Designing combination therapies for cancer treatment: application of a mathematical framework combining CAR T-cell immunotherapy and targeted radionuclide therapy.

Introduction: Cancer combination treatments involving immunotherapies with targeted radiation therapy are at the forefront of treating cancers. However, dosing and scheduling of these therapies pose a challenge. Mathematical models provide a unique way of optimizing these therapies. Methods: Using a preclinical model of multiple myeloma as an example, we demonstrate the capability of a mathematical model to combine these therapies to achieve maximum response, defined as delay in tumor growth. Data from mice studies with targeted radionuclide therapy (TRT) and chimeric antigen receptor (CAR)-T cell monotherapies and combinations with different intervals between them was used to calibrate mathematical model parameters. The dependence of progression-free survival (PFS), overall survival (OS), and the time to minimum tumor burden on dosing and scheduling was evaluated. Different dosing and scheduling schemes were evaluated to maximize the PFS and optimize timings of TRT and CAR-T cell therapies. Results: Therapy intervals that were too close or too far apart are shown to be detrimental to the therapeutic efficacy, as TRT too close to CAR-T cell therapy results in radiation related CAR-T cell killing while the therapies being too far apart result in tumor regrowth, negatively impacting tumor control and survival. We show that splitting a dose of TRT or CAR-T cells when administered in combination is advantageous only if the first therapy delivered can produce a significant benefit as a monotherapy. Discussion: Mathematical models are crucial tools for optimizing the delivery of cancer combination therapy regimens with application along the lines of achieving cure, maximizing survival or minimizing toxicity.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.

Comparison of IL-2-antibody to IL-2-Fc with or without stereotactic radiation therapy in CEA immunocompetent mice with CEA positive tumors.

BACKGROUND: The potent immune effects of interleukin-2 (IL-2) for cancer therapy can be increased by genetic fusion of IL-2 to the Fc domain of an antibody (IL-2-Fc) or tumor targeted by genetic fusion to a whole antibody known as an immunocytokine (ICK). METHODS: An anti-CEA ICK (M5A-IL-2) was compared to an IL-2-Fc fusion protein using tumor therapy and PET imaging in CEA transgenic immunocompetent mice bearing CEA positive colon or breast tumors. Combination with stereotactic radiation therapy (SRT) was performed with either ICK or IL-2-Fc. RESULTS: ICK and IL-2-Fc had comparable antitumor effects in both tumor models, although ICK had higher tumor uptake and slower blood clearance than an IL-2-Fc. Analysis of IFNγ+ /CD8+ and FoxP3+ /CD4+ T cells revealed higher levels of IFNγ-producing CD8+ T cells in ICK treated mice versus more efficient Treg elimination in IL-2-Fc treated mice. No significant or lasting toxicity was detected for either agent. Combination therapies with SRT revealed comparable efficacy and induction of immune memory for both ICK and IL-2-Fc when mice were rechallenged post-therapy. CONCLUSIONS: IL-2-Fc had comparable antitumor efficacy to CEA-targeted M5A-IL-2 ICK, while both fusion proteins induced immune memory when combined with SRT. Differences in the therapeutic mechanisms of both agents were observed.

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

Ex-vivo CS1-OKT3 dual specific bivalent antibody-armed effector T cells mediate cellular immunity against multiple myeloma.

Bispecific T cell engaging antibodies (bsAbs) have emerged as novel and powerful therapeutic agents for redirecting T cells towards antigen-specific tumor killing. The cell surface glycoprotein and SLAM family member, CS1, exhibits stable and high-level expression on malignant plasma cells including multiple myeloma, which is indicative of an ideal target for bsAb therapy. Here, we developed a CS1 bsAb (CS1-dbBiTE) using Click chemistry to conjugate intact anti-CS1 antibody (Elotuzumab) and anti-huOKT3 antibody at their respective hinge regions. Using a cellular therapy approach, human T cells were armed ex-vivo with CS1-dbBiTE prior to examining effector activity. Our data indicates that arming T cells with CS1-dbBiTE induced T cell activation and expansion and subsequent cytotoxic activity against CS1-bearing MM tumors, demonstrated by significant CD107a expression as well as inflammatory cytokine secretion. As expected, CS1-dbBiTE armed T cells showed significantly reduced effector activity in the absence of CS1 expression. Similarly, in MM mouse xenograft studies, armed T cells exhibited effective anti-tumor efficacy highlighted by reduced tumor burden in MM.1S tumor-bearing mice compared to controls. On the basis of these findings, the rationale for CS1 targeting by human T cells armed with CS1-dbBiTE presents a potentially effective therapeutic approach for targeting MM.

