Accurate Co-Localization of Luciferase Expression and Fluorescent Anti-CEA Antibody Targeting of Liver Metastases in an Orthotopic Mouse Model of Colon Cancer.

BACKGROUND: The present study aimed to validate the accuracy of a tumor-specific antibody to target liver metastases of colorectal cancer. METHODS: A humanized anti-CEA antibody conjugated to a fluorescent dye (M5A-IR800) was tested for targeting human colorectal cancer liver metastases (CRLMs) expressing luciferase in an orthotopic mouse model. Orthotopic mouse models of CRLMs were established by implanting fragments of a luciferase-expressing human colorectal cancer cell line, LS174T, in the liver of nude mice. Mice received 50 µg M5A-IR800 72 h prior to imaging. To test co-localization, bioluminescence imaging was performed using D-luciferin, which was given via intraperitoneal injection just prior to imaging. RESULTS: Tumors were able to be visualized non-invasively through the skin with the luciferase-luciferin signal. Intra-abdominal imaging showed accurate labeling of CRLMs with M5A-IR800, which co-localized with the luciferase-luciferin signal. CONCLUSIONS: The present results validate the accuracy of a tumor-specific anti-CEA antibody in targeting liver metastases of colorectal cancer.

Yttrium-90 anti-CD25 BEAM conditioning for autologous hematopoietic cell transplantation in Peripheral T-cell lymphoma.

ABSTRACT: Peripheral T-cell lymphomas (PTCLs) have a poor prognosis with current treatments. High-dose chemotherapy followed by autologous hematopoietic cell transplant (AHCT) is used as a consolidation strategy after achieving clinical remission with first-line therapy, as well as in chemotherapy-sensitive relapse if allogeneic transplant is not an option. CD25 is a targetable protein often highly expressed in PTCLs. In this phase 1 clinical trial, we tested the addition of ß-emitting 90yttrium (90Y)-labeled chimeric anti-CD25 basiliximab (aTac) to BEAM (carmustine, etoposide, cytarabine, and melphalan) as conditioning for AHCT for patients with PTCL. Twenty-three AHCT-eligible patients were enrolled, and 20 received therapeutic 90Y-aTac-BEAM AHCT. Radiation doses of 0.4, 0.5, and 0.6 mCi/kg were tested. With no observed dose-limiting toxicities, 0.6 mCi/kg was deemed the recommended phase 2 dose. The most prevalent adverse effect, grade 2 mucositis, was experienced by 80% of patients. As of this report, 6 (30%) of the treated patients had died, 5 due to progressive disease and 1 due to multiple organ failure (median time of death, 17 months [range, 9-21]) after AHCT. Median follow-up was 24 months (range, 9-26) overall and 24 months (range, 13-26) for surviving patients. For patients who received therapeutic 90Y-aTac-BEAM AHCT, the 2-year progression-free and overall survival were 59% (95% confidence interval [CI], 34-77) and 68% (95% CI, 42-84), respectively. 90Y-aTac-BEAM appears to be safe as an AHCT conditioning regimen for PTCL, with no increased toxicity over the toxicities historically seen with BEAM alone in this patient population. This trial was registered at www.ClinicalTrials.gov as #NCT02342782.

Targeting Patient-Derived Orthotopic Gastric Cancers with a Fluorescent Humanized Anti-CEA Antibody.

BACKGROUND: Gastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival. METHODS: Two patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue. RESULTS: M5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800. CONCLUSIONS: Humanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

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.

Humanized Anti-Carcinoembryonic Antigen Antibodies Brightly Target and Label Gastric Cancer in Orthotopic Mouse Models.

INTRODUCTION: Gastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models. METHODS: MKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 µg) response curves were performed in subcutaneous models. Orthotopic models received 50 µg of M5A-IR800 or 50 µg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System. RESULTS: In subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors. CONCLUSIONS: Humanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection.

Pilot study of HER2 targeted 64 Cu-DOTA-tagged PET imaging in gastric cancer patients.

