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Immune checkpoint inhibitors (ICI) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from these therapies. Thus, there is a clinical need for reliable tools that allow for the early assessment of response to ICIs, as well as a preclinical need for imaging tools that aid in the future development and understanding of immunotherapies. Here we demonstrate that CD69, a canonical early-activation marker expressed on a variety of activated immune cells, including cytotoxic T cells and natural killer (NK) cells, is a promising biomarker for the early assessment of response to immunotherapies. We have developed a PET probe by radiolabeling a highly specific CD69 mAb, H1.2F3, with Zirconium-89 (89Zr), [89Zr]-deferoxamine (DFO)-H1.2F3. [89Zr]-DFO-H1.2F3 detected changes in CD69 expression on primary mouse T cells in vitro and detected activated immune cells in a syngeneic tumor immunotherapy model. In vitro uptake studies with [89Zr]-DFO-H1.2F3 showed a 15-fold increase in CD69 expression for activated primary mouse T cells, relative to untreated resting T cells. In vivo PET imaging showed that tumors of ICI-responsive mice had greater uptake than the tumors of nonresponsive and untreated mice. Ex vivo biodistribution, autoradiography, and IHC analyses supported the PET imaging findings. These data suggest that the CD69 PET imaging approach detects CD69 expression with sufficient sensitivity to quantify immune cell activation in a syngeneic mouse immunotherapy model and could allow for the prediction of therapeutic immune responses to novel immunotherapies.

Immune checkpoint inhibitors (ICI) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from these therapies. Thus, there is a clinical need for reliable tools that allow for the early assessment of response to ICIs, as well as a preclinical need for imaging tools that aid in the future development and understanding of immunotherapies. Here we demonstrate that CD69, a canonical early-activation marker expressed on a variety of activated immune cells, including cytotoxic T cells and natural killer (NK) cells, is a promising biomarker for the early assessment of response to immunotherapies. We have developed a PET probe by radiolabeling a highly specific CD69 mAb, H1.2F3, with Zirconium-89 (89Zr), [89Zr]-deferoxamine (DFO)-H1.2F3. [89Zr]-DFO-H1.2F3 detected changes in CD69 expression on primary mouse T cells in vitro and detected activated immune cells in a syngeneic tumor immunotherapy model. In vitro uptake studies with [89Zr]-DFO-H1.2F3 showed a 15-fold increase in CD69 expression for activated primary mouse T cells, relative to untreated resting T cells. In vivo PET imaging showed that tumors of ICI-responsive mice had greater uptake than the tumors of nonresponsive and untreated mice. Ex vivo biodistribution, autoradiography, and IHC analyses supported the PET imaging findings. These data suggest that the CD69 PET imaging approach detects CD69 expression with sufficient sensitivity to quantify immune cell activation in a syngeneic mouse immunotherapy model and could allow for the prediction of therapeutic immune responses to novel immunotherapies.

Supplementary Data from Using CD69 PET Imaging to Monitor Immunotherapy-Induced Immune Activation

Supplementary Data from Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Purpose:Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response.Experimental Design:We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.Results:[18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses.Conclusions:This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy.See related commentary by Weber et al., p. 5241

Supplementary Figure from Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Supplementary Date from ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Supplementary Table from ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiotherapy, chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated fibroblasts (CAF) are abundant in PDAC tumors, the effects of radiotherapy on CAFs and the response of PDAC cells to radiotherapy are unknown. Using patient samples and orthotopic PDAC biological models, we showed that radiotherapy increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in radiotherapy-activated tumor cells, iNOS expression and nitric oxide (NO) secretion were significantly increased in CAFs secretome following radiotherapy. Culture of PDAC cells with conditioned media from radiotherapy-activated CAFs increased iNOS/NO signaling in tumor cells through NF-κB, which, in turn, elevated the release of inflammatory cytokines by the tumor cells. Increased NO after radiotherapy in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP). In murine orthotopic PDAC models, pancreatic tumor growth was delayed when iNOS inhibition was combined with radiotherapy. These data show the important role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors.Significance:A radiolabeled pH-targeted peptide can be used as a PET imaging tool to assess therapy response within PDAC and blocking iNOS/NO signaling may improve radiotherapy outcomes.

