Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice.

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.

Aligning physics and physiology: Engineering antibodies for radionuclide delivery.

The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy.

Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG1 antibodies.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Potent and selective binders of the E3 ubiquitin ligase ZNRF3 stimulate Wnt signaling and intestinal organoid growth.

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

[89Zr]A2cDb Immuno-PET of Prostate Cancer in a Human Prostate Stem Cell Antigen Knock-in (hPSCA KI) Syngeneic Model.

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.

Tri-functional platform for construction of modular antibody fragments for in vivo 18F-PET or NIRF molecular imaging.

Positron emission tomography (PET) molecular imaging is a powerful tool for interrogating physiological and biochemical processes to understand the biology of disease and advance therapeutic developments. Near-infrared fluorescence (NIRF) optical imaging has become increasingly popular for intraoperative staging to enable cellular resolution imaging of tumor margins during surgical resection. In addition, engineered antibody fragments have emerged as promising molecular imaging agents given their exquisite target selectivity, rapid systemic clearance and site-selective chemical modification. We report a tri-functional platform for construction of a modular antibody fragment that can rapidly be labeled with radionuclides or fluorophores for PET or NIRF molecular imaging of prostate stem cell antigen (PSCA).

Evaluation of [131I]I- and [177Lu]Lu-DTPA-A11 Minibody for Radioimmunotherapy in a Preclinical Model of PSCA-Expressing Prostate Cancer.

PURPOSE: Radioimmunotherapy uses tumor-specific antibodies to deliver therapeutic radionuclides, but hematological toxicity due to the long serum half-life of intact antibodies remains a challenge. We evaluated a smaller antibody fragment, the minibody, with faster kinetics and a potentially improved therapeutic index. PROCEDURES: The anti-prostate stem cell antigen (PSCA) minibody (A11 Mb) was radiolabeled with iodine-124 ([124I]I-A11 Mb) or conjugated with deferoxamine (DFO) and labeled with zirconium-89 ([89Zr]Zr-DFO-A11 Mb) for surrogate immunoPET to profile pharmacokinetics in a human prostate cancer xenograft model. Subsequently, minibodies labeled with two therapeutic beta emitters, directly iodinated [131I]I-A11 Mb (non-residualizing) and 177Lu chelated using DTPA ([177Lu]Lu-DTPA-A11 Mb) (residualizing), were compared for in vitro antigen-specific cytotoxicity. Full biodistribution studies (in 22Rv1-PSCA tumor bearing and hPSCA knock-in mice) were conducted for dosimetry calculations. Finally, the lead candidate [131I]I-A11 Mb was evaluated in a radioimmunotherapy experiment. Escalating single doses (3.7, 11, or 37 MBq) and saline control were administered to 22Rv1-PSCA tumor bearing mice and anti-tumor effects (tumor volume) and toxicity (body weight) were monitored. RESULTS: Minibodies radiolabeled with therapeutic beta emitters [131I]I-A11 Mb and [177Lu]Lu-DTPA-A11 Mb exhibited comparable tumor cell growth inhibition in vitro. In vivo surrogate immunoPET imaging using [89Zr]Zr-DFO-A11 Mb showed activity retention in liver and kidney up to 72 h, while [124I]I-A11 Mb cleared from liver, kidney, and blood by 48 h. Based on full biodistribution and dosimetry calculations, administering 37 MBq [131I]I-A11 Mb was predicted to deliver a favorable dose to the tumor (35 Gy), with a therapeutic index of 22 (tumor:bone marrow). For [177Lu]Lu-DTPA-A11 Mb, the kidneys would be dose-limiting, and the maximum tolerated activity (7.4 MBq) was not predicted to deliver an effective radiation dose to tumor. Radioimmunotherapy with a single dose of [131I]I-A11 Mb showed dose-dependent tumor inhibition with minimal off-target toxicity and improved median survival (19 and 24 days, P < 0.001) compared with untreated mice (12 days). CONCLUSIONS: These findings show the potential of the anti-PSCA minibody for targeted radioimmunotherapy with minimal toxicity, and the application of immunoPET and dosimetry for personalized treatment.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

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.

