Recombinant Anti-CD20 Antibody Fragments for Small-Animal PET Imaging of B-Cell Lymphomas (original) (raw)
ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)
Protein Engineering Design and Selection, 2010
Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V L -V H orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with 124 I for PET imaging, and biodistribution of 131 I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both 124 I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of 131 I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas.
Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma
Molecular cancer therapeutics, 2017
Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAb) might circumvent some of the limitations of radiolabeled full antibodies. In this study, a set of hCD20-targeting sdAbs was generated, and their capacity to bind hCD20 was evaluated in vitro and in vivo. A lead sdAb, sdAb 9079, was selected on the basis of its specific tumor targeting and significant lower kidney accumulation compared with other sdAbs. SdAb 9079 was then radiolabeled with 68Ga and 177Lu for PET imaging and targeted therapy. The therapeutic potential of 177Lu-DTPA-sdAb was compared with that of 177Lu-DTPA-rituximab and unlabeled rituximab in mice bearing hCD20pos tumors. Radiolabeled with 68Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in nontarget organs, except kidneys. The tumor uptake ...
European Journal of Nuclear Medicine and Molecular Imaging, 2009
Purpose 131I- and 90Y-labelled anti-CD20 antibodies have been shown to be effective in the treatment of low-grade, B-cell non-Hodgkin’s lymphoma (NHL). However, the most appropriate radionuclide in terms of high efficiency and low toxicity has not yet been established. In this study we evaluated an immunoconjugate formed by the anti-CD20 antibody rituximab and the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA-rituximab was
The Journal of Nuclear Medicine, 2015
The CD30-specific antibody-drug conjugate, brentuximab vedotin, is approved for the treatment of relapsed, refractory Hodgkin lymphomas and systemic anaplastic large T-cell lymphomas. Multiple ongoing clinical trials are investigating brentuximab vedotin efficacy in other CD30-positive hematologic malignancies. Because CD30 expression varies among different types of lymphoma and can also change during the course of treatment, companion diagnostic imaging of CD30 could be a valuable tool in optimizing patient-specific brentuximab vedotin treatment regimens. Methods: The mouse antihuman CD30 antibody AC-10 was radiolabeled with the positron-emitting radionuclide 89 Zr. The stability and specificity of 89 Zr-desferrioxamine (DFO)-labeled CD30-specific AC-10 antibody (89 Zr-DFO-AC-10) was evaluated in vitro. The pharmacokinetics of 89 Zr-DFO-AC-10 was studied in BALB/c nude mice bearing subcutaneous human Karpas 299 tumors (CD30-positive model) or A-431 tumors (CD30-negative model) using PET/CT imaging, biodistribution studies, and autoradiography. Results: AC-10 was conjugated with a DFO B chelator and radiolabeled with 89 Zr to give formulated 89 Zr-DFO-AC-10 with a radiochemical yield of 80%, radiochemical purity greater than 99%, and specific activity of 111-148 MBq/mg. 89 Zr-DFO-AC-10 was stable in mouse and human sera and preserved the immunoreactivity toward CD30. Biodistribution data showed the highest tissue accumulation of 89 Zr-DFO-AC-10 in CD30-positive tumors, with 37.9% ± 8.2% injected activity per gram of tissue at 72 h after injection, whereas uptake in CD30-negative tumors was 11.0% ± 0.4%. The specificity of 89 Zr-DFO-AC-10 binding to CD30 in vivo was confirmed by blocking studies. Time-activity curves showed that between 24 and 144 h after injection, tumor-to-muscle ratios increased from 18.9 to 51.8 in the CD30positive model and from 4.8 to 8.7 in the CD30-negative model. Tumor-to-blood ratios also increased, from 3.2 to 13.6 and from 1 to 2 in the CD30-positive and-negative models, respectively. Conclusion: Our results demonstrate that for measuring CD30 expression, 89 Zr-DFO-AC-10 is a sensitive PET agent with high tumor-to-normal-tissue contrast. 89 Zr-DFO-AC-10 is a promising CD30-imaging radiotracer for clinical translation in patients with various lymphomas and other diseases.
