This work supported by research grant funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Ontario Institute for Cancer Research (OICR, #P.SI.015.8), and the Canadian Cancer Society (CCS, #703857). The authors acknowledge the contributions of Dr. Denis Snider who provided assistance in preparing the manuscript.

동물 실험은 동물 관리에 캐나다위원회 (CCAC) 지침에 따라 맥 매스터 대학의 동물 연구 윤리위원회의 승인을했다. 99m Tc를 가진 Tz 일 - 세자리 리간드 1. 방사성 표지 주의 : 다음 절차 방사성 화합물의 사용을 요구한다. 작업은 안전 및 폐기 규정을 준수하여 허가 된 실험실에서 수행해야합니다. 전자 레인지 반응은 특히 화학 합성을 위해 설계된 전자 레인지에 수행되어야한다. <ol><li> 의 합성 99m Tc를 (CO) 3 (H 2 O) 3] + 19 20 <ol><li> 마이크로파 바이알에 결합 8 mg의 K 2 BH 3 CO 2, 15 mg의 나트륨 2 CO 3, 20 mg의 나트륨이 B 4 O 7 · 10H 2 O, 및 25 mg의 KOCO [CH (OH)]COOH를 2 · 4H 2 O 퍼지 아르곤 가스로 10 분 동안 바이알. </li><li> 유리 병에 0.9 % 식염수 (~ 1100 MBq의, ~ 30 MCI) - TCO 4 99m의 4 ML을 추가합니다. </li><li> 반응물의 완전한 혼합을 보장하기 위해 10 초 동안 교반 한 후 110 ° C에서 3.5 분 동안 극초단파에서 반응물을 가열 하였다. </li><li> ~ 400 μL, 1 M HCl을 사용 3.5-4 상기 용액의 pH를 조정한다. 산도 용지를 사용하여 확인합니다. </li></ol></li><li> 테트라의 방사성 표지는 1-5 리간드 <ol><li> 250 μL 메탄올 (21)의 각 리간드의 2 밀리그램 (화합물 1-5)을 녹인다. </li><li> 250 μL를 추가 [99m Tc를 (CO) 3 (H 2 O) 3] + (~ 74 MBq의, ~ 2 MCI) 각 솔루션. </li><li> 60 ° C에서 20 분 동안 마이크로 웨이브를 이용하여 반응 혼합물을 가열한다. 참고 :이 단계는 모두 5 tetrazines에 대한 동일했다. </li><li> 화합물 2-5의 경우, 용매를 다시 증발 dissol1 V / V의 DCM : TFA (1) 1 mL에 생성 된 제품을했습니다. </li><li> 6 분 (2-4) 또는 10 분 (5)를위한 전자 레인지에 60 ° C에서 용해 반응 생성물 (2-5)를 가열한다. </li><li> 실온으로 냉각시킨 후, 증발기를 이용하여 용매 (36 ° C, 8 mbar에서 3 분, 6,000 RPM) 증발 1 건조 화합물을 용해 : 1 ACN : H 2 O 또는 1 : 1 메탄올 : H 2 O를 HPLC 정제에 앞서. </li><li> HPLC (C 18 역상)를 사용하여, 표지 된 테트라 리간드로부터 표지 생성물 분리를 포함하는 99m Tc를 표지 된 화합물 (1-5) 정제. 