Near-Infrared Dye-Labeled Anti-Prostate Stem Cell Antigen Minibody Enables Real-Time Fluorescence Imaging and Targeted Surgery in Translational Mouse Models.

PURPOSE: The inability to intraoperatively distinguish primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery. EXPERIMENTAL DESIGN: A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW. The integrity and binding of the probe to PSCA were confirmed by gel electrophoresis, size-exclusion chromatography, and flow cytometry, respectively. The ability of the probe to detect tumor-infiltrated lymph nodes and metastatic lesions was evaluated in 2 xenograft models, as well as in transgenic mice expressing human PSCA (hPSCA). An invasive intramuscular model was utilized to evaluate the efficacy of the A11 Mb-IRDye800CW-guided surgery. RESULTS: A11 Mb was successfully conjugated with IRDye800CW and retained specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 48 hours. The A11 Mb-IRDye800CW specifically detected PSCA-positive primary tumors, tumor-infiltrated lymph nodes, and distant metastases with high contrast. Fluorescence guidance facilitated more complete tumor resection, reduced tumor recurrence, and improved overall survival, compared with conventional white light surgery. The probe successfully identified primary orthotopic tumors and metastatic lesions in hPSCA transgenic mice. CONCLUSIONS: Real-time fluorescence image-guided surgery with A11 Mb-IRDye800CW enabled detection of lymph node metastases and positive surgical margins, facilitated more complete tumor removal, and improved survival, compared with white light surgery. These results may be translatable into clinical practice to improve surgical and patient outcomes.

Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis.

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.

Dual-Modality Immuno-PET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using an Anti-Prostate Stem Cell Antigen Cys-Diabody.

Pancreatic cancer has a high mortality rate due to late diagnosis and the tendency to invade surrounding tissues and metastasize at an early stage. A molecular imaging agent that enables both presurgery antigen-specific PET (immuno-PET) and intraoperative near-infrared fluorescence (NIRF) guidance might benefit diagnosis of pancreatic cancer, staging, and surgical resection, which remains the only curative treatment. Methods: We developed a dual-labeled probe based on A2 cys-diabody (A2cDb) targeting the cell-surface prostate stem cell antigen (PSCA), which is expressed in most pancreatic cancers. Maleimide-IRDye800CW was site-specifically conjugated to the C-terminal cys-tag (A2cDb-800) without impairing integrity or affinity (half-maximal binding, 4.3 nM). Direct radioiodination with 124I (124I-A2cDb-800) yielded a specific activity of 159 ± 48 MBq/mg with a radiochemical purity exceeding 99% and 65% ± 4.5% immunoreactivity (n = 3). In vivo specificity for PSCA-expressing tumor cells and biodistribution of the dual-modality tracer were evaluated in a prostate cancer xenograft model and compared with single-labeled 124I-A2cDb. Patient-derived pancreatic ductal adenocarcinoma xenografts (PDX-PDACs) were grown subcutaneously in NSG mice and screened for PSCA expression by immuno-PET. Small-animal PET/CT scans of PDX-PDAC-bearing mice were obtained using the dual-modality 124I-A2cDb-800 followed by postmortem NIRF imaging with the skin removed. Tumors and organs were analyzed ex vivo to compare the relative fluorescent signals without obstruction by other organs. Results: Specific uptake in PSCA-positive tumors and low nonspecific background activity resulted in high-contrast immuno-PET images. Concurrent with the PET studies, fluorescent signal was observed in the PSCA-positive tumors of mice injected with the dual-tracer 124I-A2cDb-800, with low background uptake or autofluorescence in the surrounding tissue. Ex vivo biodistribution confirmed comparable tumor uptake of both 124I-A2cDb-800 and 124I-A2cDb. Conclusion: Dual-modality imaging using the anti-PSCA cys-diabody resulted in high-contrast immuno-PET/NIRF images of PDX-PDACs, suggesting that this imaging agent might offer both noninvasive whole-body imaging to localize PSCA-positive pancreatic cancer and fluorescence image-guided identification of tumor margins during surgery.