OBJECTIVE: Human epidermal growth factor receptor 2 (HER2) is an important biomarker for targeted gastric cancer (GC) immunotherapy. However, heterogeneous HER2 overexpression in GC, loss of HER2 expression during therapy, and inability to non-invasively identify HER2 overexpressing tumors impede effective targeting therapies. Improved HER2-specific functional imaging can address these challenges. Trastuzumab is a HER2-directed mAb to treat HER2 overexpressing cancers. The 64 Cu-DOTA-trastuzumab radiotracer is used to detect HER2+ metastatic breast cancer. We aimed to develop 64 Cu-DOTA-trastuzumab PET-CT to detect and characterize tumor uptake in HER2+ or - GC patients. METHODS: We conducted a single-arm phase II pilot study exploring the feasibility of 64 Cu-DOTA-trastuzumab for PET imaging of HER2 overexpressing GC compared to HER2 non-expressing tumors. Eight patients with biopsy-confirmed gastric adenocarcinoma were included. Immunohistochemistry was used to evaluate primary tumor biopsies for HER2 overexpression. Patients were injected with 45 mg of cold trastuzumab followed by 5 mg of 64 Cu-DOTA-trastuzumab. PET-CT scans were performed 24-48 h post radiotracer injection and compared to standard staging CT scans. RESULTS: We observed limited toxicity following 64 Cu-DOTA-trastuzumab injections. While there was uptake of the radiotracer in portions of HER2+ lesions, there was no statistically significant distinction between tumor and background by standardized uptake value analysis. CONCLUSION: Despite the potential of 64 Cu-DOTA-trastuzumab PET imaging of HER2+ metastatic breast cancer, a 5 mg dose of this radiotracer injected 24-48 h before imaging was insufficient to identify HER2+ GC. These results inform future GC imaging studies to optimize biomarker-targeted therapies based on dosage and timing for more clinically relevant imaging.

Enhanced Therapeutic Effects of 177Lu-DOTA-M5A in Combination with Heat Shock Protein 90 Inhibitor Onalespib in Colorectal Cancer Xenografts.

Carcinoembryonic antigen (CEA) has emerged as an attractive target for theranostic applications in colorectal cancers (CRCs). In the present study, the humanized anti-CEA antibody hT84.66-M5A (M5A) was labeled with 177Lu for potential CRC therapy. Moreover, the novel combination of 177Lu-DOTA-M5A with the heat shock protein 90 inhibitor onalespib, suggested to mediate radiosensitizing properties, was assessed in vivo for the first time. M5A antibody uptake and therapeutic effects, alone or in combination with onalespib, were assessed in human CRC xenografts and visualized using SPECT/CT imaging. Although both 177Lu-DOTA-M5A and onalespib monotherapies effectively reduced tumor growth rates, the combination therapy demonstrated the most substantial impact, achieving a fourfold reduction in tumor growth compared to the control group. Median survival increased by 33% compared to 177Lu-DOTA-M5A alone, and tripled compared to control and onalespib groups. Importantly, combination therapy yielded comparable or superior effects to the double dose of 177Lu-DOTA-M5A monotherapy. 177Lu-DOTA-M5A increased apoptotic cell levels, indicating its potential to induce tumor cell death. These findings show promise for 177Lu-DOTA-M5A as a CRC therapeutic agent, and its combination with onalespib could significantly enhance treatment efficacy. Further in vivo studies are warranted to validate these findings fully and explore the treatment's potential for clinical use.

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.

Pretargeting with Cucurbituril-Adamantane Host-Guest Pair in Xenograft Models.

The goal of reducing the total-body radiation dose of macromolecule-based nuclear medicine with a 2-step pretargeting strategy has been achieved with several pretargeting methodologies in preclinical and clinical settings. However, the lack of modularity, biocompatibility, and in vivo stability in existing pretargeting agents obstructs their respective platforms' wide clinical use. We hypothesized that host-guest chemistry would provide an optimal pretargeting methodology. A cucurbit[7]uril host and an adamantane guest molecule form a high-affinity host-guest complex (association constant, ∼1014 M-1), and in this work, we explored the use of this noncovalent interaction as the basis for antibody-based pretargeted PET. Along with the straightforward modularity of these agents, cucurbit[7]uril and adamantane are recognized to have high in vivo stability and suitability for human use, which is why we proposed this methodology as the ideal approach for pretargeted nuclear medicine. Methods: Three 64Cu-labeled adamantane guest radioligands were developed, and their in vitro stability, lipophilicity, and in vivo blood half-lives were compared. The adamantane radioligands were analyzed for pretargeting using a cucurbit[7]uril-modified carcinoembryonic antigen-targeting full-length antibody, hT84.66-M5A, as the macromolecule pretargeting agent with 2 different dosing schedules. These molecules were evaluated for pretargeting in human pancreatic cancer BxPC3 and MIAPaCa-2 mouse xenografts using PET and in vivo biodistribution studies. The dosimetry of the cucurbit[7]uril-adamantane (CB7-Adma) pretargeting approach in men was calculated and compared with that of the directly 89Zr-labeled hT84.66-M5A. Results: The adamantane radioligands possessed high in vitro stability up to 24 h (>90%). Pretargeted PET with CB7-Adma methodology resulted in specific tumor uptake (P < 0.05) with low background signal. The in vivo formed CB7-Adma complex was demonstrated to be stable, with high tumor uptake up to 24 h after radioligand injection (12.0 ± 0.9 percentage injected dose/g). The total-body radiation dose of the pretargeting strategy was only 3.3% that of the directly 89Zr-labeled hT84.66-M5A. Conclusion: The CB7-Adma strategy is highly suitable for pretargeted PET. The exceptional stability of the pretargeting agents and the specific and high tumor uptake of the pretargeted adamantane radioligands provide great potential for the platform.