Supplementary Methods Supplementary Figure 1. iNOS staining in non-tumor and PDAC tumors obtained before treatment and after RT. Supplementary Figure 2. Western blot of iNOS in the total lysates of FC1245 orthotopic PDAC tumors obtained 2 days post-RT therapy. Supplementary Figure 3. Immunohistochemistry of trichrome in FC1245 orthotopic PDAC tumors obtained at 2 days post-RT therapy. Supplementary Figure 4. CAFs isolation and expansion from FC1245 orthotopic PDAC tumors. Supplementary Figure 5. Timeline for FC1245 culture with CM from CAFs or co-cultured with CAFs. Supplementary Figure 6. Cell growth of FC1245 cultured in the presence or absence of conditioned media from irradiated CAFs or co-cultured with irradiated CAFs. Supplementary Figure 7. NO2- levels in supernatants of human pancreatic cancer cell lines in the presence and absence of supernatants from human CAFs and treated with iNOS inhibitor 1400W. Supplementary Figure 8. IL-6, LIF, RANTES, and IL-13 concentrations in the supernatants of FC1245 PDAC cells, FC1245 incubated with conditioned medium from non-RT CAFs, and FC1245 incubated with conditioned medium from RT CAFs. Supplementary Figure 9. Gene expression heatmap of non-RT and RT fibroblasts. Supplementary Figure 10. Confocal images and quantification (mean {plus minus} S.E.M, n = 3) of immunofluorescence staining of α-SMA (green) in FC1245 orthotopic PDAC tumors at 14 days after cells' implantation. Supplementary Figure 11. FC1245 migration images and quantification using the wound healing assay. Supplementary Figure 12. Liver and pancreatic tumors at 6 days after surgery. Supplementary Figure 13. Liver, stomach, spleen, and pancreatic tumors at 14 days after surgery. Supplementary Figure 14. FC1245 cell growth and NO2- levels in supernatants of FC1245 with or without DETA-NONOate, 1400W or with the combination of DETA-NONOate/1400W. Supplementary Figure 15. FC1245 cell growth and NO2- levels in supernatants of FC1245 transfected with iNOS CRISPR activation plasmid or control CRISPR activation plasmid. Supplementary Figure 16. Cell growth of FC1245 cultured in the presence or absence of irradiated and non-irradiated CAFs. Supplementary Figure 17. Survival of mice implanted with FC1245 orthotopic PDAC tumors and treated with RT. Supplementary Figure 18. Tumor weight in PDAC tumors from RT, 1400W, and 1400W/RT treated mice on day 14 post-therapy. Supplementary Figure 19. Glucose uptake and lactate production of FC1245 PDAC cells treated with or without DETA-NO, 1400W, and the combination of DETA-NO/1400W. Supplementary Figure 20. Biodistribution data at 2 h after injection of 18F-FDG in mice bearing FC1245 orthotopic tumors. Supplementary Figure 21. Biodistribution data at 24 h after injection of 67Ga-labeled pHLIP in mice bearing FC1245 orthotopic tumors. Supplementary Table 1. RT administrations, time-points, and human PDAC sample collection time-points. Supplementary Table 2. Quantification of iNOS+/α-SMA+ in PDAC RT-treated samples obtained from patient #3. Supplementary Table 3. Quantification of iNOS+/cytokeratin+ in PDAC RT-treated samples obtained from patient #3. Supplementary Table 4. Quantification of iNOS+/α-SMA+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 5. Quantification of iNOS+/α-SMA+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Supplementary Table 6. Quantification of iNOS+/cytokeratin+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 7. Quantification of iNOS+/cytokeratin+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Supplementary Table 8. Quantification of GLUT1+/α-SMA+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 9. Quantification of GLUT1+/α-SMA+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors. Supplementary Table 10. Quantification of GLUT1+/cytokeratin+ in PDAC CT samples obtained from FC1245 orthotopic tumors. Supplementary Table 11. Quantification of GLUT1+/cytokeratin+ in PDAC RT-treated samples obtained from FC1245 orthotopic tumors.

Supplementary Figure from ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Purpose:Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response.Experimental Design:The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer.Results:Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors.Conclusions:The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

A critical benchmark in the development of antibody-based therapeutics is demonstration of efficacy in preclinical mouse models of human disease, many of which rely on immunodeficient mice. However, relatively little is known about how the biology of various immunodeficient strains impacts the in vivo fate of these drugs. Here we used immunoPET radiotracers prepared from humanized, chimeric, and murine mAbs against four therapeutic oncologic targets to interrogate their biodistribution in four different strains of immunodeficient mice bearing lung, prostate, and ovarian cancer xenografts. The immunodeficiency status of the mouse host as well as both the biological origin and glycosylation of the antibody contributed significantly to the anomalous biodistribution of therapeutic monoclonal antibodies in an Fc receptor-dependent manner. These findings may have important implications for the preclinical evaluation of Fc-containing therapeutics and highlight a clear need for biodistribution studies in the early stages of antibody drug development.Significance: Fc/FcγR-mediated immunobiology of the experimental host is a key determinant to preclinical in vivo tumor targeting and efficacy of therapeutic antibodies. Cancer Res; 78(7); 1820–32. ©2018 AACR.

Immuno-positron emission tomography (immuno-PET) is a powerful tool to noninvasively characterize the in vivo biodistribution of engineered antibodies (Abs). Methods L1 cell adhesion molecule (L1CAM)-targeting HuE71 immunoglobulin G1 (IgG1) and immunoglobulin G4 (IgG4) Abs bearing identical variable heavy and light chain sequences but different fragment crystallizable (Fc) portions were radiolabeled with Zirconium-89 (89Zr) and the in vivo biodistribution was studied in SKOV3 ovarian cancer xenografted nude mice. Results In addition to showing uptake in L1CAM-expressing SKOV3 tumors like its parental counterpart HuE71 IgG1, the afucosylated variant having enhanced Fc-receptor (FcR) affinity showed high nonspecific uptake in lymph nodes. On the other hand, aglycosylated HuE71 IgG1 with abrogated FcR binding did not show lymphoid uptake. The use of IgG4 subclass showed high nonspecific uptake in the kidneys, which was prevented by mutating serine at position 228 to proline (S228P) in the hinge region of the IgG4 Ab to mitigate in vivo fragment antigen-binding (Fab) arm exchange. Conclusion Our findings highlight the influence of Fc-modifications and the choice of IgG subclass on the in vivo biodistribution of Abs and the potential outcomes thereof.

Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted 89Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of 89Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting 89Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, 89Zr-DFOPODS-DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to 89Zr-DFOMal-DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a 89Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited ∼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.

