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BackgroundNuclear medicine is on the constant search of precision radiopharmaceutical approaches to improve patient management. Although discordant expression of the estrogen receptor (ER) and the human epidermal growth factor receptor 2 (HER2) in breast cancer is a known dilemma for appropriate patient management, traditional tumor sampling is often difficult or impractical. While 2-deoxy-2[18F]fluoro-D-glucose (18F-FDG)-positron emission tomography (PET) is an option to detect subclinical metastases, it does not provide phenotype information. Radiolabeled antibodies are able to specifically target expressed cell surface receptors. However, their long circulating half-lives (days) require labeling with long-lived isotopes, such as89Zr, in order to allow sufficient time for tracer clearance from the blood compartment and to accumulate adequately in target tumors and, thus, generate high-quality PET images. The aim of this study was to develop a dual-tracer PET imaging approach consisting of a fast-clearing small molecule and a slow-clearing antibody. This approach was evaluated in a model consisting of mice harboring separate breast cancer xenografts with either an ER+/HER2− or ER−/HER2+ phenotype, comparable to human metastatic disease with intertumor heterogeneity. Lastly, the aim of our study was to determine the feasibility of specifically identifying these two important phenotypes in an acceptable time window.MethodsFemale nude mice were subcutaneously implanted on opposite shoulders with the ER+/HER2− and ER−/HER2+ MCF-7 and JIMT-1 tumor cell lines, respectively. A second model was developed consisting of mice implanted orthotopically with either MCF-7 or JIMT-1 cells. Pharmacokinetic analysis, serial PET imaging, and biodistribution were first performed for [89Zr]Zr-DFO-trastuzumab (89Zr-T) up to 8 days post-injection (p.i.) in JIMT-1 bearing mice. Region-of-interest (ROI) and biodistribution-derived uptake (% injected-activity/gram of tissue [%IA/g]) values and tumor-to-background ratios were obtained. Results were compared in order to validate ROI and identify early time points that provided high contrast tumor images. For the dual-tracer approach, cohorts of tumor-bearing mice were then subjected to sequential tracer PET imaging. On day 1, mice were administered 4-fluoro-11β-methoxy-16α-[18F]-fluoroestradiol (4FMFES) which targets ER and imaged 45 min p.i. This was immediately followed by the injection of89Zr-T. Mice were then imaged on day 3 or day 7. ROI analysis was performed, and uptake was calculated in tumors and selected healthy organs for all radiotracers. Quality of tumor targeting for all tracers was evaluated by tumor contrast visualization, tumor and normal tissue uptake, and tumor-to-background ratios.Results89Zr-T provided sufficiently high tumor and low background uptake values that furnished high contrast tumor images by 48 h p.i. For the dual-tracer approach, 4FMFES provided tumor uptake values that were significantly increased in MCF-7 tumors. When89Zr-T-PET was combined with18F-4FMFES-PET, the entire dual-tracer sequential-imaging procedure provided specific high-quality contrast images of ER+/HER2− MCF-7 and ER−/HER2+ JIMT-1 tumors for 4FMFES and89Zr-T, respectively, as short as 72 h from start to finish.ConclusionsThis protocol can provide high contrast images of tumors expressing ER or HER2 within 3 days from injection of 4FMFES to final scan of89Zr-T and, hence, provides a basis for future dual-tracer combinations that include antibodies.

Biologically-based therapies increasingly rely on the endocytic cycle of internalization and exocytosis of target receptors for cancer therapies. However, receptor trafficking pathways (endosomal sorting (recycling, lysosome localization) and lateral membrane movement) are often dysfunctional in cancer. Antibody-drug conjugates (ADCs) have revitalized the concept of targeted chemotherapy by coupling inhibitory antibodies to cytotoxic payloads. Significant advances in ADC technology and format, and target biology have hastened the FDA approval of nine ADCs (four since 2019). Although the links between aberrant endocytic machinery and cancer are emerging, the impact of dysregulated internalization processes of ADC targets and response rates or resistance have not been well studied. This is despite the reliance on ADC uptake and trafficking to lysosomes for linker cleavage and payload release. In this review, we describe what is known about all the target antigens for the currently approved ADCs. Specifically, internalization efficiency and relevant intracellular sorting activities are described for each receptor under normal processes, and when complexed to an ADC. In addition, we discuss aberrant endocytic processes that have been directly linked to preclinical ADC resistance mechanisms. The implications of endocytosis in regard to therapeutic effectiveness in the clinic are also described. Unexpectedly, information on endocytosis is scarce (absent for two receptors). Moreover, much of what is known about endocytosis is not in the context of receptor-ADC/antibody complexes. This review provides a deeper understanding of the pertinent principles of receptor endocytosis for the currently approved ADCs.

