Radioimmunotherapy Agents Research Articles

177Lu Anti-Angiogenic Radioimmunotherapy Targeting ATP Synthase in Gastric Cancer Model.

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate synthase (ATPS) monoclonal antibody (mAb) with the radioisotope 177Lu using DOTA as a chelating agent. 177Lu-DOTA-ATPS mAb demonstrated high labeling efficiency (99.0%) and stability in serum. MKN-45 cancer cells exhibited the highest cellular uptake, which could be specifically blocked by unlabeled ATPS mAb. In mice, 177Lu-DOTA-ATPS mAb accumulated significantly in tumors, with a tumor uptake of 16.0 ± 1.5%ID/g on day 7. 177Lu-DOTA-ATPS mAb treatment significantly reduced the viability of MKN-45 cells in a dose-dependent manner. In a xenograft tumor model, this radioimmunotherapy strategy led to substantial tumor growth inhibition (82.8%). Furthermore, combining 177Lu-DOTA-ATPS mAb with sunitinib, an anti-angiogenic drug, enhanced the therapeutic efficacy of sunitinib in the mouse model. Our study successfully developed 177Lu-DOTA-ATPS mAb, a radioimmunotherapy agent targeting tumor blood vessels. This approach demonstrates significant promise for inhibiting tumor growth, both as a single therapy and in combination with other anti-cancer drugs.

Pretargeted Radioimmunotherapy with the Novel Anti-oxMIF/HSG Bispecific Antibody ON105 Results in Significant Tumor Regression in Murine Models of Cancer.

Radioimmunotherapy (RIT) uses monoclonal antibodies to deliver radionuclides to cancer cells or the tumor microenvironment and has shown promise in treating localized and diffuse tumors. Although RIT agents have gained FDA/EMA approval for certain hematologic malignancies, effectiveness of RIT in treating solid tumors remains limited. In this study, we present PreTarg-it®, a novel approach for pretargeted RIT, providing optimized delivery of payloads in a two-step regimen. The effectiveness of PreTarg-it® is demonstrated by a powerful combination of ON105, a novel bispecific antibody against both oxidized macrophage migration inhibitory factor (oxMIF) and the histamine-succinyl-glycyl (HSG) hapten, as the first component and the radioactively labeled DOTA-di-HSG peptide as the second component in murine models of cancer. Mice bearing either subcutaneous mouse colorectal CT26 or human pancreatic CFPAC-1 tumors received an i.v. injection of ON105. After ON105 had accumulated in the tumor and cleared from circulation to approximately 1% to 3% of its peak concentration, 177Lu-DOTA-di-HSG peptide was administered. A single PreTarg-it® treatment cycle resulted in tumor regression when mice bearing CT26 tumors were given the highest treatment dose with a pretargeting delay of 3 days. Administered with a 5-day interval, the highest dose arrested tumor growth in both CT26 syngrafts and CFPAC-1 xenografts. In all cases, the highest treatment dose resulted in 100% survival at the study endpoint, whereas the control cohorts showed 0% and 60% survival in the CT26 and CFPAC-1 models, respectively. Therefore, PreTarg-it® holds potential as a novel and potent therapy for patients with hard-to-treat solid tumors, such as pancreatic cancer, as well as those with late-stage malignancies.

New Emerging Targets in Cancer Immunotherapy: The Role of B7-H3.

Immune checkpoints (ICs) are molecules implicated in the fine-tuning of immune response via co-inhibitory or co-stimulatory signals, and serve to secure minimized host damage. Targeting ICs with various therapeutic modalities, including checkpoint inhibitors/monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and CAR-T cells has produced remarkable results, especially in immunogenic tumors, setting a paradigm shift in cancer therapeutics through the incorporation of these IC-targeted treatments. However, the large proportion of subjects who experience primary or secondary resistance to available IC-targeted options necessitates further advancements that render immunotherapy beneficial for a larger patient pool with longer duration of response. B7-H3 (B7 Homolog 3 Protein, CD276) is a member of the B7 family of IC proteins that exerts pleiotropic immunomodulatory effects both in physiologic and pathologic contexts. Mounting evidence has demonstrated an aberrant expression of B7-H3 in various solid malignancies, including tumors less sensitive to current immunotherapeutic options, and has associated its expression with advanced disease, worse patient survival and impaired response to IC-based regimens. Anti-B7-H3 agents, including novel mAbs, bispecific antibodies, ADCs, CAR-T cells, and radioimmunotherapy agents, have exhibited encouraging antitumor activity in preclinical models and have recently entered clinical testing for several cancer types. In the present review, we concisely present the functional implications of B7-H3 and discuss the latest evidence regarding its prognostic significance and therapeutic potential in solid malignancies, with emphasis on anti-B7-H3 modalities that are currently evaluated in clinical trial settings. Better understanding of B7-H3 intricate interactions in the tumor microenvironment will expand the oncological utility of anti-B7-H3 agents and further shape their role in cancer therapeutics.