Recent events in America in 2020 have stimulated a worldwide movement to dismantle anti-Black racism in all facets of our lives. Anti-Black racism is, as defined by the Movement for Black Lives, a "term used to specifically describe the unique discrimination, violence, and harm imposed on and impacting Black people specifically." In science, technology, engineering, and mathematics (STEM), we have yet to achieve the goal and responsibility to ensure that the field reflects the diversity of our lived experiences. Members of the Women in Molecular Imaging Network (WIMIN) have come together to take a stand on diversity, equity, and inclusion in the field of molecular imaging. We strongly condemn oppression in all its forms and strive to identify and dismantle barriers that lead to inequities in the molecular imaging community and STEM as a whole. In this series coined "Visions" (Antiracism and Allyship in Action), we identify and discuss specific actionable items for improving diversity and representation in molecular imaging and ensuring inclusion of all members of the community, inclusive of race, disability, ethnicity, religion, or LGBTQ+ identity. Although the issues highlighted here extend to other under-recruited and equity-seeking groups, for this first article, we are focusing on one egregious and persistent form of discrimination: anti-Black racism. In this special article, Black women residing in America present their lived experiences in the molecular imaging field and give candid insights into the challenges, frustrations, and hopes of our Black friends and colleagues. While this special article focuses on the experiences of Black women, we would like the readers to reflect on their anti-Blackness toward men, transgender, nonbinary, and gender non-conforming people. From the vulnerability we have asked of all our participants, these stories are meant to inspire and invoke active antiracist work among the readership. We present strategies for dismantling systemic racism that research centers and universities can implement in the recruitment, retention, mentorship, and development of Black trainees and professionals. We would like to specifically acknowledge the Black women who took the time to be interviewed, write perspectives, and share their lived experiences in hopes that it will inspire genuine and lasting change.

Purpose: Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response. Experimental Design: We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer. Results: [18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses. Conclusions: This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241

Targeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation. To produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with 89Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts. Mice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3 % ID/g vs. 22.8 ± 3.8 % ID/g at 24 h) and increased tumor uptake at 120 h (13.8 ± 8.0 % ID/g vs. 6.0 ± 1.2 % ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5). The combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice.

Introduction Despite its limitations, CA125 remains the most widely used biomarker for the diagnosis and treatment monitoring of ovarian cancer. Targeting the unshed portion of serum biomarkers such as CA125/MUC16 may afford more specific imaging and targeting of MUC16-positive tumors in High Grade Serous Ovarian Cancer (HGSOC) patients. Methods Six monoclonal antibodies raised against the 58 amino acid sequence between the extracellular cleavage site and the transmembrane region of MUC16 were radiolabeled with [89Zr]Zr4+. The radioimmunoconjugates were evaluated in vitro for molar activities, target binding affinity, cellular internalization and serum stability. In vivo characterization was performed via longitudinal positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice bearing subcutaneous xenografts of SKOV3 cells transfected with the proximal 114 amino-acids of MUC16 carboxy-terminus (SKOV3+). Results In vitro screening identified 9C9 and 4H11 as the lead antibody candidates based on their comparable binding affinities, serum stability and cellular internalization profiles. Despite an identical molecular footprint for binding to MUC16, [89Zr]Zr-DFO-4H11 yielded a more favorable in vivo radiopharmacologic profile. Furthermore, a humanized variant of 4H11 capable of binding MUC16 in vitro also yielded excellent in vivo profile in subcutaneous xenograft models of SKOV3+, OVCAR3 tumors and a patient-derived xenograft model representative of HGSOC. Conclusion Radiopharmacologic screening of antibodies early during their development can provide crucial information pertinent to the in vitro characterization and in vivo pharmacokinetics. The favorable in vivo profile demonstrated by humanized 4H11 combined with the use of its murine predecessor for immunohistochemical staining of biopsied tumor tissues from HGSOC patients makes a unique pair of antibodies that is poised for clinical translation.

Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([89Zr]Zr4+)-labeled antibodies is a powerful tool used for preclinical and clinical molecular imaging. However, a comprehensive study evaluating the variables involved in DFO-conjugation and 89Zr-radiolabeling of antibodies and their impact on the in vitro and in vivo behavior of the resulting radioimmunoconjugates has not been adequately performed. Here, we synthesized different DFO-conjugates of the HER2-targeting antibody (Ab)-trastuzumab, dubbed T5, T10, T20, T60, and T200-to indicate the molar equivalents of DFO used for bioconjugation. Next we radiolabeled the immunoconjugates with ([89Zr]Zr4+) under a comprehensive set of reaction conditions including different buffers (PBS, chelexed-PBS, TRIS/HCl, HEPES; ± radioprotectants), different reaction volumes (0.1-1 mL), variable amounts of DFO-conjugated Ab (5, 25, 50 μg), and radioactivity (0.2-1.0 mCi; 7.4-37 MBq). We evaluated the effects of these variables on radiochemical yield (RCY), molar activity (Am)/specific activity (As), immunoreactive fraction, and ultimately the in vivo biodistribution profile and tumor targeting ability of the trastuzumab radioimmunoconjugates. We show that increasing the degree of DFO conjugation to trastuzumab increased the RCY (∼90%) and Am/As (∼194 MBq/nmol; 35 mCi/mg) but decreased the HER2-binding affinity (3.5×-4.6×) and the immunoreactive fraction of trastuzumab down to 50-64%, which translated to dramatically inferior in vivo performance of the radioimmunoconjugate. Cell-based immunoreactivity assays and standard binding affinity analyses using surface plasmon resonance (SPR) did not predict the poor in vivo performance of the most extreme T200 conjugate. However, SPR-based concentration free calibration analysis yielded active antibody concentration and was predictive of the in vivo trends. Positron emission tomography (PET) imaging and biodistribution studies in a HER2-positive xenograft model revealed activity concentrations of 38.7 ± 3.8 %ID/g in the tumor and 6.3 ± 4.1 %ID/g in the liver for ([89Zr]Zr4+)-T5 (∼1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([89Zr]Zr4+)-T200 (10.9 ± 0.7 DFOs/Ab) yielded 16.2 ± 3.2 %ID/g in the tumor versus 27.5 ± 4.1 %ID/g in the liver. Collectively, our findings suggest that synthesizing trastuzumab immunoconjugates bearing 1-3 DFOs per Ab (T5 and T10) combined with radiolabeling performed in low reaction volumes using Chelex treated PBS or HEPEs without a radioprotectant provided radioimmunoconjugates having high Am/As (97 MBq/nmol; 17.5 ± 2.2 mCi/mg), highly preserved immunoreactive fractions (86-93%), and favorable in vivo biodistribution profile with excellent tumor uptake.

