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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.

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.

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope (64)Cu (t(1/2) = 12.7 h) as an example payload in this protocol.

There is considerable interest in the development of novel and more efficient delivery systems for improving the efficacy of photodynamic therapy (PDT). The authors in this highlighted issue describe the synthesis and the photobiological characterizations of two photosensitizer (PS) conjugates based on β-carboline derivatives covalently conjugated to folic acid (FA) coupled to bovine serum albumin (BSA) as a carrier system specifically targeting cancer cells overexpressing FA receptor alpha (FRα). Accordingly, only the FA-BSA-β-carboline conjugates are internalized specifically in FRα-positive cells and are proved to be phototoxic. On the other hand, albumin-β-carboline conjugates without FA or β-carboline derivatives alone are not internalized and nontoxic. This conjugate is among the first to produce a conjugate composed of a PS and FA molecules that are directly conjugated to BSA. In addition, the in vitro studies are the first evidence that directly conjugated FA-BSA can be used as carriers to selectively enhance cytotoxicity by PDT relative to unmodified PS or nontargeted BSA-PS. This strategy is a positive step forward for the covalent design and construction of a photodynamic nanomedicine for FR-positive tumors.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed ⁶⁴Cu-A14-ChAcNLS, ⁶⁴Cu-A14-NLS, and ⁶⁴Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved ⁶⁴Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per ⁶⁴Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. ⁶⁴Cu-A14-ChAcNLS increased ⁶⁴Cu cellular accumulation in HT-1376 and HT-B9 cells relative to ⁶⁴Cu-A14 and ⁶⁴Cu-A14-NLS. In addition, we tested ⁶⁴Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that ⁶⁴Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to ⁶⁴Cu-A14. However, ⁶⁴Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to ⁶⁴Cu-A14. This resulted in ⁶⁴Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that ⁶⁴Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to ⁶⁴Cu-A14. In addition, ⁶⁴Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting. Refereed/Peer-reviewed

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 Leukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, 111 In-NLS-7G3, which recognizes the CD123 + /CD131 - phenotype uniquely displayed by LSCs. Methods The surviving fraction (SF) of CD123 + /CD131 - AML-5 cells exposed to 111 In-NLS-7G3 (33–266 nmols/L; 0.74 MBq/μg) or to γ-radiation (0.25-5 Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with 111 In-NLS-7G3 (16–66 nmols/L) or with γ-radiation (0.25–2 Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of 111 In-NLS-7G3 measured by cell fractionation. Results Binding of 111 In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1 μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123 + /CD131 - epitope. 111 In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25 Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with 111 In-NLS-7G3 (16–66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25–0.5 Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to 111 In-NLS-7G3 (66 nmols/L) delivered up to 0.6 Gy to AML-5 cells. Conclusions We conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of 111 In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.

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.

Increased expression of epidermal growth factor receptors (EGFR) in breast cancer (BC) is often associated with trastuzumab (Herceptin)-resistant forms of the disease and represents an attractive target for novel therapies. Nimotuzumab is a humanized IgG(1) monoclonal antibody that is in clinical trials for treatment of EGFR-overexpressing malignancies. We show here that nimotuzumab derivatized with benzylisothiocyanate diethylenetriaminepentaacetic acid for labelling with the subcellular range Auger electron-emitter, (111)In and modified with nuclear translocation sequence (NLS) peptides ((111)In-NLS-Bn-DTPA-nimotuzumab) was bound, internalized and transported to the nucleus of EGFR-positive BC cells. Emission of Auger electrons in close proximity to the nucleus caused multiple DNA double-strand breaks which diminished the clonogenic survival (CS) of MDA-MB-468 cells that have high EGFR density (2.4 × 10(6) receptors/cell) to less than 3 %. (111)In-Bn-DTPA-nimotuzumab without NLS peptide modification was sevenfold less effective for killing MDA-MB-468 cells. (111)In-Bn-DTPA-nimotuzumab with/without NLS peptide modification were equivalently cytotoxic to MDA-MB-231 and TrR1 BC cells that have moderate EGFR density (5.4 × 10(5) or 4.2 × 10(5) receptors/cell, respectively) reducing their CS by twofold. MDA-MB-231 cells have intrinsic trastuzumab resistance due to low HER2 density, whereas TrR1 cells have acquired resistance despite HER2 overexpression. Biodistribution and microSPECT/CT imaging revealed that (111)In-NLS-Bn-DTPA-nimotuzumab exhibited more rapid elimination from the blood and lower tumour uptake than (111)In-Bn-DTPA-nimotuzumab. Tumour uptake of the radioimmunoconjugates in mice with MDA-MB-468 xenografts was high (8-16 % injected dose/g) and was blocked by administration of an excess of unlabelled nimotuzumab, demonstrating EGFR specificity. We conclude that (111)In-Bn-DTPA-nimotuzumab with/without NLS peptide modification are promising Auger electron-emitting radioimmunotherapeutic agents for EGFR-positive BC, but (111)In-Bn-DTPA-nimotuzumab may be preferred due to its higher tumour uptake in vivo.