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.

Real-world data from yttrium-90 ibritumomab tiuxetan treatment of relapsed or refractory indolent B-cell non-Hodgkin's lymphoma: J3Zi Study.

Yttrium-90 ibritumomab tiuxetan (90YIT) is a radioimmunotherapy agent in which the radioisotope yttrium-90 is bound to ibritumomab via tiuxetan as a chelating agent, and is used for relapsed or refractory low-grade B-cell non-Hodgkin's lymphoma (rr-B-NHL). We conducted a joint study to evaluate the clinical outcome of 90YIT. The J3Zi study is composed of data from patients receiving 90YIT for rr-B-NHL from the top three institutions with 10years of 90YIT treatment experience from October 2008 to May 2018 in Japan. The efficacy, prognostic factors and safety of 90YIT were retrospectively evaluated. Data from 316 patients were analyzed; the mean age was 64.6years and the median number of prior treatments was 2. The median PFS was 3.0years, the final OS rate was over 60%, and the median OS was not reached during the study period. Significant factors influencing PFS were sIL-2R ≤ 500 (U/mL) and no disease progression within 24months of first treatment. Significant factors influencing OS were number of prior treatments ≤ 2 and sIL-2R ≤ 500 (U/mL). The PFS and OS rates were found to be significantly higher in the late half era (2013 to 2018) than in the early half era (2008 to 2013) during the study period. Prognosis following 90YIT treatment was improved in the late half era compared to the early half era. As treatment using 90YIT increased, administration of 90YIT shifted to the earlier treatment line. This may have contributed to the improvement of prognosis found in the late era. (UMIN000037105).

Treatment of Prostate Cancer with CD46-targeted 225Ac Alpha Particle Radioimmunotherapy.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. [225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity. Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi. [225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

Enhanced antitumor immune responses via a new agent [131I]-labeled dual-target immunosuppressant

Radionuclides theranostic are ideal “partners” for bispecific antibodies to explore the immune response of patients and synergistic treatment. A bispecific single-domain antibody-Fc fusion protein, KN046, exhibits a good treatment effect by binding to programmed cell death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). An ionizing-radiation stimulus mediated by a low-dose of [131I] may be used for immunopotentiation. In this study, we established [131I]-labeled KN046 as a novel radioimmunotherapy agent to treat malignant melanoma and explored the mechanism.MethodsAfter intravenous injection of [131I]-KN046, SPECT/CT imaging was applied to identify candidate targets for KN046 immunotherapy. [18F]-FDG and [68 Ga]-NOTA-GZP (granzyme B-specific PET imaging agent) micro-PET/CT imaging was used to assess the immune response in vivo after [131I]-KN046 treatment. The synergistic treatment effect of [131I]-KN046 was evaluated by exploring the [131I]-based radionuclide-induced release of tumor immunogenicity-related antigens as well as the histology and survival of tumor-bearing mice after treatment.ResultsThe constructed [131I]-KN046 exhibited high affinity and specificity for PD-L1/CTLA-4 immune targets and had excellent in vivo intratumoral retention capability so as to achieve good antitumor efficacy. More importantly, the combination of low-dose [131I] and KN046-enhanced immunosensitivity increased the immunotherapy response rates significantly. Exposure of tumor cells to [131I]-KN046 led to upregulated expression of MHC-I and Fas surface molecules and significant increases in the degree of T-cell activation and counts of tumor-infiltrating immunocytes.ConclusionUse of low-dose [131I] combined with a dual-target immunosuppressant could be exploited to identify the subset of treatment responders but also exhibited great potential for enhancing antitumor immune responses.

