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Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

PurposeMetabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging.MethodsGAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution.ResultsThe GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target.Conclusions[18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

Purpose: Noninvasive and quantitative tracking of CD8+ T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64Cu, to assess the sensitivity of PET imaging of normal and diseased tissue.Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64Cu was developed. The accumulation of 64Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8+ T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6-8/group studied with imaging and IHC or flow cytometry).Results: We demonstrate the ability of immunoPET to detect small differences in CD8+ T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), 64Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8+ T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols.Conclusions: 64Cu-169cDb imaging can spatially map the distribution of CD8+ T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8+ T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976-87. ©2018 AACR.

Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 antibody fragment derived from the GK1.5 hybridoma, was used as a PET probe for CD4+ T cells in the dextran sulfate sodium (DSS) mouse model of IBD. Methods: The DSS mouse model of IBD was validated by assessing changes in CD4+ T cells in the spleen and mesenteric lymph nodes (MLNs) using flow cytometry. Furthermore, CD4+ T cell infiltration in the colons of colitic mice was evaluated using immunohistochemistry. 89Zr-labeled GK1.5 cDb was used to image distribution of CD4+ T cells in the abdominal region and lymphoid organs of mice with DSS-induced colitis. Region-of-interest analysis was performed on specific regions of the gut to quantify probe uptake. Colons, ceca, and MLNs were removed and imaged ex vivo by PET. Imaging results were confirmed by ex vivo biodistribution analysis. Results: An increased number of CD4+ T cells in the colons of colitic mice was confirmed by anti-CD4 immunohistochemistry. Increased uptake of 89Zr-maleimide-deferoxamine (malDFO)-GK1.5 cDb in the distal colon of colitic mice was visible in vivo in PET scans, and region-of-interest analysis of the distal colon confirmed increased activity in DSS mice. MLNs from colitic mice were enlarged and visible in PET images. Ex vivo scans and biodistribution confirmed higher uptake in DSS-treated colons (DSS, 1.8 ± 0.40; control, 0.45 ± 0.12 percentage injected dose [%ID] per organ, respectively), ceca (DSS, 1.1 ± 0.38; control, 0.35 ± 0.09 %ID per organ), and MLNs (DSS, 1.1 ± 0.58; control, 0.37 ± 0.25 %ID per organ). Conclusion:89Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the colons, ceca, and MLNs of colitic mice and may prove useful for further investigations of CD4+ T cells in preclinical models of IBD, with potential to guide development of antibody-based imaging in human IBD.

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.

The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8(+) T cells and other immune cell subsets. In this study, we describe the generation of (89)Zr-desferrioxamine-labeled anti-CD8 cys-diabody ((89)Zr-malDFO-169 cDb) for noninvasive immuno-PET tracking of endogenous CD8(+) T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T-cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T-cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic.

UnlabelledThe proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole-body T lymphocyte dynamics noninvasively in vivo, we generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for (89)Zr-immuno-PET detection of helper and cytotoxic T cell populations.MethodsAnti-CD4 and -CD8 cDbs were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cDbs were site-specifically conjugated to maleimide-desferrioxamine for (89)Zr radiolabeling and subsequent small-animal PET/CT acquisition and ex vivo biodistribution in both wild-type mice and a model of hematopoietic stem cell (HSC) transplantation.ResultsImmuno-PET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild-type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 wk after HSC transplantation using the (89)Zr-radiolabeled anti-CD4 and -CD8 cDbs.ConclusionThese newly developed anti-CD4 and -CD8 immuno-PET reagents represent a powerful resource to monitor T cell expansion, localization, and novel engraftment protocols. Future potential applications of T cell-targeted immuno-PET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.

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.

MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers, including non-small cell lung cancer (NSCLC). As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment-based immunopositron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were reformatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 to 5.1 nmol/L. The candidate with the highest affinity, H2, was radiolabeled with (89)Zr for immunoPET studies targeting NSCLC xenografts: low MET-expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET overexpression. ImmunoPET at as early as 4 hours after injection produced high-contrast images, and ex vivo biodistribution analysis at 20 hours after injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (P < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer), produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, whereas no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications.

Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily cell adhesion molecule that is aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast cancer, colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This wide spectrum of human malignancies makes ALCAM a prospective pan-cancer immunoPET target to aid in detection and diagnosis in multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for (64)Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) immunoPET imaging of a fully human ALCAM cys-diabody (cDb) with a reduced linker length that retains its bivalent binding ability. ALCAM constructs with linker lengths of eight, five and three amino acids were produced to make true non-covalent site-specifically modified cDbs. Characterization by gel electrophoresis, size exclusion chromatography, flow cytometry and mass spectrometry of the various constructs was performed. To demonstrate the increased utility of targeting multiple malignancies expressing ALCAM, we compare the targeting of the site-specific versus non-site-specific conjugated cDbs to the human colorectal cancer xenograft LS174T. Interestingly, the conjugation strategy not only affects tumor targeting but also hepatic and renal uptake/clearance.

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.

Determination of treatment response to immunotherapy in glioblastoma multiforme (GBM) is a process which can take months. Detection of CD8+ T cell recruitment to the tumor with a noninvasive imaging modality such as positron emission tomography (PET) may allow for tumor characterization and early evaluation of therapeutic response to immunotherapy. In this study, we utilized 89Zr-labeled anti-CD8 cys-diabody-PET to provide proof-of-concept to detect CD8+ T cell immune response to oncolytic herpes simplex virus (oHSV) M002 immunotherapy in a syngeneic GBM model. Immunocompetent mice (n = 16) were implanted intracranially with GSC005 GBM tumors, and treated with intratumoral injection of oHSV M002 or saline control. An additional non-tumor bearing cohort (n = 4) receiving oHSV M002 treatment was also evaluated. Mice were injected with 89Zr-labeled anti-CD8 cys-diabody seven days post oHSV administration and imaged with a preclinical PET scanner. Standardized uptake value (SUV) was quantified. Ex vivo tissue analyses included autoradiography and immunohistochemistry. PET imaging showed significantly higher SUV in tumors which had been treated with M002 compared to those without M002 treatment (p = 0.0207) and the non-tumor bearing M002 treated group (p = 0.0021). Accumulation in target areas, especially the spleen, was significantly reduced by blocking with the non-labeled diabody (p < 0.001). Radioactive probe accumulation in brains was consistent with CD8+ cell trafficking patterns after oHSV treatment. This PET imaging strategy could aid in distinguishing responders from non-responders during immunotherapy of GBM. [ABSTRACT FROM AUTHOR]

Purpose: Molecular imaging of CD4+ T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4+ T cell viability, proliferation, CD4 expression, and function.Procedures: The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of 89Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed.Results: For immunoPET imaging, the lowest protein dose of 2 μg of 89Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-μg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 μg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 μg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 μg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4+ T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4+ T cells.Conclusions: Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4+ T cells in the context of preclinical disease models. Future approaches to minimizing biological effects may include the creation of monovalent fragments or selecting anti-CD4 antibodies which target alternative epitopes. [ABSTRACT FROM AUTHOR]

Purpose: Metabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging.Methods: GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution.Results: The GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target.Conclusions: [18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target. [ABSTRACT FROM AUTHOR]