041P_WOAS <140> <141> <150> EP02 000 315.8 <151> 2002-01-03 <150> US60/358702 <151> 2002-02-25 <160> 13 <170> PatentIn Ver. 2.1 <210> 1 <211> 238 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(116) <223> VH <220> <221> SITE <222> (117)..(130) <223> Linker <220> <221> SITE <222> (131)..(238) <223> VL <220> <221> VARIANT <222> (1)..(238) <223> deletion, insertion and/or substitution of up to 30 amino acids <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <220> <221> VARIANT <222> (1)..(3) <223> xaa each independently represent any naturally occuring amino acid <400> 2 Xaa Xaa Xaa Cys 1 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <220> <221> VARIANT <222> (1)..(3) <223> Xaa each independently represent any naturally occuring amino acid <220> <221> VARIANT <222> (5) <223> Xaa represents any naturally occuring amino acid <400> 3 Xaa Xaa Xaa Cys Xaa 1 5 <210> 4 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <220> <221> REPEAT <222> (1) <223> His=(His)n, wherein n stands for an integer from 4 to 6 <400> 4 His 1 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 5 Gly Gly Gly Cys 1 <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 6 Gly Cys Gly Cys 1 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 7 Gly Gly Gly Cys Ala 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 8 Gly Cys Gly Cys Ala 1 5 <210> 9 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ala Ala Leu 225 230 235 240 Glu His His His His His His 245 <210> 10 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Cys 225 230 235 240 <210> 11 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Cys 225 230 235 240 Ala <210> 12 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Cys Gly Cys 225 230 235 240 <210> 13 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Cys Gly Cys 225 230 235 240 Ala SEQUENCE LISTING <110> Schering AG <120> New methods for diagnosis and treatment of tumours <130> 27041P_WOAS <140> <141> <150> EP02 000 315.8 <151> 2002-01-03 <150> US60/358702 <151> 2002-02-25 <160> 13 <170> Patent In Ver. 2.1 <210> 1 <211> 238 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(116) <223> VH <220> <221> SITE <222> (117)..(130) <223> Linker <220> <221> SITE <222> (131)..(238) <223> VL <220> <221> VARIANT <222> (1)..(238) <223> deletion, insertion and/or substitution of up to 30 amino acids <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <220> <221> VARIANT <222> (1)..(3) <223> xaa each independently represent any naturally occuring amino acid <400> 2 Xaa Xaa Xaa Cys One <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <220> <221> VARIANT <222> (1)..(3) <223> Xaa each independently represent any naturally occuring amino acid <220> <221> VARIANT <222> (5) <223> Xaa represents any naturally occuring amino acid <400> 3 Xaa Xaa Xaa Cys Xaa 1 5 <210> 4 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <220> <221> REPEAT <222> (1) <223> His=(His)n, wherein n stands for an integer from 4 to 6 <400> 4 His One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 5 Gly Gly Gly Cys One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 6 Gly Cys Gly Cys One <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 7 Gly Gly Gly Cys Ala 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 8 Gly Cys Gly Cys Ala 1 5 <210> 9 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ala Ala Leu 225 230 235 240 Glu His His His His His His 245 <210> 10 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Cys 225 230 235 240 <210> 11 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Cys 225 230 235 240 Ala <210> 12 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Cys Gly Cys 225 230 235 240 <210> 13 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant antibody fragment <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser 115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Cys Gly Cys 225 230 235 240 Ala
Claims (1)
ab) 표 1에 보여진 상보성-결정 지역 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 및 LCDR3을 포함하는 피브로넥틴의 세포외 도메인 B (ED-B)에 대한 항원-결합 부위 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 HCDR1 지역의 3개 이하의 아미노산, HCDR2 지역의 8개 이하의 아미노산, HCDR3 지역의 5개 이하의 아미노산, LCDR1 지역의 6개 이하의 아미노산, LCDR2 지역의 4개 이하의 아미노산 및 LCDR3 지역의 6개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 그의 변이체; 또는
ac) 서열번호 1 (L19)에 따른 서열 또는 서열번호 1에 따른 펩티드와 동일한 기능을 가진 30개 이하의 아미노산의 결실, 삽입 및(또는) 치환인 서열번호 1의 변이체, 및
ba) 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys (서열번호 2) (여기서, Xaa1, Xaa2, 및 Xaa3은 각각 임의의 자연 발생의 아미노산을 독립적으로 나타냄) 또는
bb) 아미노산 서열 Xaa1-Xaa2-Xaa3-Cys-Xaa4 (서열번호 3) (여기서, Xaa1, Xaa2, Xaa3, 및 Xaa4는 각각 임의의 자연 발생의 아미노산을 독립적으로 나타냄) 또는
