Radioimmunotherapeutic Agents Research Articles

Progress on immuno-microenvironment and immune-related therapies in patients with pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is an indolent malignant syndrome. The standard treatment for PMP is cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (CRS + HIPEC). However, the high recurrence rate and latent clinical symptoms and signs are major obstacles to further improving clinical outcomes. Moreover, patients in advanced stages receive little benefit from CRS + HIPEC due to widespread intraperitoneal metastases. Another challenge in PMP treatment involves the progressive sclerosis of PMP cell-secreted mucus, which is often increased due to activating mutations in the gene coding for guanine nucleotide-binding protein alpha subunit (GNAS). Consequently, the development of other PMP therapies is urgently needed. Several immune-related therapies have shown promise, including the use of bacterium-derived non-specific immunogenic agents, radio-immunotherapeutic agents, and tumor cell-derived neoantigens, but a well-recognized immunotherapy has not been established. In this review the roles of GNAS mutations in the promotion of mucin secretion and disease development are discussed. In addition, the immunologic features of the PMP microenvironment and immune-associated treatments are discussed to summarize the current understanding of key features of the disease and to facilitate the development of immunotherapies.

Relative Biological Effectiveness (RBE) of [64Cu]Cu and [177Lu]Lu-NOTA-panitumumab F (ab')2 radioimmunotherapeutic agents vs. γ-radiation for decreasing the clonogenic survival in vitro of human pancreatic ductal adenocarcinoma (PDAC) cells

IntroductionOur objective was to compare [64Cu]Cu-NOTA-panitumumab F(ab')2 and [177Lu]Lu-NOTA-panitumumab F(ab')2 radioimmunotherapy (RIT) agents for decreasing the clonogenic survival fraction (SF) in vitro of EGFR-positive human pancreatic ductal adenocarcinoma (PDAC) cell lines and estimate the relative biological effectiveness (RBE) vs. γ-radiation (XRT). MethodsEGFR-positive PDAC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1) and EGFR-knockout PANC-1 EGFR KO cells were treated in vitro for 18 h with (0–19.65 MBq; 72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 or XRT (0–8 Gy) followed by clonogenic assay. The SF was determined after culturing single treated cells for 14 d. Cell fractionation studies were performed for cells incubated with 1 MBq (72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 for 1, 4, or 24 h to estimate the time-integrated activity (Ã) on the cell surface, cytoplasm, nucleus and medium. Radiation absorbed doses in the nucleus were calculated by multiplying à by S-factors calculated by Monte Carlo N Particle (MCNP) modeling using monolayer cell culture geometry. The SF of PDAC cells was plotted vs. dose and fitted to a linear quadratic model to estimate the dose required to decrease the SF to 0.1 (D10). The D10 for RIT agents were compared to XRT to estimate the RBE. DNA double-strand breaks (DSBs) caused by [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 continuous exposure for 5 h or 20 h were probed by immunofluorescence for γ-H2AX. Relative EGFR expression of PDAC cells was assessed by flow cytometry (scored + to +++) and cell doubling times for untreated cells were determined. ResultsThe D10 for [64Cu]Cu-NOTA-panitumumab F(ab′)2 ranged from 9.1 Gy (PANC-1) to 39.9 Gy (Capan-1). The D10 for [177Lu]Lu-NOTA-panitumumab F(ab′)2 ranged from 11.7 Gy (AsPC-1) to 170.8 Gy (Capan-1). The D10 for XRT ranged from 2.5 Gy (Capan-1) to 6.7 Gy (PANC-1 EGFR KO). D10 values were not correlated with EGFR expression over a relatively narrow range (++ to +++) or with cell doubling times. Based on D10 values, PANC-1 EGFR KO cells were 1.6-fold less sensitive than PANC-1 cells to [64Cu]Cu-NOTA-panitumumab F(ab′)2 and 1.9-fold less sensitive to [177Lu]Lu-NOTA-panitumumab F(ab′)2. The RBE for [64Cu]Cu-NOTA-panitumumab F(ab′)2 ranged from 0.06 for Capan-1 cells to 0.45 for PANC-1 cells. The RBE for [177Lu]Lu-NOTA-panitumumab F(ab′)2 ranged from 0.015 for Capan-1 cells to 0.28 for AsPC-1 cells. DNA DSBs were detected in PDAC cells exposed to [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 but were not correlated with the SF of the cells. ConclusionsWe conclude that at the same dose delivered to the cell nucleus [64Cu]Cu-NOTA-panitumumab F(ab′)2 and [177Lu]Lu-NOTA-panitumumab F(ab′)2 were less radiobiologically effective than XRT for decreasing the SF of human PDAC cells, but [64Cu]Cu-NOTA-panitumumab F(ab′)2 was more cytotoxic than [177Lu]Lu-NOTA-panitumumab F(ab′)2 except for AsPC-1 cells which were more sensitive to [177Lu]Lu-NOTA-panitumumab F(ab′)2. Advances in knowledge and implications for patient careThis study demonstrates that higher radiation doses may be required for RIT than XRT to achieve radiobiologically equivalent effects when used to treat PDAC.