Mentorship is a fundamental aspect that contributes to the success of a career in science, technology, engineering, and mathematics (STEM), particularly in academia. Research suggests that underrepresented minorities (URMs) often experience less quality mentorship and face barriers to finding successful mentor-mentee relationships. URM trainees in STEM face challenges that are not encountered by their majority peers or mentors, adding another level of complexity to establishing important relationships. Mentors of URM trainees must therefore mentor beyond general scientific training and tailor their mentorship to be more culturally appropriate and inclusive, allowing URM trainees to bring their whole selves to the table and leading to their effective socialization. Herein, we present the perspectives of group leaders and trainees from around the globe to highlight key aspects of creating successful mentor-mentee relationships that are sustainable and productive for both parties.

Racial, ethnic, and gender representation in an academic setting means that teachers, professors, and other leaders reflect the demographics of the student body in the educational and professional spaces that they serve. This form of representation, which is often intersectional, strengthens communities and improves student outcomes, from as early as primary and secondary education, through to college education and beyond. Representation matters because it can shape the reputation and self-image of women and Black, Indigenous, and People of Color (BIPOC) within environments dominated by over-represented majorities (ORMs). From the perspective of BIPOC women trainees, the lack of BIPOC faculty who are visible minorities, particularly at the most senior level positions, often conjures questions of whether academia is a realistic career path for aspiring minority students. This article focuses on the key component of representation in the United States (U.S.), highlighting our vision for a solution for the so-called “leaky pipeline” for BIPOC in science, technology, engineering, and mathematic with action items to end it.

Poly (ADP-ribose) (PAR) is a negatively charged polymer that is biosynthesized by Poly (ADP-ribose) Polymerase-1 (PARP-1) and regulates various cellular processes. Alpha-synuclein (αSyn) is an intrinsically disordered protein (IDP) that has been directly implicated with driving the onset and progression of Parkinson’s disease (PD). The mechanisms by which α-synuclein (αSyn) elicits its neurotoxic effects remain unclear, though it is well established that the main components of Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients are aggregated hyperphosphorylated (S129) forms of αSyn (pαSyn). In the present study, we used immunofluorescence-based assays to explore if PARP-1 enzymatic product (PAR) promotes the aberrant cytoplasmic accumulation of pαSyn. We also performed quantitative measurements using in situ proximity ligation assays (PLA) on a transgenic murine model of α-synucleinopathy (M83-SNCA∗A53T) and post mortem PD/PDD patient samples to characterize PAR–pαSyn interactions. Additionally, we used bioinformatic approaches and site-directed mutagenesis to identify PAR-binding regions on αSyn. In summary, our studies show that PAR–pαSyn interactions are predominantly observed in PD-relevant transgenic murine models of αSyn pathology and post mortem PD/PDD patient samples. Moreover, we confirm that the interactions between PAR and αSyn involve electrostatic forces between negatively charged PAR and lysine residues on the N-terminal region of αSyn.

Purpose: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. Experimental Design: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. Results: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. Conclusions: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

BackgroundPoly (ADP-ribose) (PAR) is a negatively charged polymer that is biosynthesized by Poly (ADP-ribose) Polymerase-1 (PARP-1) and regulates various cellular processes. Alpha-synuclein (αSyn) is an intrinsically disordered protein (IDP) that has been directly implicated with driving the onset and progression of Parkinson’s disease (PD). The mechanisms by which αSyn elicits its neurotoxic effects remain unclear. Recent findings indicate that one of the key processes driving PD pathology are oligomeric species of αSyn. Furthermore, it is well established that the main components of Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients are aggregated hyperphosphorylated (S129) forms of αSyn (pαSyn).MethodsWe used biochemical and immunofluorescence-based assays to explore if PARP-1 enzymatic product (PAR) drives the conversion of monomeric αSyn into aggregated assemblies. We performed quantitative measurements using in situ proximity ligation assays (PLA) on a transgenic murine model of α-synucleinopathy (M83-SNCA*A53T) and post-mortem PD/PDD patient samples to characterize PAR-pαSyn interactions. Additionally, we used bioinformatic approaches and site-directed mutagenesis to identify PAR-binding regions on fibrillar αSyn.ResultsOur studies show that elevated intracellular levels of PAR promote the transition of αSyn into higher molecular weight forms. We report that PAR-pαSyn interactions are predominant in pathological states. Moreover, we confirm that the interactions between PAR and αSyn involve electrostatic forces between negatively charged PAR and lysine residues on the N-terminal region of αSyn.ConclusionsPAR plays a critical role in the early stages of monomeric αSyn aggregation, thereby attributing to PD pathogenesis. Based on our results, we report that PAR seeds monomeric αSyn aggregation and directly interacts with phosphorylated αSyn in conditions that are pathologically relevant to PD.

PURPOSE: Targeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation. PROCEDURES: To produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with (89)Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts. RESULTS: Mice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3% ID/g vs. 22.8 ± 3.8% ID/g at 24 hours) and increased tumor uptake at 120 h (13.8 ± 8.0% ID/g vs 6.0 ± 1.2% ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5). CONCLUSIONS: The combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice.

Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([(89)Zr]Zr(4+))-labeled antibodies is a powerful tool used for preclinical and clinical molecular imaging. However, a comprehensive study evaluating the variables involved in DFO-conjugation and (89)Zr-radiolabeling of antibodies, and their impact on the in vitro and in vivo behavior of the resulting radioimmunoconjugates has not been adequately performed. Here, we synthesized different DFO-conjugates of the HER2-targeting antibody (Ab) – trastuzumab – dubbed T5, T10, T20, T60, and T200 – to indicate the molar equivalents of DFO used for bioconjugation. Next we radiolabeled the immunoconjugates with ([(89)Zr]Zr(4+)) under a comprehensive set of reaction conditions including different buffers (PBS, chelexed-PBS, TRIS/HCl, HEPES; ± radioprotectants), different reaction volumes (0.1–1 mL), variable amounts of DFO-conjugated Ab (5, 25, 50 μg) and radioactivity (0.2–1.0 mCi; 7.4–37 MBq). We evaluated the effects of these variables on radiochemical yield (RCY), molar activity (A(m))/ specific activity (A(s)), immunoreactive fraction, and ultimately the in vivo biodistribution profile and tumor targeting ability of the trastuzumab radioimmunoconjugates. We show that increasing the degree of DFO conjugation to trastuzumab increased the RCY (~90%) and A(m); A(s) (~194 MBq/nmol; 35 mCi/mg) but decreased the HER2-binding affinity (3.5x-4.6x) and the immunoreactive fraction of trastuzumab down to 50–64%, which translated to dramatically inferior in vivo performance of the radioimmunoconjugate. Cell-based immunoreactivity assays and standard binding affinity analyses using surface plasmon resonance (SPR) did not predict the poor in vivo performance of the most extreme T200 conjugate. However, SPR-based concentration free calibration analysis yielded active antibody concentration and was predictive of the in vivo trends. Positron emission tomography (PET) imaging and biodistribution studies in a HER2-positive xenograft model revealed activity concentrations of 38.7 ± 3.8 %ID/g in the tumor and 6.3 ± 4.1 %ID/g in the liver for ([(89)Zr]Zr(4+))-T5 (~1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([(89)Zr]Zr(4+))-T200 (10.9 ± 0.7 DFOs/Ab) yielded 16.2 ± 3.2 %ID/g in the tumor versus 27.5 ± 4.1 %ID/g in the liver. Collectively, our findings suggest that synthesizing trastuzumab immunoconjugates bearing 1–3 DFOs per Ab (T5 and T10) combined with radiolabeling performed in low reaction volumes using chelexed-PBS without a radioprotectant provided radioimmunoconjugates having high A(m); A(s) (97 MBq/nmol; 17.5 ± 2.2 mCi/mg), highly preserved immunoreactive fractions (86–93%), and favorable in vivo biodistribution profile with excellent tumor uptake.

The current standard for breast PET imaging is 18F-FDG. The heterogeneity of 18F-FDG uptake in breast cancer limits its utility, varying greatly among receptor status, histopathologic subtypes, and proliferation markers. 18F-FDG PET often exhibits nonspecific internalization and low specificity and sensitivity, especially with tumors smaller than 1 cm3 MYC is a protein involved in oncogenesis and is overexpressed in triple-negative breast cancer (TNBC). Increased surface expression of transferrin receptor (TfR) is a downstream event of MYC upregulation and has been validated as a clinically relevant target for molecular imaging. Transferrin labeled with 89Zr has successfully identified MYC status in many cancer subtypes preclinically and been shown to predict response and changes in oncogene status via treatment with small-molecule inhibitors that target MYC and PI3K signaling pathways. We hypothesized that 89Zr-transferrin PET will noninvasively detect MYC and TfR and improve upon the current standard of 18F-FDG PET for MYC-overexpressing TNBC. Methods: In this study, 89Zr-transferrin and 18F-FDG imaging were compared in preclinical models of TNBC. TNBC cells (MDA-MB-157, MDA-MB-231, and Hs578T) were treated with bromodomain-containing protein 4 (BRD4) inhibitors JQ1 and OTX015 (0.5-1 μM). Cell proliferation, gene expression, and protein expression were assayed to explore the effects of these inhibitors on MYC and TfR. Results: Head-to-head comparison showed that 89Zr-transferrin targets TNBC tumors significantly better (P < 0.05-0.001) than 18F-FDG through PET imaging and biodistribution studies in MDA-MB-231 and MDA-MB-157 xenografts and a patient-derived xenograft model of TNBC. c-Myc and TfR gene expression was decreased upon treatment with BRD4 inhibitors and c-MYC small interfering RNA (P < 0.01-0.001 for responding cell lines), compared with vehicle treatment. MYC and TfR protein expression, along with receptor-mediated internalization of transferrin, was also significantly decreased upon drug treatment in MDA-MB-231 and MDA-MB-157 cells (P < 0.01-0.001). Conclusion:89Zr-transferrin targets human TNBC primary tumors significantly better than 18F-FDG, as shown through PET imaging and biodistribution studies. 89Zr-transferrin is a useful tool to interrogate MYC via TfR-targeted PET imaging in TNBC.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiotherapy, chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated fibroblasts (CAF) are abundant in PDAC tumors, the effects of radiotherapy on CAFs and the response of PDAC cells to radiotherapy are unknown. Using patient samples and orthotopic PDAC biological models, we showed that radiotherapy increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in radiotherapy-activated tumor cells, iNOS expression and nitric oxide (NO) secretion were significantly increased in CAFs secretome following radiotherapy. Culture of PDAC cells with conditioned media from radiotherapy-activated CAFs increased iNOS/NO signaling in tumor cells through NF-κB, which, in turn, elevated the release of inflammatory cytokines by the tumor cells. Increased NO after radiotherapy in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP). In murine orthotopic PDAC models, pancreatic tumor growth was delayed when iNOS inhibition was combined with radiotherapy. These data show the important role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors. Significance: A radiolabeled pH-targeted peptide can be used as a PET imaging tool to assess therapy response within PDAC and blocking iNOS/NO signaling may improve radiotherapy outcomes.

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.