The fact that there are now five immune checkpoint inhibitor (ICI) monoclonal antibodies approved since 2016 that target programmed cell death protein 1 or programmed death ligand-1 for the treatment of metastatic and refractory bladder cancer is an outstanding achievement. Although patients can display pronounced responses that extend survival when treated with ICIs, the main benefit of these drugs compared to traditional chemotherapy is that they are better tolerated and result in reduced adverse events (AEs). Unfortunately, response rates to ICI treatment are relatively low and, these drugs are expensive and have a high economic burden. As a result, their clinical efficacy/cost-value relationship is debated. Long sought after targeted molecular therapeutics have now emerged and are boasting impressive response rates in heavily pre-treated, including ICI treated, patients with metastatic bladder cancer. The antibody-drug conjugates (ADCs) enfortumab vedotin (EV) and sacituzumab govitecan (SG) have demonstrated the ability to provide objective response rates (ORRs) of 44% and 31% in patients with bladder tumor cells that express Nectin-4 and Trop-2, respectively. As a result, EV was approved by the U.S. Food and Drug Administration for the treatment of patients with advanced or metastatic bladder cancer who have previously received ICI and platinum-containing chemotherapy. SG has been granted fast track designation. The small molecule Erdafitinib was recently approved for the treatment of patients with advanced or metastatic bladder cancer with genetic alterations in fibroblast growth factor receptors that have previously been treated with a platinum-containing chemotherapy. Erdafitinib achieved an ORR of 40% in patients including a proportion who had previously received ICI therapy. In addition, these targeted drugs are sufficiently tolerated or AEs can be appropriately managed. Hence, the early performance in clinical effectiveness of these targeted drugs are substantially increased relative to ICIs. In this article, the most up to date follow-ups on treatment efficacy and AEs of the ICIs and targeted therapeutics are described. In addition, drug price and cost-effectiveness are described. For best overall value taking into account clinical effectiveness, price and cost-effectiveness, results favor avelumab and atezolizumab for ICIs. Although therapeutically promising, it is too early to determine if the described targeted therapeutics provide the best overall value as cost-effectiveness analyses have yet to be performed and long-term follow-ups are needed. Nonetheless, with the arrival of targeted molecular therapeutics and their increased effectiveness relative to ICIs, creates a potential novel paradigm based on ‘targeting’ for affecting clinical practice for metastatic bladder cancer treatment.

Therapeutic anti-HER2 antibodies and antibody–drug conjugates (ADCs) have undoubtedly benefitted patients. Nonetheless, patients ultimately relapse—some sooner than others. Currently approved anti-HER2 drugs are expensive and their cost-effectiveness is debated. There is increased awareness that internalization and lysosomal processing including subsequent payload intracellular accumulation and retention for ADCs are critical therapeutic attributes. Although HER2 preferential overexpression on the surface of tumor cells is attractive, its poor internalization and trafficking to lysosomes has been linked to poor therapeutic outcomes. To help address such issues, this review will comprehensively detail the most relevant findings on internalization and cellular accumulation for approved and investigational anti-HER2 antibodies and ADCs. The improved clarity of the HER2 system could improve antibody and ADC designs and approaches for next-generation anti-HER2 and other receptor targeting agents.

Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.

In recent decades, therapy for acute myeloid leukemia (AML) has remained relatively unchanged, with chemotherapy regimens primarily consisting of an induction regimen based on a daunorubicin and cytarabine backbone, followed by consolidation chemotherapy. Patients who are relapsed or refractory can be treated with allogeneic hematopoietic stem-cell transplantation with modest benefits to event-free and overall survival. Other modalities of immunotherapy include antibody therapies, which hold considerable promise and can be categorized into unconjugated classical antibodies, multivalent recombinant antibodies (bi-, tri- and quad-specific), toxin-conjugated antibodies and radio-conjugated antibodies. While unconjugated antibodies can facilitate Natural Killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), bi- and tri-specific antibodies can engage either NK cells or T-cells to redirect cytotoxicity against AML targets in a highly efficient manner, similarly to classic ADCC. Finally, toxin-conjugated and radio-conjugated antibodies can increase the potency of antibody therapies. Several AML tumour-associated antigens are at the forefront of targeted therapy development, which include CD33, CD123, CD13, CLL-1 and CD38 and which may be present on both AML blasts and leukemic stem cells. This review focused on antibody therapies for AML, including pre-clinical studies of these agents and those that are either entering or have been tested in early phase clinical trials. Antibodies for checkpoint inhibition and microenvironment targeting in AML were excluded from this review.