Radioimmunotherapy in Non-Hodgkin's Lymphoma: Retrospective Adverse Event Profiling of Zevalin and Bexxar.

The development of monoclonal antibodies has dramatically changed the outcome of patients with non-Hodgkin’s lymphoma (NHL), the most common hematological malignancy. However, despite the satisfying results of monoclonal antibody treatment, only few NHL patients are permanently cured with single-agent therapies. In this context, radioimmunotherapy, the administration of radionuclides conjugated to monoclonal antibodies, is aimed to augment the single-agent efficacy of immunotherapy in order to deliver targeted radiation to tumors, particularly CD20+ B-cell lymphomas. Based on evidence from several trials in NHL, the radiolabeled antibodies 90Y-ibritumomab tiuxetan (Zevalin, Spectrum Pharmaceuticals) and 131I-tositumomab (Bexxar, GlaxoSmithKline) received FDA approval in 2002 and 2003, respectively. However, none of the two radioimmunotherapeutic agents has been broadly applied in clinical practice. The main reason for the under-utilization of radioimmunotherapy includes economic and logistic considerations. However, concerns about potential side effects have also been raised. Driven by these developments, we performed retrospective analysis of adverse events reporting Zevalin or Bexxar, extracted from the FDA’s Adverse Event Reporting System (FAERS) and the World Health Organization’s VigiBase repository. Our results indicate that the two radioimmunotherapeutic agents have both related and distinct side effect profiles and confirm their known toxicological considerations. Our work also suggests that computational analysis of real-world post-marketing data can provide informative clinical insights. While more prospective studies are necessary to fully characterize the efficacy and safety of radioimmunotherapy, we expect that it has not yet reached its full therapeutic potential in modern hematological oncology.

Validating α-particle emission from 211At-labeled antibodies in single cells for cancer radioimmunotherapy using CR-39 plastic nuclear track detectors.

Recently, 211At has received increasing attention as a potential radionuclide for cancer radioimmunotherapy. It is a α-particle emitter, which is extremely effective against malignant cells. We demonstrate a method to verify the efficiency of 211At-labeled trastuzumab antibodies (211At-trastuzumab) against HER2 antigens, which has not been determined for radioimmunotherapy. A CR-39 plastic nuclear detector is used for measuring the position and the linear energy transfer (LET) of individual 211At α- particle tracks. The tracks and 211At-trastuzumab-binding cells were co-visualized by using the geometric information recorded on the CR-39. HER2-positive human gastric cancer cells (NCI-N87), labelled with 211At-trastuzumab, were dropped on the centre of the CR-39 plate. Microscope images of the cells and the corresponding α-tracks acquired by position matching were obtained. In addition, 3.5 cm × 3.5 cm macroscopic images of the whole plate were acquired. The distribution of number of α-particles emitted from single cells suggests that 80% of the 211At-trastuzumab-binding cells emitted α-particles. It also indicates that the α-particles may strike the cells several times along their path. The track-averaged LET of the α-particles is evaluated to be 131 keV/μm. These results will enable quantitative evaluation of delivered doses to target cells, and will be useful for the in vitro assessment of 211At-based radioimmunotherapeutic agents.

Metal-chelating polymers developed for mass cytometry as a potential route to high activity radioimmunotherapeutic agents

Metal-chelating polymers developed for mass cytometry as a potential route to high activity radioimmunotherapeutic agents

111In-Bn-DTPA-nimotuzumab with/without modification with nuclear translocation sequence (NLS) peptides: an Auger electron-emitting radioimmunotherapeutic agent for EGFR-positive and trastuzumab (Herceptin)-resistant breast cancer

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.