Evaluation of CD20, CD22, and HLA-DR Targeting for Radioimmunotherapy of B-Cell Lymphomas
Cancer Research, 2007
Despite the promise of radioimmunotherapy using anti-CD20 antibodies (Ab) for the treatment of relapsed patients with indolent non-Hodgkin lymphoma (NHL), most patients treated with conventional doses of 131 I-tositumomab or 90 Y-ibritumomab eventually relapse. We did comparative assessments using conventional radioimmunotherapy targeting CD20, CD22, and HLA-DR on human Ramos, Raji, and FL-18 lymphoma xenografts in athymic mice to assess the potential for improving the efficacy of radioimmunotherapy by targeting other NHL cell surface antigens. Results of biodistribution studies showed significant differences in tumor localization consistent with variable antigenic expression on the different lymphoma cell lines. Interestingly, the radioimmunoconjugate that yielded the best tumor-to-normal organ ratios differed in each tumor model. We also explored administering all three 111 In-1,4,7,10-tetra-azacylododecane N,N ¶,N ¶ ¶,NØ-tetraacetic acid antibodies in combination, but discovered, surprisingly, that this approach did not augment the localization of radioactivity to tumors compared with the administration of the best single radiolabeled Ab alone. These data suggest that conventional radioimmunotherapy using anti-CD20, anti-HLA-DR, or anti-CD22 Abs is effective when used singly and provides targeted uptake of radiolabel into the tumor that is dependent on the levels of antigen expression. Improvements in tumorto-normal organ ratios of radioactivity cannot be achieved using directly labeled Abs in combination but may be afforded by novel pretargeting methods.
The status of radioimmunotherapy in CD20+ non-Hodgkin's lymphoma
Targeted oncology, 2015
Rituximab, the CD20-directed antibody, has become a standard component of treatment regimens for patients with B cell non-Hodgkin's lymphoma (NHL). The use of rituximab has resulted in greatly improved response and survival rates with less toxicity relative to standard chemotherapeutic regimes. However, relapse and recurrence is common, particularly in indolent varieties which remain incurable, requiring alternate therapeutic options. The subsequent coupling of β-emitting isotopes such as (131)I and (90)Y to anti-CD20 monoclonal antibodies (mAbs), including rituximab, has been steadily growing over the last decade and demonstrates even greater therapeutic efficacy with more durable responses. (177)Lutetium-labelled rituximab offers a number of convenient advantages over (131)I and (90)Y anti-CD20 mAbs for treatment of NHL, and a number of alpha-emitting isotopes lie at the frontier of consolidation therapy for residual, micrometastatic disease.
Bioconjugate Chemistry, 2012
Positron emission tomography (PET) is an attractive imaging tool to localize and quantify tracer biodistribution. ImmunoPET with an intact mAb typically requires two to four days to achieve optimized tumor-to-normal ratios. Thus, a positron emitter with a half-life of two to four days such as zirconium-89 [ 89 Zr] (t 1/2 : 78.4 h) is ideal. We have developed an antibody-based, long-lived immunoPET tracer 89 Zr-Desferrioxamine-p-SCN (Df-Bz-NCS)-rituximab (Zr-iPET) to image tumor for longer durations in a humanized CD20-expressing transgenic mouse model. To optimize the radiolabeling efficiency of 89 Zr with Df-Bz-rituximab, multiple radiolabelings were performed. Radiochemical yield, purity, immunoreactivity and stability assays were carried out to characterize the Zr-iPET for chemical and biological integrity. This tracer was used to image transgenic mice that express the human CD20 on their B cells (huCD20TM). Each huCD20TM mouse received a 7.4MBq /dose. One group (n=3) received 2 mg/kg pre-dose (blocking) of cold rituximab 2 h prior to 89 Zr-iPET; the other group (n=3) had no pre-dose (non-blocking). Small animal PET/CT was used to image mice at 1, 4, 24, 48, 72, and 120 h. Quality assurance of the 89 Zr-iPET demonstrated NCS-Bz-Df: antibody ratio (c/a: 1.5 ± 0.31), specific activity (0.44-1.64 TBq/mol), radiochemical yield (>70%), and purity (>98%). The Zr-iPET immunoreactivity was >80%. At 120 h, Zr-iPET uptake (% ID/g) as mean ± STD for blocking and non-blocking groups in spleen was 3.2 ± 0.1 % and 83.3 ± 2.0 % (p value < 0.0013.). Liver uptake was 1.32 ± 0.05% and 0.61 ± 0.001% (p value < 0.0128) for blocking and non-blocking, respectively. The small animal PET/CT image shows the spleen specific uptake of Zr-iPET in mice at 120 h after tracer injection. Compared to the liver, the spleen specific uptake of Zr-iPET is very high due to the expression of huCD20. We optimized the radiolabeling efficiency of 89 Zr with Df-Bzrituximab. These radioimmunoconjugate lots were stable up to 5 days in serum in vitro. The present study showed that 89 Zr is well suited for mAbs to image cancer over extended period of time (up to 5 days).