Radioimmunotherapy (RIT) with anti-CD123 monoclonal antibody CSL360 modified with nuclear translocation sequence (NLS) peptides and labeled with the Auger electron-emitter, (111)In ((111)In-NLS-CSL360) was studied in the prevalent NOD/SCID mouse AML engraftment assay. Significant decreases in CD123(+) leukemic cells and impairment of leukemic stem cell self-renewal were achieved with high doses of RIT. However, NOD/SCID mice were very radiosensitive to these doses. At low non-toxic treatment doses, (111)In-NLS-CSL360 demonstrated a trend towards improved survival associated with decreased spleen/body weight ratio, an indicator of leukemia burden, and almost complete eradication of leukemia from the bone marrow in some mice.

Despite the high interest and concern due to an increasing incidence and death rate, patients who develop muscle invasive bladder cancer (MIBC) have few options available. However, the past decade has produced many candidate bladder tumor-specific markers but further development of these markers is still needed for creating effective targeted medications to solve this urgent need. Interleukin-5 receptor α-subunit (IL-5Rα) has recently been reported to be involved in MIBC progression. Thus, we aimed to validate IL-5Rα as a target for antibody-conjugates to better manage patients with MIBC. Patients were recruited and their tumors were processed for IL-5Rα immunohistochemical analysis. NOD/SCID mice were also heterotopically implanted with the human MIBC HT-1376 and HT-B9 cell lines and established xenografts immunohistochemically evaluated for IL-5Rα and compared against patient tumors. Using the mAb A14, an antibody-drug conjugate (ADC) and a radiolabeled immunoconjugate (RIC) were developed by conjugating to vinblastine and to the positron emitter copper-64 ( 64 Cu), respectively. As a proof-of-concept for ADC and RIC efficacy, in vitro cytotoxicity and in vivo positron emission tomography (PET) imaging in tumor-bearing mice were performed, respectively. In addition, as rapid internalization and accumulation are important components for effective antibody-conjugates, we evaluated these aspects in response to IL-5 and 64 Cu-A14 treatments. Our findings suggest that although IL-5Rα protein expression is preferentially increased in MIBC, it is rapid IL-5Rα-mediated internalization allowing vinblastine-A14 to have cytotoxic activity and 64 Cu-A14 to detect MIBC tumors in vivo. This is the first report to elucidate the potential of IL-5Rα as an attractive MIBC target for antibody-conjugate applications. Refereed/Peer-reviewed

Introduction 111 In-DTPA-NLS-CSL360 radioimmunoconjugates (RIC) recognize the overexpression of the interleukin-3 receptor α-subchain (CD123) relative to the β-subchain (CD131) on leukemia stem cells (LSC). Our aim was to study Auger electron radioimmunotherapy (RIT) of acute myeloid leukemia (AML) with 111 In-DTPA-NLS-CSL360 in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice or NOD-Rag1 null IL2rγ null (NRG) mice engrafted with CD123 + human AML-5 cells. Methods The toxicity of three doses of 111 In-DTPA-NLS-CSL360 (3.3–4.8MBq; 11–15μg each) injected i.v. every two weeks was studied in non-engrafted NOD/SCID or NRG mice pre-treated with 200cGy of γ-radiation required for AML engraftment. Engraftment efficiency of (1–5)×10 6 cells AML-5 cells inoculated i.v. into NOD/SCID or NRG mice was assessed by flow cytometric analysis for human CD45 + (hCD45 + ) cells in the bone marrow (BM) and spleen. AML-5 engrafted mice were treated with two or three doses (3.7MBq; 10μg each) every two weeks of 111 In-DTPA-NLS-CSL360, non-specific 111 In-DTPA-NLS-hIgG, unlabeled CSL360 (10μg) or normal saline. The percentage of hCD45 + cells in the BM and spleen were measured at one week after completion of treatment. Results 111 In-DTPA-NLS-CSL360 in combination with 200cGy of γ-radiation caused an initial transient decrease in body weight in NOD/SCID but not in NRG mice. There were no hematological, liver or kidney toxicities. The spleen exhibited 13-fold lower engraftment efficiency than the BM in NOD/SCID mice inoculated with 1×10 6 cells but both organs were highly (>85%) engrafted in NRG mice. Unexpectedly, 111 In-DTPA-NLS-CSL360 or non-specific 111 In-DTPA-NLS-hIgG caused a paradoxical 1.5-fold increase ( P + cells in the BM of NOD/SCID mice compared to normal saline treated mice. 111 In-DTPA-NLS-CSL360 reduced hCD45 + cells in the spleen by 3.0-fold compared to 111 In-DTPA-NLS-hIgG ( P =0.0015) but the proportion of hCD45 + cells was not significantly different than in normal saline treated mice. Unlabeled CSL360 decreased the percentage of hCD45 + cells in the BM ( P =0.004) or spleen ( P =0.007) in NOD/SCID mice by 1.6-fold and 2.5-fold, respectively. 111 In-DTPA-NLS-CSL360 or unlabeled CSL360 did not decrease the proportion of hCD45 + cells in the BM or spleen of NRG mice, due to a much higher leukemic burden. Conclusion 111 In-DTPA-NLS-CSL360 and 111 In-DTPA-NLS-hIgG caused a paradoxical increase in the proportion of hCD45 + cells in the BM of NOD/SCID mice. This may be due to a priming effect on the BM niche that promotes expansion of engrafted hCD45 + cells, analogous to γ-radiation required for AML engraftment. There appears to be a competition between this effect and the cytotoxic effects of the Auger electrons on leukemia cells. The effectiveness of 111 In-DTPA-NLS-CSL360 on reducing hCD45 + cells in the BM or spleen of NOD/SCID and NRG mice was dependent on the leukemic burden. Advances in knowledge and implications for patient care This study demonstrates for the first time a paradoxical radiation priming effect of RIT on enhancing the hCD45 + cell population in the BM and spleen of NOD/SCID or NRG mice. Our results have important implications for preclinical evaluation of radioimmunotherapies for patients with AML.