Vascular endothelial growth factor-A (VEGF-A) acts via 2 vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, that play important and distinct roles in tumor biology. We reasoned that selective imaging of these receptors could provide unique information for diagnostics and for monitoring and optimizing responses to anticancer therapy, including antiangiogenic therapy. Herein, we report the development of 2 first-in-class 89Zr-labeled PET tracers that enable the selective imaging of VEGFR-1 and VEGFR-2. Methods: Functionally active mutants of scVEGF (an engineered single-chain version of pan-receptor VEGF-A with an N-terminal cysteine-containing tag for site-specific conjugation), named scVR1 and scVR2 with enhanced affinity to, respectively, VEGFR-1 and VEGFR-2, were constructed. Parental scVEGF and its receptor-specific mutants were site-specifically derivatized with the 89Zr chelator desferroxamine B via a 3.4-kDa PEG linker. 89Zr labeling of the desferroxamine B conjugates furnished scV/Zr, scVR1/Zr, and scVR2/Zr tracers with high radiochemical yield (>87%), high specific activity (≥9.8 MBq/nmol), and purity (>99%). Tracers were tested in an orthotopic breast cancer model using 4T1luc-bearing syngeneic BALB/c mice. For testing tracer specificity, tracers were coinjected with an excess of cold proteins of the same or opposite receptor specificity or pan-receptor scVEGF. PET imaging, biodistribution, and dosimetry studies in mice, as well as immunohistochemical analysis of harvested tumors, were performed. Results: All tracers rapidly accumulated in orthotopic 4T1luc tumors, allowing for the successful PET imaging of the tumors as early as 2 h after injection. Blocking experiments with an excess of pan-receptor or receptor-specific cold proteins indicated that more than 80% of tracer tumor uptake is VEGFR-mediated, whereas uptake in all major organs is not affected by blocking within the margin of error. Critically, blocking experiments indicated that VEGFR-mediated tumor uptake of scVR1/Zr and scVR2/Zr was mediated exclusively by the corresponding receptor, VEGFR-1 or VEGFR-2, respectively. In contrast, uptake of pan-receptor scV/Zr was mediated by both VEGFR-1 and VEGFR-2 at an approximately 2:1 ratio. Conclusion: First-in-class selective PET tracers for imaging VEGFR-1 and VEGFR-2 were constructed and successfully validated in an orthotopic murine tumor model.

Human epidermal growth factor receptor 2 (HER2) gene amplification and/or protein overexpression in tumors is a prerequisite for initiation of trastuzumab therapy. Although HER2 is a cell membrane receptor, differential rates of endocytosis and recycling engender a dynamic surface pool of HER2. Since trastuzumab must bind to the extracellular domain of HER2, a depressed HER2 surface pool hinders binding. Using in vivo biological models and cultures of fresh human tumors, we find that the caveolin-1 (CAV1) protein is involved in HER2 cell membrane dynamics within the context of receptor endocytosis. The translational significance of this finding is highlighted by our observation that temporal CAV1 depletion with lovastatin increases HER2 half-life and availability at the cell membrane resulting in improved trastuzumab binding and therapy against HER2-positive tumors. These data show the important role that CAV1 plays in the effectiveness of trastuzumab to target HER2-positive tumors., Trastuzumab binding to tumor cells depends on the availability of HER2 at the cell membrane. Here the authors show that caveolin-1 (CAV1) regulates HER2 density at the cell membranes and that CAV1 gene knockdown or protein depletion via the cholesterol modulator lovastatin, increases trastuzumab binding and anti-tumor activity.

The hepatocyte growth factor (HGF) binding antibody rilotumumab (AMG102) was modified for use as a 89Zr-based immuno-PET imaging agent to noninvasively determine the local levels of HGF protein in tumors. Because recent clinical trials of HGF-targeting therapies have been largely unsuccessful in several different cancers (e.g., gastric, brain, lung), we have synthesized and validated 89Zr-DFO-AMG102 as a companion diagnostic for improved identification and selection of patients having high local levels of HGF in tumors. To date, patient selection has not been performed using the local levels of HGF protein in tumors. Methods: The chelator p-SCN-Bn-DFO was conjugated to AMG102, radiolabeling with 89Zr was performed in high radiochemical yields and purity (>99%), and binding affinity of the modified antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA)-type binding assay. PET imaging, biodistribution, autoradiography and immunohistochemistry, and ex vivo HGF ELISA experiments were performed on murine xenografts of U87MG (HGF-positive, MET-positive) and MKN45 (HGF-negative, MET-positive) and 4 patient-derived xenografts (MET-positive, HGF unknown). Results: Tumor uptake of 89Zr-DFO-AMG102 at 120 h after injection in U87MG xenografts (HGF-positive) was high (36.8 ± 7.8 percentage injected dose per gram [%ID/g]), whereas uptake in MKN45 xenografts (HGF-negative) was 5.0 ± 1.3 %ID/g and a control of nonspecific human IgG 89Zr-DFO-IgG in U87MG tumors was 11.5 ± 3.3 %ID/g, demonstrating selective uptake in HGF-positive tumors. Similar experiments performed in 4 different gastric cancer patient-derived xenograft models showed low uptake of 89Zr-DFO-AMG102 (∼4-7 %ID/g), which corresponded with low HGF levels in these tumors (ex vivo ELISA). Autoradiography, immunohistochemical staining, and HGF ELISA assays confirmed that elevated levels of HGF protein were present only in U87MG tumors and that 89Zr-DFO-AMG102 uptake was closely correlated with HGF protein levels in tumors. Conclusion: The new immuno-PET imaging agent 89Zr-DFO-AMG102 was successfully synthesized, radiolabeled, and validated in vitro and in vivo to selectively accumulate in tumors with high local levels of HGF protein. These results suggest that 89Zr-DFO-AMG102 would be a valuable companion diagnostic tool for the noninvasive selection of patients with elevated local concentrations of HGF in tumors for planning any HGF-targeted therapy, with the potential to improve clinical outcomes.