일반적으로 30:70 ACN의 용출 그라데이션 사용 : H 2 O를 (모두 0.1 % TFA와) 40:60 ACN에 : H 2 O 20 분 (18 분)와 C (18) 분석 4.6 × 100 밀리미터 열 이상. UV (254 nm의)과 감마 검출 모두를 사용합니다. <ol><li> 각 표지 된 생성물의 소량의 샘플을 받아 공동 INJ의 그것 HPLC 체류 시간과 비교반사된다 비 - 방사성 표준 재 분류 (20 % 0.125 mg의 메탄올 H 2 O). 재 분류 기준은 UV HPLC 트레이스에서 식별되고, γ-HPLC 트레이스에서 99m Tc를 표지 된 화합물을 동시에 용출된다. 이 공동 분사 화합물 Tc를이 표시된-99m의 신원을 확인하고, 비교 체류 시간에서의 피크를 나타낸다. </li></ol></li><li> 증발기 (36 ℃, 8 mbar에서 3 분, 6,000 RPM)를 사용하여 HPLC 분획으로부터 용매를 증발시켰다. </li><li> 0.5 % BSA 및 0.01 % 트윈 80을 함유하는 7.4 kBq / PBS에서 μL의 농도로 정제하여 화합물을 제형. </li><li> 표지 화합물이 안정 보장하기 위해 시험 관내 안정성 연구를 수행합니다. 안정성을 평가하기 위해 각 시점에서 HPLC의 혼합물을 소량 (3.7 MBq의)를 주입, 1, 4 및 6 시간 동안 37 ℃에서 인큐베이션 제형 화합물. </li></ol></li></ol> 2. 사전 대상으로 바이오 유통 연구 <ol><l동물의 난&gt; 준비 <ol><li> 7-9주 세, 여성을 Balb / c 마우스를 사용하여 (N = 3), 꼬리 정맥 주사를 통해 TCO-BP는 식염수 (20 ㎎ / ㎏) (5 μg의 / μL)에 공식화 관리 할 수 ​​있습니다. </li><li> 물리적 구속 장치에 마우스를 놓고 꼬리의 측면에있는 혈관을 확인하고 알코올 면봉으로 닦아. 꼬리의 끝에서 약 2cm에서 정맥에 얕은 각도 병렬에서 30 게이지 바늘을 삽입합니다. 천천히, 주사 바늘을 제거하고 출혈이 멈출 때까지 약간의 압력으로 주사 부위에 깨끗한 거즈 스폰지를 적용하는 플런저를 우울. </li><li> TCO-BP의 1 시간 후 분사에서 관리 ~ 0.5 % BSA 100 μL로 제형 99m Tc를-테트라의 0.74 MBq의 (20 μCi를), 0.01 % 트윈 80 PBS에서, 꼬리 정맥 주사를 통해. </li></ol></li><li> 생물 분포 연구 <ol><li> 원하는 시점 (t = 6 H)에서, 3 % 이소 플루 란 2 % 산소 혼합 가스를 사용하여 마우스를 마취시키다. 에 시연마취 된 마우스에 전자 핀치 철수는 마취의 수술 비행기 아래에 확인합니다. </li><li> 주사기 헤파린 전처리를 사용하여 혈액을 심장 천자를 통해 (1 ml)에 수집합니다. 계속 마취의 코 콘에서 코와의 뒷면에 놓고 마우스 및 동물에 칼 모양의 프로세스를 찾습니다. <ol><li> 20 ° 각도로 약간 칼 모양의 과정에서 동물의 정중선의 왼쪽에, 25 G 바늘을 삽입합니다. 완전히 바늘을 삽입하고 천천히 심장에 손상이 된 경우 바늘 허브에 피를 볼 플런저에 다시 당깁니다. 필요한 경우 심장에 구멍을 위해, 플런저를 누른 상태에서 약간 바늘을 다시 조정. 