One-Step Automatic Radiosynthesis and Evaluation of [18F]TM-30089 as GPR44 Radiotracer.

Recently, a G-protein coupled receptor 44 (GPR44) was discovered to play a significant role in the process of inflammation-related diseases, including cancer and diabetes. However, the precise role of GPR44 has yet to be fully elucidated. Currently, there is a strong and urgent need for the development of GPR44 radiotracers as a non-invasive methodology to explore the exact mechanism of GPR44 on inflammation-related diseases and monitor the progress of therapy. TM-30089 is a potent GPR44 antagonist that exhibits a high specificity and selectivity for GPR44. Its structure contains a fluorine nuclide, which could potentially be replaced with 18F. In the present study, we successfully took a highly effective synthesis strategy that pretreated the unprotected carboxylic acid group of the precursor and developed a feasible one-step automatic radiosynthesis strategy for [18F]TM-30089 with a high radiochemical purity and a good radiochemical yield. We further evaluated this radiotracer using mice models implanted with 1.1 B4 cell lines (GPR44-enriched cell lines) and human islets (high GPR44 expression), respectively. The results revealed the persistent and specific uptake of [18F]TM-30089 in GPR44 region, indicating that [18F]TM-30089 is a promising candidate for targeting GPR44. Further evaluation is ongoing.

Indole-Based and Cyclopentenylindole-Based Analogues Containing Fluorine Group as Potential 18F-Labeled Positron Emission Tomography (PET) G-Protein Coupled Receptor 44 (GPR44) Tracers.

Recently, growing evidence of the relationship between G-protein coupled receptor 44 (GPR44) and the inflammation-cancer system has garnered tremendous interest, while the exact role of GPR44 has not been fully elucidated. Currently, there is a strong and urgent need for the development of non-invasive in vivo GPR44 positron emission tomography (PET) radiotracers that can be used to aid the exploration of the relationship between inflammation and tumor biologic behavior. Accordingly, the choosing and radiolabeling of existing GPR44 antagonists containing a fluorine group could serve as a viable method to accelerate PET tracers development for in vivo imaging to this purpose. The present study aims to evaluate published (2000-present) indole-based and cyclopentenyl-indole-based analogues of the GPR44 antagonist to guide the development of fluorine-18 labeled PET tracers that can accurately detect inflammatory processes. The selected analogues contained a crucial fluorine nuclide and were characterized for various properties including binding affinity, selectivity, and pharmacokinetic and metabolic profile. Overall, 26 compounds with favorable to strong binding properties were identified. This review highlights the potential of GPR44 analogues for the development of PET tracers to study inflammation and cancer development and ultimately guide the development of targeted clinical therapies.

PEGylated Fluorescent Anti-carcinoembryonic Antigen Antibody Labels Colorectal Cancer Tumors in Orthotopic Mouse Models.

INTRODUCTION: Colorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models. METHODS: The hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System. RESULTS: M5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75 µg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96 h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver. CONCLUSIONS: M5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors.

Generation of IL-2-Fc-antibody conjugates by click chemistry.

BACKGROUND: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine. METHODS: We now show that antibodies conjugated by click chemistry to interleukin-2 (IL-2)-Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK. RESULTS: An IL-2-Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL-2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148. The IL-2-Fc fusion protein with K35E and C125S mutations with 3 intact hinge cysteines, designated as IL-2-Fc Par, was selected based on its minimal tendency to aggregate. IL-2-Fc-antibody clicked conjugates retained high IL-2 activity and bound target antigens comparable to parent antibodies. An IL-2-Fc-anti-CEA click conjugate showed comparable anti-tumor activity to an anti-CEA-IL-2 ICK in immunocompetent CEA transgenic mice bearing CEA positive orthotopic breast tumors. Significant increases in IFNγ+ /CD8+ and decreases in FoxP3+ /CD4+ T-cells were found for the clicked conjugate and ICK therapies, suggesting a common mechanism of tumor reduction. CONCLUSION: The production of antibody targeted IL-2 therapy via a click chemistry approach is feasible with comparable activity to genetically produced ICKs with the added advantage of multiplexing with other monoclonal antibodies.