Antibody-Drug Conjugates (ADC) represent a promising therapeutic modality for improving the clinical management of cancer. However, there now exists resistant mechanisms by cancer cells that are effective at ultimately reducing the cellular accumulation of the delivered drug and hence, reducing cytotoxic effectiveness. Kadcyla® (trastuzumab-emtansine [T-DM1]) is a benchmark as it is the first solid-tumor approved ADC. Despite this new therapeutic option, approximately 43% of patients achieve complete or partial response and most who initially respond develop resistance soon after. In order to transform current ADCs to more effective products improved intracellular delivery strategies could provide solutions. Our group developed is a natural composite compound (termed Accum) that conjugates to surface lysines and enables antibodies to escape endosomal entrapment followed by active routing to the nucleus. Modified antibodies retain high tumor cell selectivity, enhanced cellular accumulation, and improved tumor delivery of molecular payloads in vivo. We asked whether the modification of Accum-modified T-DM1 maintained its intracellular delivery mechanism and cytotoxic effectiveness in T-DM1-resistant cells. The cancer cell lines N87, OE19 (gastric), JIMT1, SK-BR-3 (breast), SKOV3 and SKOV3.IP1 (ovarian) were treated with increasing concentrations of T-DM1 in a 3 day on, 10 days off fashion for a period of 1 to 2 months. Resistant clones were identified. Parental and resistant cells were placed and passaged in SILAC (Stably Labeled Amino acid for Cell culture) media for proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Cells were also treated with Accum-T-DM1 and mechanistic underpinnings determined. The IC50 of Accum-T-DM1 was increased 50- and 10-fold relative to T-DM1 in parental OE19, and JIMT1 cells, respectively. T-DM1 resistant cell lines were successfully generated for each tumor type. Typically, the T-DM1 IC50 values were increased ≥100-fold in resistant cells compared to parental cells. Preliminary proteomic studies revealed HER2 expression is marginally reduced in resistant cells. IP of Accum-T-DM1 co-precipitated showed the interaction with α-importin. This study is a work in progress of modifying T-DM1 with Accum and demonstrating enhanced cytotoxicity in T-DM1 resistant HER2-positive tumor cells. Ongoing studies will determine DM1 accumulation levels, potential resistance mechanisms, and ultimately cytotoxic effectiveness. Citation Format: Vincent Lacasse, Jeffrey V. Leyton, Simon Beaudoin, Mark Barok, Heikki Joensuu. Development of next-generation antibody-drug conjugates for resistant HER2-positive tumors [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 756.

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.

PurposeProstate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies.MethodsYeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors.ResultsThe A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo.ConclusionOf the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study.

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.

Chimeric IgG1 monoclonal antibody CSL360 recognizes the CD123(+)/CD131(-) phenotype expressed by leukemic stem cells (LSC). Auger electron-emitting (111)In-DTPA-NLS-CSL360 radioimmunoconjugates incorporating nuclear translocation sequence (NLS) peptides bound specifically to Raji cells transfected with CD123 and exhibited a KD of 11nmols/L in a competition receptor-binding assay using CD123-transfected CHO cells. (111)In-DTPA-NLS-CSL360 was bound, internalized and transported to the nucleus of human AML-5 myeloid leukemia cells. The clonogenic survival of AML-5 cells was reduced by (111)In-DTPA-NLS-CSL360 up to 3.7-fold. Isotype control (111)In-DTPA-chIgG1 was 2-fold less cytotoxic, and unlabeled CSL360, DTPA-NLS-CSL360 or free (111)In acetate did not decrease cell survival. These results are promising for further evaluation of (111)In-DTPA-NLS-CSL360 for Auger electron radioimmunotherapy of AML targeting the critical LSC subpopulation.