천천히 주사기로 피를 그립니다. </li></ol></li><li> 마취 동안, 경부 탈구에 의해 동물을 안락사. </li><li> 플라스틱 백에 각각의 동물을 배치하고 전신 활성 수준을 측정하는 용량 교정기 (99m Tc를 설정)을 사용한다. </li><li> 에 다음과 같은 조직과 체액 수집 프리 - 무게혈액, 뼈 (무릎 및 어깨), 담낭, 신장, 간, (내용) 위 (내용) 소장, 대장과 (내용) 맹장, 갑상선 및 기관, 소변과 방광 : 에드 튜브를 계산 그리고 꼬리. </li><li> 적절한 계산 튜브에 조직을 배치하기 전에 혈액을 제거하고 건조시킨다하는 PBS에서 (혈액, 담즙 방광과 방광 제외) 해당 조직을 씻어. </li><li> 비닐 봉지에 동물의 시체를 놓고 용량 교정기를 사용하여 잔류 몸 전체의 활동을 측정합니다. </li><li> 조직 샘플을 포함하는 각 튜브의 무게. 튜브의 초기 중량을 빼기 조직의 덩어리를 얻었다. </li><li> 각 마우스에 대한 주입시의 시료 (100 μL)의 활동량을 측정하기위한 도즈 교정기 (99m Tc를 설정)을 사용한다. 주 :이 시험 샘플 따라서 주입시 활성 수를 제공 주입 부피와 동일하다. </li><li> 조직을 측정하는시기이전에 사용 된 시료의 liquot 5 μL. 다중 검출기 감마 카운터 (99m Tc를 설정)를 사용하여 5 μL의 시료에 대한 분 (CPM) 당 개수를 구하는 계산. </li><li> 변환 계수 (CPM μCi를 -1)를 얻었다 식 1을 사용하여 활성 및 CPM 관계를 계산 2.2.9 및 2.2.10에서 얻어진 두 개의 값을 사용한다. (1) <img alt="식 (1)" src="/files/ftp_upload/55188/55188eq1.jpg" /></li><li> 각 조직 또는 유체의 샘플에서 방사능의 양을 측정하기 위해, 감마 카운터를 사용한다. </li><li> 총 주입 선량에 대해 측정시, 각 조직 또는 유체 활동의 양을 계산하는 수학 식 1을 사용한다. 이 값은 장기 중량 정규화 및 조직의 그램 당 %에 주입 용량 (즉, %의 ID / g)로보고됩니다. </li><li> absen에서 99m Tc를 테트라 표지 리간드를 이용한 대조군 실험을 수행하는 단계 2.2.13에 따라 2.1.2TCO-BP의 가전. 희생 마우스 (N = 3), 0.5, 1, 4, 6 시간 포스트 분사로하는 조직 또는 전술 한 바와 같은 액체를 얻었다. </li></ol></li></ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Molecule+Inhibitors+of+PSMA+Incorporating+Technetium-99m+for+Imaging+Prostate+Cancer:+Effects+of+Chelate+Design+on+Pharmacokinetics.">Small Molecule Inhibitors of PSMA Incorporating Technetium-99m for Imaging Prostate Cancer: Effects of Chelate Design on Pharmacokinetics.</a> Inorg. Chim. Acta. 389, 168-175 (2012).</li><li>Bartholom&#228;, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+Mode+of+Action+of+Re(CO)3+Thymidine+Complexes.">Insight into the Mode of Action of Re(CO)3 Thymidine Complexes.</a> ChemMedChem. 5 (9), 1513-1529 (2010).</li></ol>