bc) 아미노산 서열 (His)n (서열번호 4) (여기서 n은 4 내지 6의 정수를 나타냄)
을 포함하고, 여기서 aa), ab) 또는 ac)의 C-말단이 펩티드 결합을 통해 서열번호 2, 서열번호 3, 또는 서열번호 4의 서열들 중의 하나의 N-말단에 결합되는 펩티드의 방사성동위원소를 결합하기 위한 용도. aa) an antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and / or LCDR3 shown in Table 1 or an HCDR3 region having the same function as a peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions, and / or substitutions of up to 5 amino acids of and up to 6 amino acids of the LCDR3 region;
ab) the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or the peptide according to SEQ ID NO: 1 Less than 3 amino acids in the HCDR1 region, up to 8 amino acids in the HCDR2 region, up to 5 amino acids in the HCDR3 region, up to 6 amino acids in the LCDR1 region, up to 4 amino acids in the LCDR2 region and LCDR3 region Variants thereof which are the deletion, insertion and / or substitution of up to 6 amino acids of; or
ac) a variant of SEQ ID NO: 1 that is a deletion, insertion, and / or substitution of up to 30 amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and
ba) the amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys (SEQ ID NO: 2), wherein Xaa 1 , Xaa 2 , and Xaa 3 each independently represent any naturally occurring amino acid; or
bb) Amino acid sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 4 (SEQ ID NO: 3), wherein Xaa 1 , Xaa 2 , Xaa 3 , and Xaa 4 each independently represent any naturally occurring amino acid or
bc) amino acid sequence (His) n (SEQ ID NO: 4), where n represents an integer from 4 to 6
Wherein the C-terminus of aa), ab), or ac) is linked to the N-terminus of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 via a peptide bond For combining elements.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP02000315 | 2002-01-03 | ||
| EP02000315.8 | 2002-01-03 | ||
| US35870202P | 2002-02-25 | 2002-02-25 | |
| US60/358,702 | 2002-02-25 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020047010498A Division KR100998803B1 (en) | 2002-01-03 | 2003-01-02 | Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20100077052A true KR20100077052A (en) | 2010-07-06 |
Family
ID=35976951
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020107013008A KR20100077052A (en) | 2002-01-03 | 2003-01-02 | Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours |
| KR1020047010498A KR100998803B1 (en) | 2002-01-03 | 2003-01-02 | Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020047010498A KR100998803B1 (en) | 2002-01-03 | 2003-01-02 | Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours |
Country Status (8)
| Country | Link |
|---|---|
| KR (2) | KR20100077052A (en) |
| AT (1) | ATE478095T1 (en) |
| DE (1) | DE60333820D1 (en) |
| DK (1) | DK1461360T3 (en) |
| ES (1) | ES2346750T3 (en) |
| PT (1) | PT1461360E (en) |
| SI (1) | SI1461360T1 (en) |
| ZA (1) | ZA200406162B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10975477B2 (en) | 2017-10-02 | 2021-04-13 | Battelle Energy Alliance, Llc | Methods and systems for the electrochemical reduction of carbon dioxide using switchable polarity materials |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI259837B (en) * | 1998-05-11 | 2006-08-11 | Eidgenossische Tech Hochscule | Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis |
-
2003
- 2003-01-02 DE DE60333820T patent/DE60333820D1/en not_active Expired - Lifetime
- 2003-01-02 PT PT03726977T patent/PT1461360E/en unknown
- 2003-01-02 ES ES03726977T patent/ES2346750T3/en not_active Expired - Lifetime
- 2003-01-02 KR KR1020107013008A patent/KR20100077052A/en not_active Application Discontinuation
- 2003-01-02 KR KR1020047010498A patent/KR100998803B1/en not_active IP Right Cessation
- 2003-01-02 AT AT03726977T patent/ATE478095T1/en not_active IP Right Cessation
- 2003-01-02 SI SI200331895T patent/SI1461360T1/en unknown
- 2003-01-02 DK DK03726977.6T patent/DK1461360T3/en active
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2004
- 2004-08-02 ZA ZA200406162A patent/ZA200406162B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10975477B2 (en) | 2017-10-02 | 2021-04-13 | Battelle Energy Alliance, Llc | Methods and systems for the electrochemical reduction of carbon dioxide using switchable polarity materials |
Also Published As
| Publication number | Publication date |
|---|---|
| SI1461360T1 (en) | 2010-12-31 |
| DE60333820D1 (en) | 2010-09-30 |
| PT1461360E (en) | 2010-11-23 |
| ZA200406162B (en) | 2005-09-27 |
| DK1461360T3 (en) | 2010-11-15 |
| KR100998803B1 (en) | 2010-12-06 |
| KR20040073540A (en) | 2004-08-19 |
| ES2346750T3 (en) | 2010-10-20 |
| ATE478095T1 (en) | 2010-09-15 |
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