An improved approach composed of an oxidation reaction in acidic H2O2 solution and a sequential silanization reaction using neat silane reagents for surface modification of poly(dimethylsiloxane) (PDMS) substrates was developed. This solution-phase approach is simple and convenient for some routine analytical applications in chemistry and biology laboratories and is designed for intact PDMS-based microfluidic devices, with no device postassembly required. Using this improved approach, two different functional groups, poly(ethylene glycol) (PEG) and amine (NH2), were introduced onto PDMS surfaces for passivation of nonspecific protein absorption and attachment of biomolecules, respectively. X-ray electron spectroscopy and temporal contact angle experiments were employed to monitor functional group transformation and dynamic characteristics of the PEG-grafted PDMS substrates; fluorescent protein solutions were introduced into the PEG-grafted PDMS microchannels to test their protein repelling characteristics. These analytical data indicate that the PEG-grafted PDMS surfaces exhibit improved short-term surface dynamics and robust long-term stability. The amino-grafted PDMS microchannels are also relatively stable and can be further activated for modifications with peptide, DNA, and protein on the surfaces of microfluidic channels. The resulting biomolecule-grafted PDMS microchannels can be utilized for cell immobilization and incubation, semiquantitative DNA hybridization, and immunoassay.

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.

Leukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131⁻ phenotype uniquely displayed by LSCs.The surviving fraction (SF) of CD123⁺/CD131⁻ AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33-266 nmols/L; 0.74MBq/μg) or to γ-radiation (0.25-5Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) or with γ-radiation (0.25-2Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation.Binding of (111)In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131⁻ epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16-66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25-0.5Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6Gy to AML-5 cells.We conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.

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 124I-labeled hu1G8 antibody format. 124I-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 124I-hu1G8 IgG has its best time point for tumor high-contrast imaging at 168 hours postinjection. The 124I-hu1G8 minibody at 44 hours postinjection results in superior tumor high-contrast imaging compared to the other antibody formats. The 124I-hu1G8 minibody at 44 hours postinjection also has comparable percent tumor radioactivity compared to 124I-hu1G8 IgG at 168 hours postinjection. The 124I-hu1G8 minibody is the best engineered hu1G8 antibody format for imaging prostate cancer.

Fluorescence imaging (FI) provides increased sensitivity for the detection of bladder tumors compared to white light cystoscopy. However, most advanced dyes used in FI suffer from nonspecific cellular accumulation resulting in increased false-positives. In addition, these dyes have not made significant progress in detecting the most aggressive bladder tumors such as muscle invasive bladder cancer (MIBC). Our objective is to develop an antibody-conjugate (AC) PDI agent against interleukin-5 receptor α-subunit (IL-5Rα)-positive MIBC cells. IL-5Rα is overexpressed on MIBC cells relative to superficial bladder cancer or healthy bladder cells. We selected a class of fluorescent dyes termed BODIPY for its availability, facile coupling to proteins, and easy chemical modifications to accommodate required absorption and fluorescence properties. BODIPY was attached to an AC composed of the antibody A14 functionalized with a moiety termed ChAcNLS, which we have shown enables it to escape endosome entrapment and redirect its trafficking to the nucleus. This novel approach results in increased intracellular accumulation of attached molecular payloads in MIBC cells. We aim to determine if ChAcNLS-A14 will increase intracellular accumulation of BODIPY to enhance fluorescence detection of MIBC by probe-based confocal laser endomicroscopy (pCLE). In addition, we aim to develop in an improved orthotopic rat bladder tumor model of human MIBC for testing. Methods We have prepared a carboxyl BODIPY derivative (B105) that allows for facile coupling to A14-ChAcNLS using routine procedures and reagents. The number of B105 and ChAcNLS per A14 was determined by fluorescence spectroscopy and SDS-PAGE, respectively. Intracellular accumulation of B105 was determined by treating HT1376 MIBC cells with A14-B105 and ChAcNLS-A14-B105. Cells were then imaged by fluorescence confocal microscopy. For the in vivo model of MIBC, RNU rats were anesthetized and their bladder washed in mild acid to disrupt the endothelium. HT1376 cells marked with the fluorescent marker DiL and instilled in the rat bladder. Intravesical pCLE was performed 2 weeks later. Results ChAcNLS-A14-B105 conjugate contained 7 molecules of B105 and 3 ChAcNLS peptides per A14. Confocal microscopy revealed the level of B105 intracellular accumulation in IL-5Rα-positive MIBC cells was increase ≥10-fold with ChAcNLS-A14-B105 relative to A14-B105. pCLE revealed HT1376 cells survived and colonized the rat bladder urothelium. Conclusion ChAcNLS-A14-B105 was successfully constructed and shown to significantly enhance B105 intracellular accumulation in target IL-5Rα-positive MIBC cells. A novel orthotopic rat bladder tumor model of human MIBC was also generated. Future testing of ChAcNLS-A14-B105 administered intravesically in RNU rat bladders bearing orthotopic tumors of human MIBC will be performed to determine the potential clinical translation of this new FI agent. Citation Format: Laurent Fafard-Couture, Simon Beaudoin, Samira Osati, Véronique Dumoulon-Perreault, Réjean Lebel, Brigitte Guérin, Johan E. van Lier, Jeffrey Leyton. ChAcNLS-A14-B105, a novel fluorescence imaging agent for enhanced detection of muscle invasive bladder cancer. [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 4243.

