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Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivoResults:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242-52. ©2017 AACR.

PurposeProstate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. In this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to (18)F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation.Experimental designOsteoblastic, PSCA-expressing, LAPC-9 intratibial xenografts were imaged with serial (124)I-anti-PSCA A11 minibody immunoPET and (18)F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and posttreatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation.ResultsA11 minibody demonstrated improved sensitivity and specificity over (18)F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo.ConclusionsA11 minibody immunoPET has the potential to improve the sensitivity and specificity of clinical prostate cancer metastasis detection over bone scans, which are the current clinical standard-of-care. A11 minibody immunoPET additionally has the potential to image the activity of the androgen signaling axis in vivo which may help evaluate the clinical response to androgen deprivation and the development of castration resistance.

PurposeIntact antibodies have a long serum persistence resulting in high background signal that inhibits their direct translation as imaging agents. Engineering of antibody fragments through the introduction of mutations in the fragment crystallizable (Fc) region can dramatically reduce serum persistence. We sought to develop a Fc-mutated, anti-CA19-9 antibody fragment (anti-CA 19-9 scFv-Fc H310A) to provide micro-positron emission tomography (microPET) imaging of pancreatic cancer xenografts.ProceduresThe anti-CA19-9 scFv-Fc H310A was successfully expressed and purified. Biochemical characterization included size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and flow cytometry. The antibody fragment was labeled with iodine-124 ((124)I) and injected into mice containing human pancreatic cancer xenografts. MicroPET/CT images were then obtained. Blood, organ, and tumor radioactivity was measured and expressed as the percent of injected dose per gram of tissue (%ID/g).ResultsBiochemical characterization was consistent with the creation of a 105 kD dimer containing a human Fc region. Flow cytometry demonstrated antigen-specific binding, and cell-based ELISA further established a dissociation constant (K D) of 10.7 nM. (124)I-labeled scFv-Fc H310A localized to the antigen-positive tumor xenografts as detected by microPET. Objective confirmation of targeting was demonstrated by higher %ID/g in the antigen-positive tumor compared to the blood, antigen-negative tumor, and liver.ConclusionsWe successfully engineered and produced an anti-CA19-9 scFv-Fc H310A antibody fragment that retains similar affinity when compared to the parental intact murine antibody. Additionally, our engineered and mutated fragment exhibited antigen-specific microPET imaging of both subcutaneous and orthotopic pancreatic cancer xenografts at early time points secondary to decreased serum half-life.

The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.

Purpose: A great challenge in the diagnosis and treatment of prostate cancer is distinguishing between indolent or local disease and aggressive or metastatic disease. Antibody-based positron emission tomography (immuno-PET) as a cancer-specific imaging modality could improve diagnosis of primary disease, aid the detection of metastases to regional lymph nodes as well as to distant sites (e.g., bone), and monitor response to therapy.Procedure: In search for a more physiologically relevant disease model, a human prostate stem cell antigen knock-in (hPSCA KI) mouse model was generated. The use of a syngeneic prostate cancer cell line transduced to express human PSCA (RM-9-hPSCA) enabled the evaluation of anti-PSCA immuno-PET in immunocompetent mice and in the context of normal tissue expression of PSCA. Two PSCA-specific humanized antibody fragments, A11 minibody and A2 cys-diabody, were radiolabeled with positron emitters iodine-124 and zirconium-89, respectively ([124I]A11 Mb and [89Zr]A2cDb), and used for immuno-PET in wild-type, hPSCA KI and tumor-bearing mice.Results: The hPSCA KI mice express PSCA at low levels in the normal prostate, bladder and stomach, reproducing the expression pattern seen in humans. [124I]A11 Mb immuno-PET detected increased levels of PSCA expression in the stomach, and because I-124 is non-residualizing, very little activity was seen in organs of clearance (liver, kidney, spleen). However, due to the longer half-life of the 80 kDa protein, blood activity (and thus urine activity) at 20 h postinjection remains high. The smaller 50 kDa [89Zr]A2cDb cleared faster, resulting in lower blood and background activity, despite the use of a residualizing radiometal. Importantly, [89Zr]A2cDb immuno-PET showed antigen-specific targeting of PSCA-expressing tumors and minimal nonspecific uptake in PSCA-negative controls.Conclusion: Tracer biodistribution was not significantly impacted by normal tissue expression of PSCA. [89Zr]A2cDb immuno-PET yielded high tumor-to-blood ratio at early time points. Rapid renal clearance of the 50 kDa tracer resulted in an unobstructed view of the pelvic region at 20 h postinjection that would allow the detection of cancer in the prostate. [ABSTRACT FROM AUTHOR]

The noninvasive detection and quantification of CD8+ T cells in vivo are important for both the detection and staging of CD8+ lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8+ T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8+ T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for 64Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both 64Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice. [ABSTRACT FROM AUTHOR]

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20. Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing ( 89 Zr) versus non-residualizing ( 124 I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivo Results: 124 I-GAcDb and 124 I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89 Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124 I-labeled tracers. Pairwise comparison of 89 Zr- and 124 I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells. Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242-52. ©2017 AACR ., (©2017 American Association for Cancer Research.)