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).

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.

Investigation of Copper-64-Based Host-Guest Chemistry Pretargeted Positron Emission Tomography.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled in vivo at the target site using a radiolabeled small molecule (radioligand) that interacts with the macromolecule with high specificity. We report an investigation of host-guest chemistry-driven pretargeting using copper-64 radiolabeled ferrocene (Fc; guest) compounds and a cucurbit[7]uril (CB7; host) molecule functionalized carcinoembryonic antigen targeting hT84.66-M5A monoclonal antibody (CB7-M5A). Two novel ferrocene-based radioligands ([64Cu]Cu-NOTA-PEG3-Fc and [64Cu]Cu-NOTA-PEG7-Fc) were prepared, and their in vitro stability, pharmacokinetic in vivo profile in healthy mice, and pretargeting performance in a subcutaneous BxPC3 human pancreatic cancer cell xenograft mouse model were compared. The antibody dosing was optimized using a zirconium-89 radiolabeled M5A antibody ([89Zr]Zr-DFO-M5A) in a BxPC3 xenograft model, and the dosimetry of [89Zr]Zr-DFO-M5A and the pretargeting approach were compared. Finally, the effects of varying lag times up to 9 days between CB7-M5A and radioligand injection were investigated. In vivo pretargeting studies with both ferrocene radioligands resulted in specific tumor uptake (p = 0.0006 and p = 0.003) and also showed that the host-guest-based pretargeting approach excels with extended lag times up to 9 days with good tumor localization, suggesting that host-guest pretargeting may be suitable for use without clearing agents which have complicated clinical application of this technique. To our knowledge, the reported lag time of 9 days is the longest investigated lag time in any reported pretargeting studies.

Pre-clinical data supporting immunotherapy for HIV using CMV-HIV-specific CAR T cells with CMV vaccine.

T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120. In this study, we use a GMP-compliant platform to engineer CMV-specific T cells to express a second-generation CAR derived from the N6 broadly neutralizing antibody, one of the broadest anti-gp120 neutralizing antibodies. These CMV-HIV CAR T cells exhibit dual effector functions upon in vitro stimulation through their endogenous CMV-specific T cell receptors or the introduced CARs. Using a humanized HIV mouse model, we show that CMV vaccination during ART accelerates CMV-HIV CAR T cell expansion in the peripheral blood and that higher numbers of CMV-HIV CAR T cells were associated with a better control of HIV viral load and fewer HIV antigen p24+ cells in the bone marrow upon ART interruption. Collectively, these data support the clinical development of CMV-HIV CAR T cells in combination with a CMV vaccine in HIV-infected individuals.

In Vitro Characterization of 177Lu-DOTA-M5A Anti-Carcinoembryonic Antigen Humanized Antibody and HSP90 Inhibition for Potentiated Radioimmunotherapy of Colorectal Cancer.