Purpose: Prostate stem cell antigen (PSCA) is a cell surface glycoprotein that is overexpressed in prostate cancer, including hormone refractory disease. Previous preclinical studies showed the intact anti-PSCA antibodies, 1G8 and hu1G8, localized specifically to PSCA-expressing xenografts. Optimal micro positron emission tomography (microPET) imaging using hu1G8, however, required a delay of 168 hours postinjection. In this study, the 2B3 minibody (an 80-kDa engineered antibody fragment) has been produced for rapid targeting and imaging.Experimental Design: A gene encoding a PSCA-specific minibody, VL-linker-VH-hinge-huIgG1 CH3, was assembled. The minibody was expressed by secretion from mammalian cells and purified by cation exchange chromatography. Relative affinity and specificity were determined by competition ELISA and flow cytometry. Serial microPET imaging using a 124I-labeled minibody was conducted at 4 and 21 hours in mice bearing LAPC-9 AD, LAPC-9 AI, PC-3, and LNCaP-PSCA human prostate cancer xenografts. Tumor and tissue biodistribution was determined, and region of interest analysis of the images was conducted.Results: Yields of 20 mg/L purified 2B3 minibody were obtained that showed specific binding to LNCaP-PSCA cells. Purified 2B3 minibody showed specific binding to LNCaP-PSCA cells with an apparent affinity of 46 nmol/L. Radioiodinated 2B3 minibody showed rapid nontarget tissue and blood clearance kinetics (t1/2β = 11.2 hours). MicroPET scanning using the 124I-2B3 minibody showed both androgen-dependent and -independent tumors as early as 4 hours and excellent high contrast images at 21 hours postinjection.Conclusions: Imaging PSCA-positive prostate cancer is feasible using an intermediate size antibody fragment at 21 hours.

We have previously demonstrated preclinical in vivo targeting of prostate stem cell antigen (PSCA) using a humanized anti-PSCA 2B3 monoclonal antibody (mAb). However, humanization resulted in 5-fold loss of apparent affinity relative to the parental mAb (1 nM). In this study, diabodies (scFv dimers of 55 kDa) were generated from 2B3 including variants with different linker lengths as well as back-mutations to original murine residues to improve affinity. Parental 2B3 (p2B3) and back-mutated 2B3 (bm2B3) diabodies (Dbs) with five- or eight-amino acid linkers (p2B3-Db5, p2B3-Db8, bm2B3-Db5 and bm2B3-Db8) were evaluated for binding to PSCA by flow cytometry and affinities were determined by surface plasmon resonance. Back-mutation restored the affinity from 5.4 to 1.9 nM. Stability, evaluated by size exclusion, revealed that diabodies with eight-residue linkers existed as a mixture of dimeric and monomeric species at low concentrations (