The authors declare they have no competing financial interests.

다양한 극성의 테트라 링크 세자리 킬레이트의 컬렉션을 준비하고, 생체 내에서 TCO 유도체와 IEDDA 반응에서 자신의 99m Tc를 단지의 유틸리티를 평가 하였다. 효과적이고 재현 99m Tc를 라벨링 방법은 리간드 농도가 라벨링 단계 (화합물 2-5) T- 부틸 기의 탈 보호 하였다 10-3 M.이었다 다섯 테트라 - 킬레이트 위해 개발되었다. 리간드의 농도는 높은 방...

99m Tc를 전 세계적으로 올해 1, 2, 3 당 실시 만 50 이미징 절차에 진단 핵 의학에서 사용되는 지배적 인 방사성 동위 원소, 남아있다. 임상 적으로 사용되는 99m Tc를 제의 대부분은 관류 형 방사성 의약품이다. 99m Tc를가 표적화 구조물에 라이 게이션을 통해 특정 바이오 마커에 결합하도록 지시하는 능동적 표적 화합물의 제한된 수있다. 타겟 99m Tc를 방사성 의약품의 생성은 종종 관심의 바이오 마커에 결합하는 표적 분자의 능력 99m Tc를 리간드 복합체의 영향에 의해 방해하거나 동위 원소 반감기 고 분자량 생체 분자와 함께 사용하도록 충분히 길게하지 않다 이러한 항체. 이미지가 담배 마는 비 대상에서 삭제하는 생체 분자의 순서를 인수하기 전에 후자는 일반적으로 몇 일이 필요합니다<html> 단말. 사전 타겟팅은 이러한 문제를 극복하기위한 대안적인 접근 방식을 제공합니다. bioorthogonal 화학 결합 사전 타겟팅 모두 형광 무선 촬상 4, 5, 6, 7, 8에 대한 새로운 분자 이미징 프로브를 개발하는 효과적인 방법으로 밝혀졌다. 도 1에 도시 된 바와 같이, 1,2,4,5- 테트라 (Tz 일) 및 트랜스 -cyclooctene (TCO) 유도체의 역 전자 수요 딜스 - 알더 (IEDDA) 반응은 6 특히 효과적인 것으로 밝혀졌다. 이러한 구성 요소와 IEDDA 반응은 높은 선택성, 생체 사전 타겟팅 응용 프로그램 (9), (10)에 이상적 (2 ≈ 6,000 M -1의 -1 k)를 PBS에 빠른 반응 속도를 나타낼 수있다. e_content &quot;&gt; 사용되는 가장 일반적인 방법은 TCO 파생 대상 벡터를 관리하고 충분한 지연 기간에 따라 포함하는 방사성 표지 테트라 투여한다. 방사성 표지 tetrazines을왔다 11 C, 18 F, 64 구리, 89 ZR, 111 년을 기준으로 13, 12, 11 내지 14, 15를보고 하였다. 반면, 생체 내에서 결합 단백질 및 열화를 방지하기 위해 CO 리간드의 사용을 필요로하는 HYNIC 형 리간드를 사용하여 제조 하였다 Tz 일 Tc를이 - 표식 99m, 단지 하나의 보고서가있다 16. 다른 방법으로, 우리는 + 코어 [3 99m Tc를 (CO)]을 안정 세자리 복합체를 형성 리간드 제품군을 사용하여 tetrazines를 표시 여기 99m Tc를 (I)의 합성을보고합니다. 그림 1 : 테트라 트랜스 -cyclooctene 사이의 bioorthogonal IEDDA 반응. <a href="http://ecsource.jove.com/files/ftp_upload/55188/55188fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 준비 리간드의 가족은 극성과 금속 결합 영역과의 Tz (그림 2) 사이의 링커 그룹의 성격에 차이가 세자리 킬레이트를 포함한다. 목표는 고속 목표 대 비 표적 비율을 수득하기 위해, 테크네튬 테트라가 효과적으로 지역화 및 TCO 표지 된 생체 부위와 결합하지 않을 때 빠르게 클리어와 반응 할 수있는 구성 99m을 확인 하였다. 리간드를 테스트하기 위해, 비스포스포네이트의 TCO 파생 (TCO-BP)는 17을 사용 하였다. 우리는 TCO-BP 활성 골대사의 영역에 편재와 반응 할 수 있음을 미리 보여생체 (18)에서 방사성 표지 tetrazines. 이 단일 단계로 제조 될 수 있으며, 실험 지역화 주로 관절 (무릎, 어깨)에 발생하는 정상 마우스에서 수행 될 수 있기 때문에, 새로운 tetrazines 테스트하는 편리한 시약이다.