Multimodality PET and Near-Infrared Fluorescence Intraoperative Imaging of CEA-Positive Colorectal Cancer.

PURPOSE: Molecular imaging is a major diagnostic component for cancer management, enabling detection, staging of disease, targeting therapy, and monitoring the therapeutic response. The coordination of multimodality imaging techniques further enhances tumor localization. The development of a single agent for real-time non-invasive targeted positron emission tomography (PET) imaging and fluorescence guided surgery (FGS) will provide the next generation tool in the surgical management of cancer. PROCEDURES: The humanized anti-CEA M5A-IR800 "sidewinder" (M5A-IR800-SW) antibody-dye conjugate was designed with a NIR 800 nm dye incorporated into a PEGylated linker and conjugated with the metal chelate p-SCN-Bn-deferoxamine (DFO) for zirconium-89 PET imaging (89Zr, half-life 78.4 h). The dual-labeled 89Zr-DFO-M5A-SW-IR800 was evaluated for near infrared (NIR) fluorescence imaging, PET/MRI imaging, terminal tissue biodistribution, and blood clearance in a human colorectal cancer LS174T xenograft mouse model. RESULTS: The 89Zr-DFO-M5A-SW-IR800 NIR fluorescence imaging showed high tumor targeting with normal liver uptake. Serial PET/MRI imaging was performed at 24 h, 48 h, and 72 h and showed tumor localization visible at 24 h that persisted throughout the experiment. However, the PET scans showed higher activity for the liver than the tumor, compared to the NIR fluorescence imaging. This difference is an important finding as it quantifies the expected difference due to the sensitivity and depth of penetration between the 2 modalities. CONCLUSIONS: This study demonstrates the potential of a pegylated anti-CEA M5A-IR800-Sidewinder for NIR fluorescence/PET/MR multimodality imaging for intraoperative fluorescence guided surgery.

Sequential CAR T cell and targeted alpha immunotherapy in disseminated multiple myeloma.

Multiple myeloma (MM) is still an incurable disorder despite improved antibody and cellular therapies against different MM antigens. Single targeted antigens have so far been ineffective against MM with most patients relapsing after initial response. Hence, sequential immunotherapies directed at different targets are expected to perform better than monotherapy alone. Here, we optimized and established in preclinical studies the therapeutic rationale of using targeted alpha therapy (TAT) directed against CD38 antigen (225Ac-DOTA-daratumumab) with CAR T cell therapy directed at CS1 antigen in a systemic MM model. The sequential therapies compared CAR T therapy followed by TAT to TAT followed by CAR T therapy. CAR T cell monotherapy increased median survival from 49 days (d) in untreated controls to 71d with a modest improvement to 89d for 3.7 kBq of TAT given 14d later. When CAR T was followed by 7.4 kBq of TAT 29d later, sequential therapy increased median survival from 47d in untreated controls to 106d, compared to 68d for CAR T monotherapy. When CAR T therapy was followed by untargeted alpha immunotherapy using 7.4 kBq of 225Ac-DOTA-trastuzumab (anti-HER2) antibody 29d later, there was only a slight improvement in response over CAR T monotherapy demonstrating the role of tumor targeting. TAT (7.4 kBq) followed by CAR T therapy was also effective when CAR T therapy was delayed for 21d vs 14d or 28d post TAT, highlighting the importance of timing sequential therapies. Sequential targeted therapies using CS1 CAR T or 225Ac-DOTA-CD38 TAT in either order shows promise over monotherapies alone.

First-In-Human Pilot PET Immunoimaging Study of 64Cu-Anti-Carcinoembryonic Antigen Monoclonal Antibody (hT84.66-M5A) in Patients with Carcinoembryonic Antigen-Producing Cancers.