Abstract 1909 Introduction: Patients with acute myeloid leukemia (AML) in remission relapse frequently from residual leukemic stem cells (LSCs) that are CD34+CD38-CD123+. NK-92 is an infusible CD16- malignant NK cell line cytotoxic against AML and after irradiation for prevention of NK cell malignancy has minimal adverse events in Phase I trials. Here, we tested irradiated NK-92 (iNK-92) against primary AML in a NOD/SCID gamma null (NSG) xenograft model. To optimize killing of LSCs in vitro we utilized a gene modified CD16+NK-92 against the CD123+ leukemia cell line OCI/AML5 treated with and without an anti-CD123 monoclonal antibody (7G3) to facilitate antibody dependent cell mediated cytotoxicity (ADCC). Methods: NSG mice were irradiated with 225 or 325 cGy before infusion with primary AML blasts (1 or 3×10e6 cells). iNK-92 was started 1 or 10 days post AML infusion and given 1–3 times weekly (15 or 20×10e6 cells/dose) to a total dose of 60–75 ×10e6 cells/mouse. In vitro cytotoxicity was measured by the chromium release assay. OCI/AML5 or primary AML was pretreated with 7G3 or anti-Class I antibody respectively at 10 ug/ml 2 hours before ADCC assays. Results: We developed an NSG AML xenograft model using a CD34+CD38+ primary AML sample containing a small fraction of CD34+CD38- cells that were predominantly CD123+. 3×10e6 primary cells induced leukemia in NSG mice by 6 weeks and maintained comparable potency up to quaternary transplantations with a stable immunophenotype. In vitro cytotoxicity against LSCs was assessed by treating 1×10e6 first passage BM derived primary AML +/− 5×10e6 iNK-92 ×4 hours and injecting into 2 cohorts of 5 mice. At 6 weeks mice were sacrificed and bone marrow harvested. Average leukemic engraftment in the iNK-92 group (79.8, 3.48, 92.1, 81.3, 86.1, Av= 68.6%) was less than untreated AML inoculated group (95.0, 93.4, 19.4, 95, 97.3, Av=80.0%), but not statistically significant (p=0.62). Removing one poorly engrafted outlier mouse from each group yielded a higher engraftment in the control (Av=95.1%) versus the treatment group (Av=84.8%) and was significant (p=0.011). To test iNK-92 to treat engrafted leukemia, NSG mice were infused with 3×10e6 AML cells starting day 10 with 20×10e6 iNK-92 weekly ×6 doses. Survival was improved in the treatment group to near statistical significance (p=0.055), but all mice ultimately succumbed to disease. Addition of IL-2 to iNK-92 did not improve outcomes (p=0.13). 3×10e6 primary AML cells were also infused into 2 cohorts of 4 mice and treated +/− iNK-92 from day 2 and given 15×10e6 cells twice weekly to 75×10e6 total dose. BM (1×10e6 cells) from each of 4 primary recipients in control and treatment was serially transplanted 1:1 into four new NSG mice. BM engraftment occurred in all AML only cohort secondary mice (80.8, 93.3, 80.4, 96.4 Av=87.7%) while one mouse from iNK-92 group was leukemia free with engraftment at background levels of non-injected mice (96.4, 94.7, 1.8, 95.7 Av=72.2%). Proportion of LSCs in secondary transplanted mice was: AML (8.01, 9.48, 8.66, 5.25, Av=7.85%) and AML + iNK-92 (7.13, 3.46, 0.03, 4.0 Av=3.66%) which was statistically significant (p=0.05). CD16+NK-92 killed CD123+ OCI/AML5 cells at effector:target (E:T) ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 35.0 +/−4.0, 9.0+/−6.6, -2.0 +/−0.1, 1.7 +/−3.3) and was significantly enhanced (2–6x) when targets were coated with anti-CD123 mAb (% lysis +/−SD: 64.3 +/−3.1, 48.5 +/−4.1, 20.9 +/−0.1, 10.1+/−3.3). Further, CD16+NK-92 also killed primary AML at E:T ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 34.7 +/−4.6, 15.6 +/− 4.7, 11.5 +/− 2.0, 7.7 +/−1.6) which was significantly enhanced (2–3x) by coating with anti-Class I monoclonal antibodies (% lysis +/−SD: 64.8 +/−10.4, 31.1 +/− 8.9, 30.2 +/− 9.4, 23.9+/−2.8). Conclusion: iNK-92 reduces engraftment of AML and improves survival in a primary AML xenograft model. CD16+NK-92 cytotoxicity against primary AML is enhanced with anti-class I antibodies via ADCC, demonstrating that CD16+NK-92 can be redirected against bulk primary leukemia using a highly expressed cell surface marker. Finally, CD16+NK-92 cytotoxicity can be improved against OCI/AML5 with anti-CD123 antibodies via ADCC, providing the first proof-of-principle for the targeting of leukemic stem cells by combining humoral and cellular approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 968 Radioimmunotherapy (RIT) of acute myelogenous