Automated Module Radiolabeling of Peptides and Antibodies with Gallium-68, Lutetium-177 and Iodine-131

Our objectives were to automate radiolabeling of therapeutic activities for safe, reliable, cost-effective, practical routine preparation of (177)Lu-radiopeptides, (131)I radioimmunotherapeutic agents, and (68)Ga-peptide PET diagnostics and, in particular, minimize radiation exposure to the radiopharmaceutical chemist. Reprogramming and adaptation of a commercially available synthetic module (IBA molecular; Synthera®) allowed high yield, fully automated, in-house radiolabeling of novel therapeutic and diagnostic radiopharmaceuticals under remote shielded sterile conditions. Radiochemical yield and purity was measured by instant thin-layer chromatography and high-performance liquid chromatography. (68)Ga-octreotate and (177)Lu-octreotate were synthesized, resulting in both radiochemical yield and radiochemical purity greater than 99%. Synthesis of (131)I-rituximab resulted in a yield of 60%, with a radiochemical purity greater than 99%. Using 400 MBq (68)GaCl(3) per synthesis, the estimated absorbed body and hand dose for a manual synthesis was 2 and 27 μ Sv, contrasting with automated synthesis exposure of 1.3 and 7.9 μ Sv. Using 8000 MBq (177)LuCl(3) per synthesis, the estimated absorbed body and hand dose for a manual synthesis was 44.7 and 75 μ Sv, contrasting with automated synthesis exposure of 2.5 and 20 μ Sv. Using 6000 MBq (131)I per synthesis, the estimated absorbed body and hand dose for a manual synthesis was 83.7 and 335 μ Sv, contrasting with automated synthesis exposure of 10.9 and 54.7 μ Sv. The reduction in radiation exposure by automated synthesis of radiopharmaceuticals in the Synthera® module was at least five fold. Automated synthesis of therapeutic (177)Lu and (131)I radiopharmaceuticals and (68)Ga PET agents in the shielded sterile Synthera® module is simple, practical, and efficient and virtually eliminates radiation exposure to the radiopharmaceutical chemist.

Generation of Small 32P-Labeled Peptides as a Potential Approach to Colorectal Cancer Therapy

Cancers have been revealed to be extremely heterogenous in terms of the frequency and types of mutations present in cells from different malignant tumors. Thus, it is likely that uniform clinical treatment is not optimal for all patients, and that the development of individualized therapeutic regimens may be beneficial. We describe the generation of multiple, unique small peptides nine to thirty-four amino acids in length which, when labeled with the radioisotope 32P, bind with vastly differing efficiencies to cell lines derived from different colon adenocarcinomas. In addition, the most effective of these peptides permanently transfers the 32P radioisotope to colorectal cancer cellular proteins within two hours at a rate that is more than 150 times higher than in cell lines derived from other cancers or from the normal tissues tested. Currently, the only two FDA-approved radioimmunotherapeutic agents in use both employ antibodies directed against the B cell marker CD20 for the treatment of non-Hodgkin's lymphoma. By using the method described herein, large numbers of different 32P-labeled peptides can be readily produced and assayed against a broad spectrum of cancer types. This report proposes the development and use of 32P-labeled peptides as potential individualized peptide-binding therapies for the treatment of colon adenocarcinoma patients.

Development of radioimmunotherapeutic and diagnostic antibodies: an inside-out view.

Only a handful of radiolabeled antibodies (Abs) have gained US Food and Drug Administration (FDA) approval for use in clinical oncology, including four immunodiagnostic agents and two targeted radioimmunotherapeutic agents. Despite the advent of nonimmunogenic Abs and the availability of a diverse library of radionuclides, progress beyond early Phase II radioimmunotherapy (RIT) studies in solid tumors has been marginal. Furthermore, [18F]fluorodeoxyglucose continues to dominate the molecular imaging domain, underscored by a decade-long absence of any newly approved Ab-based imaging agent (none since 1996). Why has the development of clinically successful Abs for RIT been limited to lymphoma? What obstacles must be overcome to allow the FDA approval of immuno-positron emission tomography (immuno-PET) imaging agents? How can we address the unique challenges that have thus far prevented the introduction of Ab-based imaging agents and therapeutics for solid tumors? Many poor decisions have been made regarding radiolabeled Abs, but useful insight can be gained from these mistakes. The following review addresses the physical, chemical, biological, clinical, regulatory and financial limitations that impede the progress of this increasingly important class of drugs.

Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds.

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.

A dose-escalation study of tositumomab and iodine I 131 tositumomab (Bexxar) in pts with previously treated non-Hodgkin's lymphoma (NHL) with > 25% bone marrow involvement

6575 Introduction: The Bexxar therapeutic regimen can produce durable and complete responses in patients with relapsed/refractory low-grade NHL. Virtually all previous studies of radioimmunotherapeutic agents (including Bexxar therapy) for NHL excluded patients with extensive bone marrow involvement (BMI) with tumor (>25% intertrabecular space) due to risk of excess hematologic toxicity. Therefore, we evaluated Bexxar therapy specifically in this patient population to assess safety and toxicity in a dose-escalation study. Methods: Subjects were required to have baseline BMI of >25% and platelet count of 150,000/mm3. A patient-specific dose of Iodine 131 Tositumomab was administered to deliver a defined amount of total body radiation. Dose escalation began at a total body dose of 45 cGy (subsequently increased in increments of 10 cGy). Dose limiting toxicity (DLT) was defined as ANC < 500/mm3 or platelets <25,000/mm3 for >17 d; or ANC <750/mm3 or platelets <50,000/mm3 for >24 d. Results: Eleven patients were enrolled (8 at 45 cGy and 3 at 55 cGy). Estimated BMI was 30% to 65%, with median of approx 40%. Subjects had a median of 3 prior chemotherapies (range: 1–6). One of 6 evaluable patients treated at 45 cGy experienced hematologic DLT. Three patients (estimated BMI 40–60%) received 55 cGy, with 1 having hematologic DLT concurrent with lymphoma progression with extensive BMI. Three of 11 (27%) subjects received hematologic supportive care. Non-hematologic toxicities were minimal and were similar to previous studies. Two patients responded with durations of 1 and 43.6+ months. Conclusion: Treatment of patients with >25 % bone marrow involvement with Bexxar therapy was well tolerated at 45 cGy total body dose and can result in lymphoma responses. Further studies with additional patients and at higher doses are required to further characterize the MTD and to establish efficacy. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Corixa Corp. Corixa Corp. Corixa Corp. Corixa Corp.

Radiobiology of radioimmunotherapy with 90Y ibritumomab tiuxetan (zevalin)

Radioimmunotherapy represents a significant advance over unlabeled immunotherapy for the treatment of patients with B-cell non-Hodgkin’s lymphoma. The efficacy of radioimmunotherapeutic agents depends in large part on the basic biological effects associated with their components, monoclonal antibodies and radionuclides, separately and in combination. The radiobiological effects associated with yttrium 90-labeled ibritumomab tiuxetan (Zevalin; Biogen Idec Inc, Cambridge, MA) include the induction of apoptosis and cell-cycle redistribution (eg, arrest of cells in the G 2/M phase of the cell cycle). Because of dose-rate effects, tumor cells may, in some cases, be more susceptible to the low-dose-rate radiation used in radioimmunotherapy than to the high-dose-rate radiation used in external beam radiotherapy. The efficacy of radioimmunotherapy may potentially be optimized through a variety of approaches, including the use of agents that increase the expression of certain tumor antigens (thus facilitating improved biodistribution of radiolabeled monoclonal antibodies) or that sensitize tumor cells to radiation.

Physics for practitioners: The use of radiolabeled monoclonal antibodies in B-Cell non-Hodgkin's lymphoma

The limitations of conventional treatment for non-Hodgkin's lymphoma (NHL) have led to intense investigations of biological therapies for this disease, including monoclonal antibodies. Conjugation of these antibodies with radionuclides results in additional cytotoxic activity with generally acceptable levels of toxicity. Numerous antibodies and radionuclides of widely varying properties are in clinical development. Given the likelihood that one or more radioimmunotherapeutic agents will soon be commercially available, there is a need for oncologists who treat patients with NHL to become more aware of the principles of radiation physics that underlie radioimmunotherapy and the interactions among radiolabeled antibodies, the tumor, and the host.