<table><tbody><tr><td>Argon gas</td><td>Alphagaz</td><td>---</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>EMD Millipore</td><td>106395</td><td>---</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#183;10H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>S9640</td><td>---</td></tr><tr><td>KNaC&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;&amp;#183;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Anachemia</td><td>217255</td><td>---</td></tr><tr><td>Technelite &lt;sup&gt;99m&lt;/sup&gt;Tc generator</td><td>Lantheus medical imaging</td><td>---</td><td>Source of &lt;sup&gt;99m&lt;/sup&gt;TcO&lt;sup&gt;4-&lt;/sup&gt;</td></tr><tr><td>0.9% Saline</td><td>Lantheus medical imaging</td><td>---</td><td>To elute generator</td></tr><tr><td>1 M HCl</td><td>Lab Chem</td><td>---</td><td>---</td></tr><tr><td>MeOH</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>ACN</td><td>Caledon</td><td>---</td><td>HPLC grade</td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher Scientific</td><td>Barnstead Nanopure</td><td>---</td></tr><tr><td>DCM</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>TFA</td><td>Caledon</td><td>---</td><td>---</td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>pH 7.4 1x</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>---</td></tr><tr><td>Tween80</td><td>Sigma Aldrich</td><td>P8047</td><td>---</td></tr><tr><td>Isoflurane</td><td>CDMV</td><td>108737</td><td>Supplier: Fresenius Kabi Animal Health</td></tr><tr><td>HPLC</td><td>Waters</td><td>1525 Binary Pump, 2998 Photodiodde Array Detector, E-SAT/IN, Bioscan Flowcount PMT detector (item # 15590)</td><td>---</td></tr><tr><td>HPLC column for analysis and purification of compounds 2-4</td><td>Phenomenex</td><td>00G-4435-E0</td><td>Gemini&amp;reg; 5 &amp;micro;m C18 110&amp;nbsp;&amp;Aring;, LC Column 250 x 4.6 mm</td></tr><tr><td>HPLC column for analysis and purification of compounds 1 and 5</td><td>Waters&amp;nbsp;</td><td>186003115</td><td>XBridge BEH C18 Column, 130 &amp;Aring;, 5 &amp;micro;m, 4.6 mm x 100 mm</td></tr><tr><td>Microwave Reactor&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>Initiator 8</td><td>---</td></tr><tr><td>Biotage V10 Evaporator</td><td>Biotage&amp;nbsp;</td><td>Serial # V1041</td><td>---</td></tr><tr><td>Dose calibrator</td><td>Capintec, Inc.&amp;nbsp;</td><td>CRC-25R</td><td>---</td></tr><tr><td>Gamma counter</td><td>Perkin Elmer</td><td>Wizard 1470 Automatic Gamma Counter</td><td>---</td></tr><tr><td>Animal room scale&amp;nbsp;</td><td>Mettler Toledo</td><td>XP105 Delta Range</td><td>---</td></tr><tr><td>Microwave vials&amp;nbsp;</td><td>Biotage&amp;nbsp;</td><td>355629</td><td>0.5-2 mL&amp;nbsp;</td></tr></tbody></table>

preparation and evaluation of 99mtc-labeled tridentate chelates for pre-targeting using bioorthogonal chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

리간드는 시판되는 테트라 22 23 생성물의 결합에 의해 다음 간단한 환원성 아 민화 전략 (도 2)를 통해 다른 링커 및 킬레이트 제를 사용하여 합성 하였다. 방사성 표지는 모든 화합물에 대한 방법과 동일한 방법을 사용하여 수행 재현성이었다. 83 % (1) 45 % (2) 31 % 공정은 테크네튬 방사성 표?...

Watch this Scientific Journal Video about Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry at JoVE.com

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

준비 및 평가<sup&gt; 99m</sup&gt; Bioorthogonal 화학을 사용하여 사전이 타겟팅에 대한 세자리 킬레이트를 Tc를 표지

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

preparation-evaluation-99mtc-labeled-tridentate-chelates-for-pre

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

8.1K 조회수.  McMaster University. Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. 

비디오: Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

역으로 작은 분자 또는 바이오 센서 애플리케이션을위한 다중 구성 요소 구조를 무력화하는 미세 유체 온칩 캡처 - 고리 화 반응

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

연구

화학

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

바이오 콘쥬 게이션과의 Radiosynthesis<sup&gt; 89</sup&gt; ZR-DFO 표지 항체

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

를 사용하여 금 나노 입자의 효율적인 방사성 표지를위한 최적화 된 프로토콜<sup&gt; (125)</sup&gt; I-표시 아 지드 보철 그룹

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Pretargeted PET 영상에 대한 Bioorthogonal 역 전자 수요 딜스 - 알더 고리 모으고

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

생체공학

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

작은 분자 CLT1 펩타이드 표적 조영제와 전립선 암의 MR 분자 영상

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

의학

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

Pretargeted Radioimmunotherapy 역 전자 수요 Diels 오리 나무 반응에 따라

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

합성 및 Bioconjugation의 Thiol 반응 시 약 사이트에 선택적으로의 창조에 대 한 수정 Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

세포 이동의 양전자 방출 단층 촬영 이미징: 세포 방사선 표지 방법

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

암 연구학

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

세포 축적을 증가 하 고 종양을 대상으로 개선에 대 한 바이러스 성 파생 된 펩 티 드 항 체 어원이 같은 말의 초기 평가 수정

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

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