Background: Bladder cancer is one of the most prevalent cancers impacting adults worldwide.Although,successfully treated in its early stages, there are no effective therapies when the cancer has progressed into the surrounding muscle layer, clinically classified as muscle invasive bladder cancer (MIBC). CD125 was recently shown to be involved in MIBC progression and uniquely overexpressed in MIBC tumors relative to healthy urothelium or superficial tumors. Cyclin E (CNE) is a critical cell cycle protein and regulates progression of normal cells to replicate their DNA. There is a strong link between CNE dysregulation and tumorigenesis and was recently found to be overexpressed in MIBC. We characterized the monoclonal antibodies (mAbs) A14 specific for CD125, and HE-12 and HE-172 specific for CNE with a long-term purpose to develop a CD125xCNE therapeutic bispecific antibody. In order, for the antibody to efficiently target CNEour group developed a natural composite compound (termed Accum)that conjugates to surface lysines and enables mAbs to escape endosomal entrapment followed by active routing to and efficient accumulation in the nucleus. Methods: For Accum conjugation, maleimide groups were introduced into A14 by reaction with 10-to-100-fold molar excess of a PEGylated SMCC crosslinker at RT for 1 h. Purified and concentrated maleimide-derivatized A14 was reacted with 100-fold molar excess Accum for 18 h at 4 °C. Excess Accum-A14 was purified and concentrated. Conjugation was characterized by SDS-PAGE. CD125-positive MIBC cells were treated with Accum-A14 and nuclear localization efficiency determined by confocal microscopy.HE-12 and HE-172 mAbs were evaluated for binding nuclear CNE by flow cytometry using the whole cell and fractionated cell lysate along with saponin to enable the mAbs to diffuse into the cell. In addition, HE-12 and HE-172 mAb binding was evaluated by co-immunoprecipitation. PCR amplification of A14, HE-12, and HE-172 was performed. Results: A14 is readily loaded with Accum with minimal aggregation.Accum-A14 undergoesCD125-specific internalization and has 25-60-fold increased nuclear localization relative to A14 in MIBC cells.The binding of the intracellular CNE by mAb HE-12 and HE-172 was specific. Conclusion: This preliminary data demonstrates that Accum modification of A14 retains CD125 specificity and efficiently localizes to the nucleus of MIBC cells. HE-12 and HE172 recognized CNE. On going work will construct the CD125xCNE bispecific antibody. Future studies will determine the efficiency to target CNE and its impact to MIBC progression. Citation Format: Judit Hunyadkurti, Jeffrey Leyton, Laurent Fafard-Couture, Vincent Lacasse, Marc-Andre Bonin, Angel Lopez. CD125xCNE bispecific antibody development to treat bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5610.