Radiochimica Acta, 2014
In this study, zinc-62 was prepared at radiopharmaceutical grade (for 62 Zn/ 62 Cu generator production) using nat Cu( , ) reaction with the production yield of 5.9 mCi/ Ah at 30 MeV proton energy (radiochemical separation yield >95%, radionuclidic purity >99% and radiochemical purity >99%). In the next step, rituximab was successively labeled with [ 62 Zn]-ZnCl 2 after conjugation with p-SCN-Bz-DOTA followed by molecular filtration and determination of the average number of DOTA conjugated per mAb (6:1) by spectrophotometric method. Radiochemical purity (>97%, measured by ITLC and HPLC), integrity of protein after radiolabeling (gel electrophoresis) and stability of [ 62 Zn]-DOTA-rituximab (in final formulation, and human serum) were determined 1-8 h as well as biodistribution studies in wild-type rats followed by coincidence imaging for 6 h. However, the accumulation of the radiolabeled antibody was not consistent with the former reported rituximab conjugates. [ 62 Zn]-labeled monoclonal antibodies and fragments can be prepared as potential in vivo PET generators for molecular imaging however, the search for application of stable zinc complexes must be continued.
Clinical Cancer Research, 2005
Purpose: The L1 cell adhesion protein is overexpressed in tumors, such as neuroblastomas, renal cell carcinomas, ovarian carcinomas, and endometrial carcinomas, and represents a target for tumor diagnosis and therapy with anti-L1-CAM antibody chCE7. Divalent fragments of this internalizing antibody labeled with 67/64Cu and 177Lu were evaluated to establish a chCE7 antibody fragment for radioimmunotherapy and positron emission tomography imaging, which combines high-yield production with improved clearance and biodistribution properties. Experimental Design: chCE7F(ab′)2 fragments were produced in high amounts (0.2 g/L) in HEK-293 cells, substituted with the peptide-linked tetraazamacrocycle 3-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-triglycyl-l-p-isothiocyanato-phenylalanine, and labeled with 67Cu and 177Lu. In vivo bioevaluation involved measuring kinetics of tumor and tissue uptake in nude mice with SK-N-BE2c xenografts and NanoPET (Oxford Positron Syst...
Journal of Nuclear Medicine
Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C H 3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with 124 I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. Methods: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of 124 I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or Ͼ90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9 -3.1 MBq (53-85 Ci) of 124 I-minibody or with 3.1 MBq (85 Ci) of 124 I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using 18 F-FDG 2 d before imaging with 124 I-minibody. Results: PET images using 124 I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using 124 I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the 124 Idiabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the 124 I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (Ͻ3 mm in diameter) could be imaged, and 124 I-anti-CEA minibodies, compared with 18 F-FDG, demonstrated highly specific localization. Conclusion: 124 I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.