Background: PET imaging using radiolabeled immunoconstructs shows promise in cancer detection and in assessing tumor response to therapies. The authors report the first-in-human pilot study evaluating M5A, a humanized anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb), radiolabeled with 64Cu in patients with CEA-expressing malignancies. The purpose of this pilot study was to identify the preferred patient population for further evaluation of this agent in an expanded trial. Methods: Patients with CEA-expressing primary or metastatic cancer received 64Cu-DOTA-hT84.66-M5A with imaging performed at 1 and 2 days postinfusion. 64Cu-DOTA-hT84.66-M5A PET scan findings were correlated with CT, MRI, and/or FDG PET scans and with histopathologic findings from planned surgery or biopsy performed postscan. Results: Twenty patients received 64Cu-DOTA-hT84.66-M5A. Twelve patients demonstrated positive images, which were confirmed in 10 patients as tumor by standard-of-care (SOC) imaging, biopsy, or surgical findings. Four of the 8 patients with negative imaging were confirmed as true negative, with the remaining 4 patients having disease demonstrated by SOC imaging or surgery. All 5 patients with locally advanced rectal cancer underwent planned biopsy or surgery after 64Cu-DOTA-hT84.66-M5A imaging (4 patients imaged 6-8 weeks after completing neoadjuvant chemotherapy and radiation therapy) and demonstrated a high concordance between biopsy findings and 64Cu-DOTA-hT84.66-M5A PET scan results. Three patients demonstrated positive uptake at the primary site later confirmed by biopsy and at surgery as residual disease. Two patients with negative scans each demonstrated complete pathologic response. In 5 patients with medullary thyroid cancer, 64Cu-DOTA-hT84.66-M5A identified disease not seen on initial CT scans in 3 patients, later confirmed to be disease by subsequent surgery or MRI. Conclusions: 64Cu-DOTA-hT84.66-M5A demonstrates promise in tumor detection, particularly in patients with locally advanced rectal cancer and medullary thyroid cancer. A successor trial in locally advanced rectal cancer has been initiated to further evaluate this agent's ability to define tumor extent before and assess disease response after neoadjuvant chemotherapy and radiotherapy. clinical trial.gov (NCT02293954).

Heart Uptake of [18F]Fluoro-4-Thia-Oleate in a Non-Alcoholic Fatty Liver Disease Mouse Model.

The world-wide high incidence of non-alcoholic fatty liver disease (NAFLD) is of concern for its progression to insulin resistance, steatohepatitis and cardiovascular disease (CVD). The increased uptake of fatty acids in critical organs plays a major role in NAFLD progression. Male Ceacam1−/− mice that develop NAFLD, insulin resistance and CVD on normal chow are a potential model for studying the dysregulation of fatty acid uptake. [18F]fluoro-4-thia-oleate ([18F]FTO) was chosen as a fatty acid reporter because of its higher uptake and retention in the heart in an animal model of CVD. Male wild-type (WT) or Ceacam1−/− mice fasted 4−6 h were administered [18F]FTO i.v., and dynamic PET scans were conducted in an MR/PET small animal imaging system along with terminal tissue biodistributions. Quantitative heart image analysis revealed significantly higher uptake at 35 min in Ceacam1−/− (6.0 ± 1.0% ID/cc) vs. WT (3.9 ± 0.6% ID/cc) mice (p = 0.006). Ex vivo heart uptake/retention (% ID/organ) was 2.82 ± 0.45 for Ceacam1−/− mice vs. 1.66 ± 0.45 for WT mice (p < 0.01). Higher kidney and pancreas uptake/retention in Ceacam1−/− was also evident, and the excretion of [18F]FTO into the duodenum was observed for both WT and Ceacam1−/− mice starting at 10 min. This study suggests that the administration of [18F]FTO as a marker of fatty acid uptake and retention may be an important tool in analyzing the effect of NAFLD on lipid dysregulation in the heart.

Reversal of obesity development in Ceacam1-/- male mice by bone marrow transplantation or introduction of the human CEACAM1 gene.

OBJECTIVE: Although Ceacam1-/- male mice become obese on normal chow, the effect of bone marrow transplantation or introduction of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) gene has not been studied, to the knowledge of the authors. METHODS: This study analyzed Ceacam1-/- mice on normal diet or high-fat diet (HFD), including effects of bone marrow transplantation or introduction of the CEACAM1 gene. RESULTS: Male Ceacam1-/- mice on normal diet versus HFD for 24 weeks gained significantly more weight than controls, and Ceacam1-/- mice aged up to 2 years had a high frequency of liver cancer. Transplantation of wild-type bone marrow into Ceacam1-/- mice or introduction of the human CEACAM1 gene fully or partially reversed the obesity phenotype. Liver lipidomics on Ceacam1-/- versus wild-type controls on an HFD revealed a significant increase in diacyl glycerides. An increase in fatty acid transporter CD36 levels further suggests that loss of Ceacam1 leads to a major dysregulation of free fatty acid uptake. CONCLUSIONS: CEACAM1 expression in both the liver and immune cells regulates obesity and lipid storage pathways in the liver. Bone marrow reconstitution of the immune system or introduction of the human CEACAM1 gene can fully or partially reverse the phenotype.