A novel theranostic probe [111In]In-DO3A-NHS-nimotuzumab in glioma xenograft

Abstract Indium-111 (111In) has an appropriate half-life (T 1/2 = 67 h) and energy characteristics for cancer diagnosis via γ-ray imaging and cancer therapy with Auger electrons. The aim of our study is to evaluate the potential of [111In]In-DO3A-NHS-nimotuzumab as a theranostic agent for radioimmunoimaging (RII) and radioimmunotherapy (RIT) against human glioma xenografts in mice. We explored the chelators DO3A-NHS and DOTA-p-SCN-Bz to optimize 111In radiolabeling efficiency of nimotuzumab. The radiopharmaceuticals were purified by PD-10 mini-column and their in vitro stabilities were assessed. We investigated the biodistribution of [111In]In-DO3A-NHS-nimotuzumab as it had relatively superior labeling efficiency and stability in vitro. We conducted SPECT imaging on mice bearing glioma (U87MG) xenografts, which were injected with ∼3.7 MBq of [111In]In-DO3A-NHS-nimotuzumab. The in vivo radiotherapeutic effects of [111In]In-DO3A-NHS-nimotuzumab was analyzed via injecting a single 37 MBq dose, 2 × 18 MBq doses, or 2 × 37 MBq doses into mice bearing U87MG xenografts. The control groups were administered either 30 μg nimotuzumab or saline. The radiochemical yields of [111In]In-DO3A-NHS-nimotuzumab and [111In]In-DOTA-p-SCN-Bz-nimotuzumab were > 85% and > 75%, respectively. [111In]In-DO3A-NHS-nimotuzumab had > 95% radiochemical purity and was more stable in vitro than [111In]In-DOTA-p-SCN-Bz-nimotuzumab. Biodistribution study demonstrated that [111In]In-DO3A-NHS-nimotuzumab was highly stable in vivo. SPECT imaging disclosed that [111In]In-DO3A-NHS-nimotuzumab had excellent targeted tumor uptake and retained in tumors for 24 and 72 h. All [111In]In-DO3A-NHS-nimotuzumab treatments substantially inhibited tumor growth over the controls. The 2 × 37 MBq treatment was particularly efficacious, and presented with survival time prolonged by ≤66 days. In contrast, the survival time of the control group was only 30 days. In our study, we developed an optimized synthesis protocol for radiopharmaceutical 111In-DO3A-NHS-nimotuzumab and demonstrated that it is a promising theranostic agent. It could be highly efficacious in RII and RIT against EGFR-expressing glioma.

GPC3-targeted immunoPET imaging of hepatocellular carcinomas.

Early detection of hepatocellular carcinoma (HCC) remains a clinical challenge. Glypican 3 (GPC3) is a proteoglycan highly specific for HCC and is a potential diagnostic and therapeutic target for HCC. This work aims to develop GPC3-targeted immuno-positron emission tomography (immunoPET) imaging strategies and to assess the diagnostic values in preclinical HCC models. Flow cytometry was used to screen GPC3-positive HCC cell lines. The expression of GPC3 in HCCs was detected by immunohistochemistry on tissue microarray. A novel GPC3-specific single domain antibody (sdAb) was produced and labeled with gallium-68 (68Ga, T1/2 = 1.1h) and fluorine-18 (18F, T1/2 = 1.8h) to develop radiotracers with different half-lives. The diagnostic efficacies of the developed probes (i.e., [68Ga]Ga-NOTA-G2, [18F]F-G2, and [68Ga]Ga-NOTA-ABDG2) were interrogated in preclinical HCC models bearing varying GPC3 levels. GPC3 was strongly expressed on HCC cell lines and patients with poorly differentiated HCC. [68Ga]Ga-NOTA-G2 immunoPET imaging specifically delineated the subcutaneous HCC lesions, outperforming the traditional 18F-fluorodeoxyglucose PET and the nonspecific [68Ga]Ga-NOTA-NbGFP immunoPET. ImmunoPET imaging with [18F]F-G2 also efficiently diagnosed the tumors with clarity. Moreover, the fusion of G2 to an albumin-binding domain (ABD) significantly increased the tumor uptake and decreased kidney accumulation of the radiotracer when compared to [68Ga]Ga-NOTA-G2. In the work, we successfully developed sdAb-derived GPC3-targeted immunoPET imaging strategies and characterized the superior diagnostic accuracies in preclinical HCC models. Furthermore, we synthesized a fusion protein ABDG2 with improved targeting and pharmacokinetic properties, serving as a promising candidate for developing radioimmunotherapy agents.