A critical benchmark in the development of antibody-based therapeutics is demonstration of efficacy in preclinical mouse models of human disease, many of which rely on immunodeficient mice. However, relatively little is known about how the biology of various immunodeficient strains impacts the in vivo fate of these drugs. Here we used immunoPET radiotracers prepared from humanized, chimeric, and murine mAbs against four therapeutic oncologic targets to interrogate their biodistribution in four different strains of immunodeficient mice bearing lung, prostate, and ovarian cancer xenografts. The immunodeficiency status of the mouse host as well as both the biological origin and glycosylation of the antibody contributed significantly to the anomalous biodistribution of therapeutic monoclonal antibodies in an Fc receptor-dependent manner. These findings may have important implications for the preclinical evaluation of Fc-containing therapeutics and highlight a clear need for biodistribution studies in the early stages of antibody drug development. Significance: Fc/FcγR-mediated immunobiology of the experimental host is a key determinant to preclinical in vivo tumor targeting and efficacy of therapeutic antibodies. Cancer Res; 78(7); 1820–32. ©2018 AACR.

The current standard for breast PET imaging is 18F-FDG. The heterogeneity of 18F-FDG uptake in breast cancer limits its utility, varying greatly among receptor status, histopathologic subtypes, and proliferation markers. 18F-FDG PET often exhibits nonspecific internalization and low specificity and sensitivity, especially with tumors smaller than 1 cm3. MYC is a protein involved in oncogenesis and is overexpressed in triple-negative breast cancer (TNBC). Increased surface expression of transferrin receptor (TfR) is a downstream event of MYC upregulation and has been validated as a clinically relevant target for molecular imaging. Transferrin labeled with 89Zr has successfully identified MYC status in many cancer subtypes preclinically and been shown to predict response and changes in oncogene status via treatment with small-molecule inhibitors that target MYC and PI3K signaling pathways. We hypothesized that 89Zr-transferrin PET will noninvasively detect MYC and TfR and improve upon the current standard of 18F-FDG PET for MYC-overexpressing TNBC. Methods: In this study, 89Zr-transferrin and 18F-FDG imaging were compared in preclinical models of TNBC. TNBC cells (MDA-MB-157, MDA-MB-231, and Hs578T) were treated with bromodomain-containing protein 4 (BRD4) inhibitors JQ1 and OTX015 (0.5–1 μM). Cell proliferation, gene expression, and protein expression were assayed to explore the effects of these inhibitors on MYC and TfR. Results: Head-to-head comparison showed that 89Zr-transferrin targets TNBC tumors significantly better (P < 0.05–0.001) than 18F-FDG through PET imaging and biodistribution studies in MDA-MB-231 and MDA-MB-157 xenografts and a patient-derived xenograft model of TNBC. c-Myc and TfR gene expression was decreased upon treatment with BRD4 inhibitors and c-MYC small interfering RNA (P < 0.01–0.001 for responding cell lines), compared with vehicle treatment. MYC and TfR protein expression, along with receptor-mediated internalization of transferrin, was also significantly decreased upon drug treatment in MDA-MB-231 and MDA-MB-157 cells (P < 0.01–0.001). Conclusion: 89Zr-transferrin targets human TNBC primary tumors significantly better than 18F-FDG, as shown through PET imaging and biodistribution studies. 89Zr-transferrin is a useful tool to interrogate MYC via TfR-targeted PET imaging in TNBC.

Objectives To compare the radiolabeling performance, stability, and practical efficacy of the chelators CHX-A″-DTPA and H 4 octapa with the therapeutic radiometal 90 Y. Methods The bifunctional chelators p -SCN-Bn-H 4 octapa and p -SCN-Bn-CHX-A″-DTPA were conjugated to the HER2-targeting antibody trastuzumab. The resulting immunoconjugates were radiolabeled with 90 Y to compare radiolabeling efficiency, in vitro and in vivo stability, and in vivo performance in a murine model of ovarian cancer. Results High radiochemical yields (>95%) were obtained with 90 Y-CHX-A″-DTPA-trastuzumab and 90 Y-octapa-trastuzumab after 15min at room temperature. Both 90 Y-CHX-A″-DTPA-trastuzumab and 90 Y-octapa-trastuzumab exhibited excellent in vitro and in vivo stability. Furthermore, the radioimmunoconjugates displayed high tumoral uptake values (42.3±4.0%ID/g for 90 Y-CHX-A″-DTPA-trastuzumab and 30.1±7.4%ID/g for 90 Y-octapa-trastuzumab at 72h post-injection) in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. Finally, 90 Y radioimmunotherapy studies performed in tumor-bearing mice demonstrated that 90 Y-CHX-A″-DTPA-trastuzumab and 90 Y-octapa-trastuzumab are equally effective therapeutic agents, as treatment with both radioimmunoconjugates yielded substantially decreased tumor growth compared to controls. Conclusions Ultimately, this work demonstrates that the acyclic chelators CHX-A″-DTPA and H 4 octapa have comparable radiolabeling, stability, and in vivo performance, making them both suitable choices for applications requiring 90 Y.