Isolation and expansion of murine γδ T cells from mouse splenocytes.

Elucidation of the function of gamma delta T cells (γδ T cells) requires robust models that show how γδ T cells are commonly involved in inflammation, since very little is known about the factors that promote and control their development and function. There are few studies of murine γδ T cells primarily because these cells have proven difficult to isolate, expand and characterize. Here, we describe a simple method that utilizes key expansion elements to isolate and expand murine CD4-CD8-CD3+ γδ T cells typically found in secondary lymphoid tissues. Expansion of γδ T cells reached 150-fold by day 8 of culture, depended on exogenous IL-2, αCD3, and αCD28, and supported efficient and reproducible in vitro differentiation. These studies showed high production of cytokines IFNγ and Granzyme B, with the novel finding of IL-24 upregulation as well. Expression analysis of expanded γδ T cells, after treatment with IL-2, revealed high levels of Granzyme B, Granzyme D, and IFNγ. Lactate dehydrogenase (LDH) cytotoxicity assays showed that expanded γδ T cells were effective at inducing >90% cytolysis of murine MC38 colon cancer, E0771 breast cancer, and B16 melanoma cells at 10:1 effector to target ratios. These findings indicated that murine γδ T cells can be successfully isolated, expanded, and used to perform preclinical therapy studies.

Improved Tumor Responses with Sequential Targeted α-Particles Followed by Interleukin 2 Immunocytokine Therapies in Treatment of CEA-Positive Breast and Colon Tumors in CEA Transgenic Mice.

Targeted α-therapy (TAT) delivers high-linear-transfer-energy α-particles to tumors with the potential to generate tumor immune responses that may be augmented by antigen-targeted immunotherapy. Methods: This concept was evaluated in immunocompetent carcinoembryonic antigen (CEA) transgenic mice bearing CEA-positive mammary or colon tumors. Tumors were targeted with humanized anti-CEA antibody M5A labeled with 225Ac for its 10-d half-life and emission of 4 α-particles, as well as being targeted with the immunocytokine M5A-interleukin 2. Results: A dose response (3.7, 7.4, and 11.1 kBq) to TAT only, for orthotopic CEA-positive mammary tumors, was observed, with a tumor growth delay of 30 d and an increase in median survival from 20 to 36 d at the highest dose. Immunocytokine (4 times daily) monotherapy gave a tumor growth delay of 20 d that was not improved by addition of 7.4 kBq of TAT 5 d after the start of immunocytokine. However, TAT (7.4 kBq) followed by immunocytokine 10 d later led to a tumor growth delay of 38 d, with an increase in median survival to 45 d. Similar results were seen for TAT followed by immunocytokine at 5 versus 10 d. When a similar study was performed with subcutaneously implanted CEA-positive MC38 colon tumors, TAT (7.4 kBq) monotherapy gave an increase in median survival from 29 to 42 d. The addition of immunocytokine 10 d after 7.4 kBq of TAT increased median survival to 57 d. Immunophenotyping showed increased tumor-infiltrating interferon-γ-positive, CD8-positive T cells and an increased ratio of these cells to Foxp3-positive, CD4-positive regulatory T cells with sequential therapy. Immunohistochemistry confirmed there was an increase in tumor-infiltrating CD8-positive T cells in the sequential therapy group, strongly suggesting that immunocytokine augmented TAT can lead to an immune response that improves tumor therapy. Conclusion: Low-dose (7.4 kBq) TAT followed by a 4-dose immunocytokine regimen 5 or 10 d later gave superior tumor reductions and survival curves compared with either monotherapy in breast and colon cancer tumor models. Reversing the order of therapy to immunocytokine followed by TAT 5 d later was equivalent to either monotherapy in the breast cancer model.

Biodistribution of Intra-Arterial and Intravenous Delivery of Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles in a Rat Model to Guide Delivery Strategies for Diabetes Therapies.

Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UC-MSC-EVs) have become an emerging strategy for treating various autoimmune and metabolic disorders, particularly diabetes. Delivery of UC-MSC-EVs is essential to ensure optimal efficacy of UC-MSC-EVs. To develop safe and superior EVs-based delivery strategies, we explored nuclear techniques including positron emission tomography (PET) to evaluate the delivery of UC-MSC-EVs in vivo. In this study, human UC-MSC-EVs were first successfully tagged with I-124 to permit PET determination. Intravenous (I.V.) and intra-arterial (I.A.) administration routes of [124I]I-UC-MSC-EVs were compared and evaluated by in vivo PET-CT imaging and ex vivo biodistribution in a non-diabetic Lewis (LEW) rat model. For I.A. administration, [124I]I-UC-MSC-EVs were directly infused into the pancreatic parenchyma via the celiac artery. PET imaging revealed that the predominant uptake occurred in the liver for both injection routes, and further imaging characterized clearance patterns of [124I]I-UC-MSC-EVs. For biodistribution, the uptake (%ID/gram) in the spleen was significantly higher for I.V. administration compared to I.A. administration (1.95 ± 0.03 and 0.43 ± 0.07, respectively). Importantly, the pancreas displayed similar uptake levels between the two modalities (0.20 ± 0.06 for I.V. and 0.24 ± 0.03 for I.A.). Therefore, our initial data revealed that both routes had similar delivery efficiency for [124I]I-UC-MSC-EVs except in the spleen and liver, considering that higher spleen uptake could enhance immunomodulatory application of UC-MSC-EVs. These findings could guide the development of safe and efficacious delivery strategies for UC-MSC-EVs in diabetes therapies, in which a minimally invasive I.V. approach would serve as a better delivery strategy. Further confirmation studies are ongoing.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

BACKGROUND: Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. METHODS: Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. RESULTS: We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. CONCLUSION: The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

Role of lncRNA LIPE-AS1 in adipogenesis.

Recent studies have identified long non-coding RNAs (lncRNAs) as potential regulators of adipogenesis. In this study, we have characterized a lncRNA, LIPE-AS1, that spans genes CEACAM1 to LIPE in man with conservation of genomic organization and tissue expression between mouse and man. Tissue-specific expression of isoforms of the murine lncRNA were found in liver and adipose tissue, one of which, designated mLas-V3, overlapped the Lipe gene encoding hormone-sensitive lipase in both mouse and man suggesting that it may have a functional role in adipose tissue. Knock down of expression of mLas-V3 using anti-sense oligos (ASOs) led to a significant decrease in the differentiation of the OP9 pre-adipocyte cell line through the down regulation of the major adipogenic transcription factors Pparg and Cebpa. Knock down of mLas-V3 induced apoptosis during the differentiation of OP9 cells as shown by expression of active caspase-3, a change in the localization of LIP/LAP isoforms of C/EBPß, and expression of the cellular stress induced factors CHOP, p53, PUMA, and NOXA. We conclude that mLas-V3 may play a role in protecting against stress associated with adipogenesis, and its absence leads to apoptosis.

Use of 64Cu-DOTA-Trastuzumab PET to Predict Response and Outcome of Patients Receiving Trastuzumab Emtansine for Metastatic Breast Cancer: A Pilot Study.

We hypothesized that functional imaging with 64Cu-DOTA-trastuzumab PET/CT would predict the response to the antibody-drug conjugate trastuzumab-emtansine (T-DM1). Methods: Ten women with metastatic human epidermal growth factor receptor 2-positive breast cancer underwent 18F-FDG PET/CT and 64Cu-DOTA-trastuzumab PET/CT on days 1 and 2 before treatment with T-DM1. Results: T-DM1-responsive patients had higher uptake than nonresponsive patients. Day 1 minimum SUVmax (5.6 vs. 2.8, P < 0.02), day 2 minimum SUVmax (8.1 vs. 3.2, P < 0.01), and day 2 average SUVmax (8.5 vs. 5.4, P < 0.05) for 64Cu-DOTA-trastuzumab all favored responding patients. Tumor-level response suggested threshold dependence on SUVmax Patients with a day 2 minimum SUVmax above versus below the threshold had a median time to treatment failure of 28 mo versus 2 mo (P < 0.02). Conclusion: Measurement of trastuzumab uptake in tumors via PET/CT is promising for identifying patients with metastatic breast cancer who will benefit from T-DM1.