Efficacy and safety of yttrium-90 ibritumomab tiuxetan in the treatment of non-Hodgkin lymphoma.

103 Background: Yttrium 90-labeled ibritumomab tiuxetan (Zevalin) is a radioimmunotherapy agent which combines the action of a beta-emitting isotope, yttrium-90, with the anti-CD20 monoclonal antibody, rituximab. The purpose of this study was to evaluate the efficacy of Zevalin in the treatment of Non-Hodgkin Lymphoma (NHL) in terms of response to therapy, overall survival (OS) and progression free survival (PFS). Methods: A total of 171 patients with NHL referred for treatment with Zevalin between September 2005 and January 2017 were identified for retrospective analysis. Clinical characteristics, treatments, and outcomes were extracted from clinical chart review. Survival rates and survival curves were generated using Kaplan-Meier analysis. Chi-square and independent student t test were used for comparison of categorical variables. Multivariate analysis (MVA) Cox proportional hazards ratios (HR) model was used to assess OS and PFS. Results: Of the 171 patients referred for treatment, median age was 70 years old, 54% were male (n = 93), and each patient had received a median of 2 prior chemotherapy regimens. The majority of patients were treated for follicular lymphoma (n = 107) and diffuse large B-cell lymphoma (n = 34). Seventy-three patients (53%) achieved complete response, and 23 patients achieved partial response (17%). One-year progression free survival was 71%, and median PFS was 31 months. Adverse events included cytopenic events, GI side effects, and mucocutaneous toxicity. Conclusions: Use of Zevalin for treatment of NHL demonstrated a significant improvement in PFS. In addition to improved efficacy, Zevalin was well-tolerated in this study and demonstrated an adequate safety profile. Further considerations will be given as to which treatment factors are associated with a complete response and improved PFS.

Dose-dependent cell cycle arrest and apoptosis in HER2 breast cancer cells by177Lu-CHX-A"-DTPA-Trastuzumab.

Trastuzumab is a Food and Drug Administration-approved humanized monoclonal antibody which targets the extracellular domain of human epidermal growth factor receptor 2 (HER2) receptor overexpressed on HER2-positive breast cancer cells. The combination of Lutetium-177 (177 Lu) (t½= 6.7 days, Eβmax497 keV (78.6%) and trastuzumab makes it a suitable targeting agent for radioimmunotherapy. In preclinical and clinical studies,177 Lu-Trastuzumab has proven to be effective for the treatment of HER2-positive malignancies such as breast and ovarian cancer. In this study, we report the mechanism of action of177 Lu-CHX-A"-diethylenetriaminepentaacetic acid (DTPA)-trastuzumab at the cellular and molecular level by performing various in vitro assays in HER2-positive MDA-MB-453 breast cancer cells. Trastuzumab was conjugated to the bifunctional chelating agent (BFCA) para-isothiocyanatobenzyl-DTPA and radiolabeled with177 Lu. In vitro cell binding studies were carried out in MDA-MB-453 cells to confirm the specificity of the complex toward the receptor. Cellular toxicity, cell cycle, and cell death analysis were also performed for exploring the potential of the radioimmunoconjugate at cellular and molecular level. In vitro cell binding studies showed a maximum binding of 10.7 ± 0.1% which reduced to 2.9 ± 0.1% on coincubation with unlabeled antibody. Our study revealed that the cellular toxicity was dose dependent, and mode of cell death was predominantly by apoptosis. The radioimmunoconjugate retarded the cell in the S phase of cell cycle with two-fold increase in G2/M arrest which justifies the enhanced apoptosis at higher doses. The study revealed that the formulation can execute a dose-dependent cellular toxicity through induction of apoptosis.