The murine 1G8 (micro1G8) monoclonal antibody directed against prostate stem cell antigen (PSCA) prevents prostate tumor establishment, growth, and metastasis in murine models. To further delineate in vivo targeting properties, micro1G8 was radiolabeled with In-111 and evaluated in nude mice bearing PC3-PSCA xenografts. Tumor activity ranged from 11.8% to 17.1% injected dose per gram (ID/g) at 24 to 96 hours postinjection. To extend the clinical applicability of micro1G8, a chimeric 1G8 antibody was produced that exhibited specific binding to PSCA and significant antitumor effect over micro1G8 in established LAPC-9 prostate cancer xenografts (P=0.0014). However, low expression yields and instability prompted us to humanize 1G8 by grafting the complementary determining regions onto the stable, human Fv framework of anti-p185 4D5v8 (trastuzumab). Two humanized 1G8 (hu1G8) versions (A and B) that differed in the number of murine residues present in the C-terminal half of CDR-H2, were produced. Biacore binding studies demonstrated affinities of 1.47 nM for micro1G8 and 3.74 nM for hu2B3-B, representing a 2.5-fold reduction. Tumor targeting of version B radioiodinated with I was evaluated by serial microPET imaging. Specific tumor targeting of I-hu1G8-B to PC3-PSCA [12.7 (+/-1.6)% ID/g at 94 h] and LAPC-9 [6.6 (+/-0.9)% ID/g at 168 h) xenografts was observed. Inhibition of tumor growth by hu1G8-B was demonstrated in mice bearing low-expressing SW-780-PSCA bladder carcinoma xenografts. In this model, the micro1G8 was ineffective, whereas the hu1G8-B exhibited approximately 50% inhibitory effect. These data support further development of hu1G8 anti-PSCA antibody for targeted imaging and therapy for tumors of urogenital origin.

Engraftment of primary human acute myeloid leukemia (AML) specimens into the bone marrow (BM) of NOD/SCID mice has been used to study leukemia biology and new treatments for the disease. CSL360 is a chimeric IgG 1 monoclonal antibody that recognizes CD123 (IL-3 receptor α-subchain) expressed in the absence of CD131 (β-subchain), an epitope that is displayed by leukemia stem cells (LSCs). We are studying CSL360 modified with diethylenetriaminepentaacetic acid (DTPA) for complexing 111 In and 13-mer nuclear translocation sequence (NLS) peptides to enable nuclear importation in LSCs for Auger electron radioimmunotherapy (RIT) of AML. We demonstrate that microSPECT/CT imaging using 111 In-DTPA-NLS-CSL360 revealed engraftment of primary human AML specimens into the BM and spleen of NOD/SCID mice. Our results suggest that microSPECT/CT imaging is a powerful tool which enables non-invasive assessment of the engraftment of AML into NOD/SCID mice and in the current study specifically probes an epitope displayed by the LSC subpopulation. The targeting of 111 In-DTPA-NLS-CSL360 to sites of AML engraftment in the NOD/SCID mouse model is encouraging for future RIT studies. Ultimately, SPECT imaging could be applied in AML patients to assess the delivery of 111 In-DTPA-NLS-CSL360 to sites of leukemia and be combined with Auger electron RIT using the same agent targeting the LSC population as a “theranostic” pair.

The humanized antibody (hu1G8) has been shown to localize to prostate stem cell antigen (PSCA) and image PSCA-positive xenografts. We previously constructed hu1G8 anti-PSCA antibody fragments and tested them for tumor targeting and the ability to image prostate cancer at early and late time points postinjection by positron emission tomography (PET). We now then compare the PET imaging and the radioactivity accumulation properties in prostate cancer tumors and nontarget tissues to determine the superior 124 I-labeled hu1G8 antibody format. 124 I-labeled diabody, minibody, scFv-Fc, scFv-Fc double mutant (DM), and parental IgG were administered into severe combined immunodeficiency (SCID) mice bearing LAPC-9 xenografts and followed by whole-body PET imaging of mice at preselected time points. Regions of interest were manually drawn around tumor and nontarget tissues and evaluated for radioactivity accumulation. The 124 I-hu1G8 IgG has its best time point for tumor high-contrast imaging at 168 hours postinjection. The 124 I-hu1G8 minibody at 44 hours postinjection results in superior tumor high-contrast imaging compared to the other antibody formats. The 124 I-hu1G8 minibody at 44 hours postinjection also has comparable percent tumor radioactivity compared to 124 I-hu1G8 IgG at 168 hours postinjection. The 124 I-hu1G8 minibody is the best engineered hu1G8 antibody format for imaging prostate cancer.