Introduction: Although heterogeneous, the extracellular tumor environment is generally marked by decreased pH relative to nontumor tissues due to cancer cell metabolism. Using a pH-sensitive peptide to deliver diagnostic PET isotopes can provide clinicians with improved diagnostic tools for cancer-specific imaging and an alternative to receptor-based cancer targeting. pH (low) Insertion Peptides (pHLIPs) have previously been shown to target cancerous tissues and are able to deliver PET metals to the cancer site utilizing a NOTA-based chelator. Methods: The thiol-containing cysteine moiety on the pHLIP peptide sequence was conjugated to the NOTA chelator. The NOTA-derivatized peptides were labeled with 64CuCl2 in 100 mM NH4OAc (pH 5) solution; the [18F]-fluoride was reacted with AlCl3 before forming the [18F]-AlF-NOTA-pHLIP complexes in NH4OAc buffered reaction mixtures (pH ∼ 4.1). After purification and formulation, the radiolabeled peptides were injected intravenously into 4T1 breast, PC-3 prostate, LNCaP prostate, or B16-F10 melanoma tumor-bearing mice. Results: The 4T1 tumor-bearing mice showed significant uptake > 10%ID/g by 4 h p.i. and continued to show increased uptake of the 64Cu-NOTA-cysVar3 over 20 h. Tumors were visible in the 4T1 tumor-bearing mice as early as 2 h, but had the greatest tumor-to-background ratios at 24 h. Initial studies with B16-F10 tumor-bearing mice showed > 9%ID/g uptake in the tumor by 4 h p.i. with slight increase of 64Cu-NOTA-cysVar3 uptake over 24 h. Due to the placement of the melanoma tumors away from internal organs, tumors were visible by 1 h p.i. Additionally, the size of the melanoma tumors did not affect the%ID/g of radiotracer uptake. Conclusions: Our studies indicate that the NOTA-cys(pHLIP), specifically NOTA-cysVar3, radiolabeled with 64Cu or 18F is a viable imaging tool for detecting highly metabolic tumors in preclinical mouse models. Funding: NIH F32 CA186721 (D.W.D.), NIH R01 CA138468 (J.S.L.), NIH MSKCC Center Grant (P30-CA08748). Citation Format: Dustin W. Demoin, Kimberly J. Edwards, Linden C. Wyatt, Mirkka Sarparanta, Jacob Pourat, Oleg A. Andreev, Yana K. Reshetnyak, Nerissa Viola-Villegas, Jason S. Lewis. Targeting a variety of cancers with NOTA-derivatized pH (low) insertion peptide (pHLIP) complexes with 64Cu and 18F: What cancers are targetable. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4205.

OBJECTIVES: The PET imaging isotope 89Zr (t1/2 = ∼3.3 d) is the most commonly used isotope with antibodies (Abs) (immuno-PET). The chelator desferrioxamine (DFO) has been conjugated to many Abs, radiolabeled with 89Zr, and imaged in many animals and humans. It is generally known that conjugating too many chelators onto Abs will decrease binding affinity and immunoreactivity. We have conjugated DFO to the HER2/neu targeting Abs trastuzumab in varying quantities, and then radiolabeled with 89Zr under a comprehensive set of conditions and evaluated their effects on radiochemical yields (RCY), specific activity (SA), immunoreactivity, and ultimately in vivo tumor targeting. We believe a comprehensive study on these variables has not been sufficiently performed and would be of substantial value to anyone attaching isotopes to antibodies. METHODS: The chelator p-SCN-Bn-DFO was conjugated to trastuzumab (5, 10, 20, 40, 60, 100, 150, 200 equivalents), purified, and the number of attached chelators determined by MALDI-TOF mass spectrometry. These different modifications of DFO-trastuzumab were then radiolabeled and RCY were monitored from 15-60 min by iTLC (25 °C and 37 °C, n = 3, 50 mM EDTA mobile phase). The immunoreactivity of these different conjugates was determined by a cell binding assay with SKOV3 cells, and binding affinity by surface plasmon resonance. This was followed by 89Zr radiolabeling with varying quantities of Abs (5, 25, 50 μg), different buffers (PBS, chelexed PBS, TRIS/HCl, HEPES), different volumes (100, 300, 1000 μL), and activities (0.2, 1.0 mCi). To test radiolysis effects, 89Zr-DFO-trastuzumab (∼200 μCi) was radiolabeled and then placed in 100 μL of different buffers (PBS, TRIS/HCl, saline, HEPES, PBS + ascorbic acid, and PBS + gentisic acid) for 48 h before determining immunoreactivity. Representative samples (5, 10, 20, and 200 equiv. DFO conjugations) were radiolabeled and injected in mice bearing subcutaneous SKOV3 xenografts (n = 4 per group), and imaged by PET at 24, 72, and 120 h p.i. with biodistributions done at 120 h. RESULTS: Regardless of the equiv. chelator used during conjugations, a maximum average number of chelates/Abs of only ∼10-12 could be attached. To achieve maximum SA, a small quantity of Abs was used (5 μg), achieving RCY of ∼50-90% and SA of ∼15-35 mCi/mg (depending on chelates/Abs). The optimal conjugation level and radiolabeling conditions were 1-3 DFO per Abs (5-10 equiv. conjugation), chelexed PBS as buffer, minimum buffer volume, and no radioprotectant (gentisic acid or ascorbic acid). The in vivo effects of these chelate conjugations were dramatic, revealing a trend where higher amounts of attached DFO correlated to lower tumor uptake and higher liver and spleen uptake. The 89Zr-DFO-Trastuzumab that was prepared by reacting 5 equiv. of chelator (∼1.4 ± 0.5 chelates/Abs) had tumor and liver uptake at 120 h p.i. of 38.7 ± 3.8 and 6.3 ± 4.1%ID/g, respectively, where the 200 equiv. conjugation (∼10.9 ± 0.7 chelates/Abs) had values of 16.2 ± 3.2 and 27.5 ± 4.1%ID/g, respectively (p < 0.05). CONCLUSIONS: Increasing the degree of DFO attachment to trastuzumab resulted in a concomitant increase in maximum SA values (up to ∼35 mCi/mg), and a decrease in immunoreactivity/binding affinity, which translated to inferior in vivo performance. Optimized radiolabeling conditions provided very high SA of 17.5 ± 2.2 mCi/mg with only ∼1.4 ± 0.5 chelates/Abs. Citation Format: Eric W. Price, Jonathan M. Glaser, Kimberly J. Edwards, Jason S. Lewis. Optimized antibody modification and radiolabeling conditions for zirconium-89 immuno-PET. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-182.