Yttrium-90-Labeled Anti-Glypican 3 Radioimmunotherapy Halts Tumor Growth in an Orthotopic Xenograft Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a 89Zr-conjugated monoclonal anti-GPC3 antibody (αGPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated 90Y, a high-energy beta-particle-emitting radionuclide, to our αGPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to αGPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200 μCi/mouse or 300 μCi/mouse. Control mice received DOTA-αGPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200 μCi only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300 μCi experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of 90Y-αGPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC.

Therapeutic efficacy evaluation of radioimmunotherapy with 90 Y-labeled anti-podoplanin antibody NZ-12 for mesothelioma.

Podoplanin is a type I transmembrane sialomucin‐like glycoprotein that is highly expressed in malignant mesothelioma. The rat‐human chimeric antibody NZ‐12 has high affinity for human podoplanin and antibody‐dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ‐12 and the antitumor effect of RIT with 90Y‐labeled NZ‐12 in an NCI‐H226 (H226) malignant mesothelioma xenograft mouse model. 111In‐labeled NZ‐12 bound specifically to H226 cells with high affinity, and accumulation was high in H226 tumors but low in major organs. RIT with 90Y‐labeled NZ‐12 significantly suppressed tumor growth and prolonged survival without body weight loss and obvious adverse effects. Higher podoplanin expression levels were observed in human mesothelioma specimens, suggesting higher tumor accumulation of 90Y‐labeled NZ‐12 in patients compared with the H226 tumor xenografts. Our findings suggest that 90Y‐labeled NZ‐12 is a promising RIT agent as a new therapeutic option for malignant mesothelioma that warrants further clinical studies to evaluate the dosimetry and efficacy in patients.

Radiolabeling, Characteristics and NanoSPECT/CT Imaging of 188Re-cetuximab in NCI-H292 Human Lung Cancer Xenografts.

Cetuximab has exhibited high EGFR-targeting specificity and clinical promise in previous studies. In this study, we formulated unit dose kits for preparation of high specific activity 188Re-cetuximab for imaging and treatment of EGFR-positive cancer. 188Re-cetuximab was prepared by adding 0.37-0.74 GBq/0.5 ml of 188Re-perrhenate for 4 h at 37°C. Cell surface expression of EGFR, cell binding and cytotoxic effects were evaluated in vitro using both EGFR-positive (NCI-H292, A431) and EGFR-negative (BT483) tumors. A nanoSPECT/CT imaging study was performed in mice bearing EGFR-expressing NCI-H292 tumors. 188Re-cetuximab bound specifically to EGFR-expressing cells and labeling of radionuclides to cetuximab preserved the binding ability of the antibody. Besides, the cytotoxic effect of 188Re-cetuximab was increased dose-dependently. NanoSPECT/CT imaging revealed that 188Re-cetuximab could continually target the tumor region for at least 48 h. The highly specific targeted property of 188Re-cetuximab suggested that it is suitable as a diagnostic tool and maybe a potent radioimmunotherapy agent in EGFR-positive cancers.

Biodistribution and Dosimetry of Indigenously Produced131I-Rituximab in B-Cell Lymphoma: Pilot Study Estimating Patient-Specific Dose Comparing 2 Different Dosimetric Methods