leukemia (AML) is a promising strategy that could improve the long-term outcome of patients. A limitation of current therapies is their inability to eradicate the leukemic stem cell (LSC), which is responsible for causing and propagating the disease and the failure to achieve long-term survival. Monoclonal antibody (mAb) 7G3 recognizes CD123 (IL-3R α) in the absence of CD131 (IL-3R β) a LSC characteristic but not of normal hematopoietic stem cells. Our laboratory is developing 111In-labeled 7G3 modified with nuclear localization sequence (NLS) peptides for Auger electron RIT of AML aimed at the LSC. These electrons have only nanometer-micrometer range but are lethal when released inside the cell nucleus. Our objective was to examine the ability of 111In-NLS-7G3 to localize at sites of AML engraftment in NOD-scid mice taking advantage of the concurrent gamma emissions of 111In for microSPECT/CT imaging. These properties were examined in NOD-scid mice engrafted with CD123+/CD131- AML cell lines which avoids the challenges of engrafting primary AML specimens including their variability in engraftment efficiency and heterogeneity in CD123 expression. Alternatively, mice bearing s.c. CD123-transfected Raji (Raji-CD123) tumors were studied. 7G3 was reacted with sulfo-SMCC to introduce maleimide groups for conjugation to NLS peptides (CGYGPKKKRKVGG) then directly derivatized with DTPA for complexing 111In. The pharmacokinetics (PK) of elimination from the blood of 111In-NLS-7G3 intact IgG2a and F(ab’)2 fragments were studied in NOD-scid and Balb/c mice. The effect of pre-administration of a 10-fold excess of unlabeled BM4 IgG2a or IgG1 irrelevant mAbs on the PK was assessed. Imaging and biodistribution studies were performed at 48 to 72 h p.i. of 111In-NLS-7G3 IgG2a and F(ab’)2 in AML-engrafted mice. Sites of engraftment visualized on the images were analyzed for AML by flow cytometry. PK studies revealed an unusually rapid elimination of 111In-NLS-7G3 IgG2a but not F(ab’)2 in NOD-scid compared to Balb/c mice with blood radioactivity levels at 18 h 4-fold lower (3.9 ± 0.9 vs. 17.0 ± 4.4% i.d./ml, respectively). This rapid elimination prevented uptake into Raji-CD123 xenografts and did not allow their visualization by microSPECT/CT. The elimination of 111In-NLS-7G3 F(ab’)2 was not significantly different in NOD-scid or Balb/c mice (0.60 ± 0.17 vs. 0.87 ± 0.06 %i.d./ml, respectively at 72 h p.i.). Pre-administration of unlabeled BM4 improved the retention of 111In-NLS-7G3 in the blood by 3.2-fold and allowed for high uptake (14.7 ± 2.8 % i.d./g) and imaging of tumors. Xenografts also had high uptake (7.2 ± 3.8 % i.d./g) and imaged with 111In-NLS-7G3 F(ab’)2. AML-3, -4 and -5 cells engrafted into bone marrow (BM) and NOD-scid mice assessed by the proportion of CD45+ cells (12%, 0.5% and 63%, respectively). CD45+ cells in the spleen for AML-5 engraftment was 83%. Uptake of 111In-NLS-7G3 F(ab’)2 in the spleen and BM of AML-3 engrafted mice were 5-fold and 1.5-fold higher than 111In-NLS-BM4 control F(ab’)2. The spleen was visualized by microSPECT/CT in engrafted mice receiving 111In-NLS-7G3 F(ab’)2 but not with 111In-NLS-BM4 F(ab’)2. In AML-3 engrafted mice imaged with 111In-NLS-7G3 IgG2a, multiple intense foci of radioactivity were noted in the spine, pelvis and femur, representing leukemia in the marrow. Extramedullary (EM) disease was also imaged in the brain and lymph nodes. Uptake in the femur was 2-fold higher in engrafted mice compared to non-engrafted mice. In AML-5 engrafted mice, outgrowths of AML at the femoral heads was imaged with 111In-NLS-7G3 IgG2a following pre-administration of an excess of unlabeled BM4. No visualization of these AML growths was noted for mice receiving 111In-NLS-BM4. We conclude that microSPECT/CT allowed sensitive detection of the specific uptake of 111In-NLS-7G3 and its F(ab’)2 fragments in AML engrafted mice. PK and Raji-CD123 xenograft microSPECT/CT studies identified a tumor delivery barrier. A robust AML engraftment model was established with cells displaying the CD123+/CD131- phenotype. These results are promising for preclinical evaluation of 111In-NLS-7G3 for Auger electron RIT of AML and suggest that imaging is a useful tool to assess engraftment in NOD-scid mice as well as for evaluating response to 111In-NLS-7G3. Preliminary results of studies to examine 111In-NLS-7G3 for RIT of AML will also be presented. Disclosures: No relevant conflicts of interest to declare.