Carcinoembryonic antigen (CEA) is an antigen that is highly expressed in colorectal cancers and widely used as a tumor marker. 131I and 90Y-radiolabeled anti-CEA monoclonal antibodies (mAbs) have previously been assessed for radioimmunotherapy in early clinical trials with promising results. Moreover, the heat shock protein 90 inhibitor onalespib has previously demonstrated radiotherapy potentiation effects in vivo. In the present study, a 177Lu-radiolabeled anti-CEA hT84.66-M5A mAb (M5A) conjugate was developed and the potential therapeutic effects of 177Lu-DOTA-M5A and/or onalespib were investigated. The 177Lu radiolabeling of M5A was first optimized and characterized. Binding specificity and affinity of the conjugate were then evaluated in a panel of gastrointestinal cancer cell lines. The effects on spheroid growth and cell viability, as well as molecular effects from treatments, were then assessed in several three-dimensional (3D) multicellular colorectal cancer spheroid models. Stable and reproducible radiolabeling was obtained, with labeling yields above 92%, and stability was retained at least 48 h post-radiolabeling. Antigen-specific binding of the radiolabeled conjugate was demonstrated on all CEA-positive cell lines. Dose-dependent therapeutic effects of both 177Lu-DOTA-M5A and onalespib were demonstrated in the spheroid models. Moreover, effects were potentiated in several dose combinations, where spheroid sizes and viabilities were significantly decreased compared to the corresponding monotherapies. For example, the combination treatment with 350 nM onalespib and 20 kBq 177Lu-DOTA-M5A resulted in 2.5 and 2.3 times smaller spheroids at the experimental endpoint than the corresponding monotreatments in the SNU1544 spheroid model. Synergistic effects were demonstrated in several of the more effective combinations. Molecular assessments validated the therapy results and displayed increased apoptosis in several combination treatments. In conclusion, the combination therapy of anti-CEA 177Lu-DOTA-M5A and onalespib showed enhanced therapeutic effects over the individual monotherapies for the potential treatment of colorectal cancer. Further in vitro and in vivo studies are warranted to confirm the current study findings.

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.

The Use of Fluorescent Anti-CEA Antibodies to Label, Resect and Treat Cancers: A Review.

A major barrier to the diagnosis and effective treatment of solid-tumor cancers is the difficulty in detection and visualization of tumor margins in primary and metastatic disease. The use of fluorescence can augment the surgeon's ability to detect cancer and aid in its resection. Several cancer types express carcinoembryonic antigen (CEA) including colorectal, pancreatic and gastric cancer. Antibodies to CEA have been developed and tagged with near-infrared fluorescent dyes. This review article surveyed the use of CEA antibodies conjugated to fluorescent probes for in vivo studies since 1990. PubMed and Google Scholar databases were queried, and 900 titles and abstracts were screened. Fifty-nine entries were identified as possibly meeting inclusion/exclusion criteria and were reviewed in full. Forty articles were included in the review and their citations were screened for additional entries. A total of 44 articles were included in the final review. The use of fluorescent anti-CEA antibodies has been shown to improve detection and resection of tumors in both murine models and clinically. The cumulative results indicate that fluorescent-conjugated anti-CEA antibodies have important potential to improve cancer diagnosis and surgery. In an emerging technology, anti-CEA fluorescent antibodies have also been successfully used for photoimmunotherapy treatment for cancer.

Anti-CD25 radioimmunotherapy with BEAM autologous hematopoietic cell transplantation conditioning in Hodgkin lymphoma.

High-risk relapsed or refractory (R/R) classical Hodgkin lymphoma (HL) is associated with poor outcomes after conventional salvage therapy and autologous hematopoietic cell transplantation (AHCT). Post-AHCT consolidation with brentuximab vedotin (BV) improves progression-free survival (PFS), but with increasing use of BV early in the treatment course, the utility of consolidation is unclear. CD25 is often expressed on Reed-Sternberg cells and in the tumor microenvironment in HL, and we hypothesized that the addition of 90Y-antiCD25 (aTac) to carmustine, etoposide, cytarabine, melphalan (BEAM) AHCT would be safe and result in a transplantation platform that is agnostic to prior HL-directed therapy. Twenty-five patients with high-risk R/R HL were enrolled in this phase 1 dose-escalation trial of aTac-BEAM. Following an imaging dose of 111In-antiCD25, 2 patients had altered biodistribution, and a third developed an unrelated catheter-associated bacteremia; therefore, 22 patients ultimately received therapeutic 90Y-aTac-BEAM AHCT. No dose-limiting toxicities were observed, and 0.6 mCi/kg was deemed the recommended phase 2 dose, the dose at which the heart wall would not receive >2500 cGy. Toxicities and time to engraftment were similar to those observed with standard AHCT, though 95% of patients developed stomatitis (all grade 1-2 per Bearman toxicity scale). Seven relapses (32%) were observed, most commonly in patients with ≥3 risk factors. The estimated 5-year PFS and overall survival probabilities among 22 evaluable patients were 68% and 95%, respectively, and non-relapse mortality was 0%. aTac-BEAM AHCT was tolerable in patients with high-risk R/R HL, and we are further evaluating the efficacy of this approach in a phase 2 trial. This trial was registered at www.clinicaltrials.gov as #NCT01476839.