Cost containment through indigenous production of radioimmunotherapy agents for non-Hodgkin lymphoma (NHL) would be a pivotal step toward wider clinical availability, especially in developing countries. We examined the biodistribution and dosimetry of indigenously developed and radiolabeled 131I-rituximab, using the monoclonal antibody of chimeric origin, in patients with B-cell lymphoma for potential use in radioimmunotherapy. Methods: This prospective study included 13 patients with B-cell NHL who underwent low-dose diagnostic scanning for dosimetric and biodistribution studies. Soon after rituximab infusion, a diagnostic dose of radioiodinated rituximab was administered. Serial planar whole-body γ-camera images were taken soon afterward and on days 1, 2, 4, and 6. A source of 131I with known activity was used as a reference standard for dosimetry calculations. Results: The patient-specific administered dose that would give a whole-body absorbed radiation dose of 75 cGy, calculated by the MIRD schema, ranged from 3,095.42 to 6,330.33 MBq (83.66-171.09 mCi), with a mean of 3,986.01 ± 863.95 MBq (107.73 ± 23.35 mCi) and a median of 3,697.41 MBq (99.93 mCi). The mean residence time was 69.54 h. Within the first 48 h at least 50% of the injected activity was cleared, and by 144 h at least 80% was cleared. The patient-specific administered dose that would give a whole-body absorbed radiation dose of 75 cGy, calculated by mean residence time and activity-hours, ranged from 2,654.75 to 6,210.45 MBq (71.75-167.85 mCi), with a mean of 3,576.42 ± 927.59 MBq (96.66 ± 25.07 mCi) and a median of 3,421.02 MBq (92.46 mCi). With respect to organ-specific dosimetry, the mean absorbed doses to organs (apart from blood pool [3.77 Gy] and spleen [4.02 Gy]) were 0.97 Gy to the lungs, 0.69 Gy to the liver, and 0.7 Gy to the kidneys. Conclusion: The indigenous product had kinetics similar to commercial radiopharmaceuticals, with the advantage of a lower human antimouse antibody response because of the pharmaceutical's being a chimeric antibody rather than a murine antibody. Hence, clinical administration was safe. In none of the organs did dose-limiting radiation exposure occur at the proposed therapeutic dose.

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen.

5D3 is a new high-affinity murine monoclonal antibody specific for prostate-specific membrane antigen (PSMA). PSMA is a target for the imaging and therapy of prostate cancer. 111In-labeled antibodies have been used as surrogates for 177Lu/90Y-labeled therapeutics. We characterized 111In-DOTA-5D3 by SPECT/CT imaging, tissue biodistribution studies, and dosimetry. Methods: Radiolabeling, stability, cell uptake, and internalization of 111In-DOTA-5D3 were performed by established techniques. Biodistribution and SPECT imaging were done on male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing human PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer xenografts on the upper right and left flanks, respectively, at 2, 24, 48, 72, and 192 h after injection. Biodistribution was also evaluated in tumor-free, healthy male CD-1 mice. Blocking studies were performed by coinjection of a 10-fold and 50-fold excess of 5D3 followed by biodistribution at 24 h to determine PSMA binding specificity. The absorbed radiation doses were calculated on the basis of murine biodistribution data, which were translated to a human adult man using organ weights as implemented in OLINDA/EXM. Results:111In-DOTA-5D3 was synthesized with specific activity of approximately 2.24 ± 0.74 MBq/μg (60.54 ± 20 μCi/μg). Distribution of 111In-DOTA-5D3 in PSMA(+) PC3 PIP tumor peaked at 24 h after injection and remained high until 72 h. Uptake in normal tissues, including the blood, spleen, liver, heart, and lungs, was highest at 2 h after injection. Coinjection of 111In-DOTA-5D3 with a 10- and 50-fold excess of nonradiolabeled antibody significantly reduced PSMA(+) PC3 PIP tumor and salivary gland uptake at 24 h but did not reduce uptake in kidneys and lacrimal glands. Significant clearance of 111In-DOTA-5D3 from all organs occurred at 192 h. The highest radiation dose was received by the liver (0.5 mGy/MBq), followed by the spleen and kidneys. Absorbed radiation doses to the salivary and lacrimal glands and bone marrow were low. Conclusion:111In-DOTA-5D3 is a new radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA.

Evaluation of 177Lu[Lu]-CHX-A″-DTPA-6A10 Fab as a radioimmunotherapy agent targeting carbonic anhydrase XII

IntroductionDue to their infiltrative growth behavior, gliomas have, even after surgical resection, a high recurrence tendency. The approach of intracavitary radioimmunotherapy (RIT) is aimed at inhibiting tumor re-growth by directly administering drugs into the resection cavity (RC). Direct application of the radioconjugate into the RC has the advantage of bypassing the blood-brain barrier, which allows the administration of higher radiation doses than systemic application. Carbonic anhydrase XII (CA XII) is highly expressed on glioma cells while being absent from normal brain and thus an attractive target molecule for RIT. We evaluated a CA XII-specific 6A10 Fab (fragment antigen binding) labelled with 177Lu as an agent for RIT. Methods6A10 Fab fragment was modified and radiolabelled with 177Lu and characterized by MALDI-TOF, flow cytometry and radio-TLC. In vitro stability was determined under physiological conditions. Biodistribution studies, autoradiography tumor examinations and planar scintigraphy imaging were performed on SCID-mice bearing human glioma xenografts. ResultsThe in vitro CA XII binding capacity of the modified Fab was confirmed. Radiochemical purity was determined to be >90% after 72 h of incubation under physiological conditions. Autoradiography experiments proved the specific binding of the Fab to CA XII on tumor cells. Biodistribution studies revealed a tumor uptake of 3.0%ID/g after 6 h and no detectable brain uptake. The tumor-to-contralateral ratio of 10/1 was confirmed by quantitative planar scintigraphy. ConclusionThe radiochemical stability in combination with a successful in vivo tumor uptake shows the potential suitability for future RIT applications with the 6A10 Fab.

Abstract 5204: Preclinical development of MVT-1075 as radioimmunotherapy for pancreatic cancer and other CA19-9 positive malignancies

Abstract Rationale: The CA19-9 antigen is frequently overexpressed in pancreatic and other GI tumors. MVT-5873 (HuMab-5B1), a fully human monoclonal antibody currently in phase I study, targets the sialyl Lewis A (sLea) epitope on CA19-9, and is a promising platform for development of a targeted radioimmunotherapy (RIT). MVT-5873 was conjugated with the chelator CHX-A″-DTPA and radiolabeled with the beta-emitting isotopes Lutetium -177 (177Lu) or Yttrium-90 (90Y) to form the RIT agents MVT-1075 (177Lu- CHX-A″-DTPA-HuMAb-5B1) and MVT-1916 (90Y- CHX-A″-DTPA-HuMAb-5B1), respectively. The antitumor efficacy of each of the constructs was studied in nude mice bearing BxPC3 human pancreatic tumor xenografts, known to express CA19-9. Methods: The initial dose-finding studies utilized doses of MVT-1075 of 75-450 μCi and MVT-1916 of 25-250 μCi, administered to groups of mice (n = 8) bearing subcutaneous (subQ) BxPC3 tumors (~ 150 mm3). Further studies focused on MVT-1075 and assessed antitumor effect in an orthotopic xenograft model, the effect of dose fractionation, and biodistribution in nontumor bearing (normal) and BxPC3 tumor-bearing mice. Results: A single dose of MVT-1075 at 75, 150, 300, or 450 μCi significantly inhibited subQ BxPC3 tumor growth at all dose levels, with sustained suppression with higher doses. MVT-1916 produced similar results. MVT-1075 was selected based on the favorable half-life of 177Lu (6.7 d) and its utility for clinical biodistribution assessments. In an orthotopic BxPC3 tumor model, treatment with a single dose of MVT-1075 at 300 μCi significantly inhibited tumor growth, with Day 20 tumor volume approximately 50% that of the initial starting volume. A third BxPC3 xenograft study evaluated fractionated dosing schedules, (150 μCi x 1, 75 μCi x 2, 50 μCi x3), with both single-dose and fractionated schedules effectively inhibiting subQ BxPC3 tumor growth. Biodistribution studies in normal mice showed an expected gradually decreasing activity in blood, heart, and lungs, with low uptake in normal pancreas. In subQ BxPC3 tumor-bearing mice, tumor uptake was rapid, reaching 69% ID/g by 24 h and 86% ID/g by 120 h. Otherwise, the biodistribution pattern paralleled that of normal mice, with relative %ID/g values within about ± 25% of normal mice across all time points comparing blood, heart, lungs, kidneys, and pancreas, with slightly higher uptake in liver and slightly lower uptake in spleen. Conclusions: MVT-1075 demonstrates promising antitumor activity in a human pancreatic cancer xenograft model, with efficacy shown in both single dose and fractionated schedules. Biodistribution shows rapid and substantial tumor uptake, with much lower uptake in normal organs. These findings support the phase I clinical trial of MVT-1075 in patients with CA19-9 positive pancreatic cancers planned to begin in early 2017. Citation Format: Jacob L. Houghton, Ryan Lanning, Dayla Abdel-atti, Toni Jun, Christine M. Kearns, Michael Schlosser, Wolfgang Scholz, Jason S. Lewis, Paul W. Maffuid. Preclinical development of MVT-1075 as radioimmunotherapy for pancreatic cancer and other CA19-9 positive malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2017-5204

Radiolabeled Antibody Fragment for Preparation of (<sup>177</sup>Lu-DOTA)<sub>m</sub>-PAMAM G3.0-F(ab’)<sub>2</sub> trastuzumab as a Radiopharmaceutical for Cancer Therapy

Several radiolabeled monoclonal antibodies (mAbs) have been used as radioimmunotherapy (RIT) agents for cancer therapy. The use of mAbs as RIT agents is due to their ability to carry effectors, in the form of radionuclides which emit alpha (α) particles, beta (β) particles, or auger electrons, and bind specifically to cancer expressed receptor. This paper reports the preparation of radiolabelled trastuzumab in form of ( 177 Lu-DOTA) m -PAMAM G3-F(ab') 2 -trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed human epidermal growth factor receptor 2 (HER2). Due to its reduced molecular weight, the use of F(ab') 2 -trastuzumab on the aforementioned RIT agent candidate is expected to reach its target much faster compared to the intact trastuzumab. Meanwhile, the role of PAMAM G3 is to increase the specific activity of the radiotherapeutic agent of Lu-177 due to the ability of its 32 –NH 2 functional groups that are able to bind many DOTAs ( £ 31) which in turn can bind a large number of 177 Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer with a pH of 4.5 to produce F(ab') 2 -trastuzumab with a purity of 95 % after purification with PD-10 column. The F(ab') 2 -trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to produce SMCC-F(ab') 2 -trastuzumab. The next reaction was to conjugate SMCC-F(ab') 2 -trastuzumab with DOTA-PAMAM G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAM G3.0 and followed by reacting it with 2-iminothiolane to give (DOTA) m -PAMAM G3.0-F(ab') 2 -trastuzumab. Finally, the (DOTA) m -PAMAM G3.0-F(ab') 2 -trastuzumab was radiolabelled with 177 Lu to produce ( 177 Lu-DOTA) m -PAMAM G3.0-F(ab') 2 -trastuzumab, resulting in a radiochemical purity of 98 % after purification with PD-10 column . Received: 31 October 2015 ; Revised: 30 June 2016 ; Accepted: 25 September 2016

177Lu-DOTA-Bevacizumab: Radioimmunotherapy Agent for Melanoma.

Vascular endothelial growth factor (VEGF) is one of the classic factors to tumor-induced angiogenesis in several types, including melanoma. Bevacizumab is a humanized monoclonal antibody directed against VEGF. To radiolabel Bevacizumab with 177-Lutetium as a potential radioimmunotherapy agent for melanoma. Bevacizumab was derivatized with DOTA-NHS-ester at 4 ºC for 18 h. DOTABevacizumab was radiolabeled with 177LuCl3 (15 MBq/mg) at 37 ºC for 1 h. The studies were performed in healthy and B16F1 tumor-bearing C57BL/6J mice at 24 and 48 h (n = 5). Scinthigraphic imaging studies were performed at 24 h to determine the radiochemical stability, targeting specificity and pharmacokinetics of the 177Lutetium-labeled antibody. DOTA-Bevacizumab was efficiently labeled with 177LuCl3 at 37 °C. The in-vitro stability of labeled product was optimal over 72 h. In-vivo biodistribution studies showed a high liver and tumor uptake of 177Lu-DOTA-Bevacizumab, with tumor-to-muscle ratios of 11.58 and 6.37 at 24 and 48 h p.i. Scintigraphic imaging of melanoma tumor-bearing C57BL/6J mice showed liver and a high tumor selective uptake of 177Lu-DOTA-Bevacizumab at 24 h. Our results support the potential role of 177Lu-DOTA-Bevacizumab as a novel radioimmunotherapy agent for melanoma. We hope that these novel molecular imaging agents will open the path to new diagnostic and therapeutic strategies for Melanoma disease.