Radiotherapy has extensively been employed as a curative or palliative intervention against cancer throughout the last century, with a varying degree of success. For a long time, the antineoplastic activity of X- and γ-rays was entirely ascribed to their capacity of damaging macromolecules, in particular DNA, and hence triggering the (apoptotic) demise of malignant cells. However, accumulating evidence indicates that (at least part of) the clinical potential of radiotherapy stems from cancer cell-extrinsic mechanisms, including the normalization of tumor vasculature as well as short- and long-range bystander effects. Local bystander effects involve either the direct transmission of lethal signals between cells connected by gap junctions or the production of diffusible cytotoxic mediators, including reactive oxygen species, nitric oxide and cytokines. Conversely, long-range bystander effects, also known as out-of-field or abscopal effects, presumably reflect the elicitation of tumor-specific adaptive immune responses. Ionizing rays have indeed been shown to promote the immunogenic demise of malignant cells, a process that relies on the spatiotemporally defined emanation of specific damage-associated molecular patterns (DAMPs). Thus, irradiation reportedly improves the clinical efficacy of other treatment modalities such as surgery (both in neo-adjuvant and adjuvant settings) or chemotherapy. Moreover, at least under some circumstances, radiotherapy may potentiate anticancer immune responses as elicited by various immunotherapeutic agents, including (but presumably not limited to) immunomodulatory monoclonal antibodies, cancer-specific vaccines, dendritic cell-based interventions and Toll-like receptor agonists. Here, we review the rationale of using radiotherapy, alone or combined with immunomodulatory agents, as a means to elicit or boost anticancer immune responses, and present recent clinical trials investigating the therapeutic potential of this approach in cancer patients.

Radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) have been exclusively studied for a dozen or so years, but few clinical RIS and RIT have been successful due to some limitations. In this review, two approaches are introduced to exceed the limitations; One is the modification of physiological and biochemical properties of tumors and the other is the modification of radiolabeled antibodies such as the development of labeling procedures and the production of new antibodies and fragments. The effect of interferon and vasoactive agents on the uptake of antibodies in tumors, and comparative biodistribution and pharmacokinetics of different radiolabeled antibodies and fragments which are produced in the new generation are reviewed. We believe that RIS and RIT could be evaluated precisely when these approaches are well applied to clinical patients.

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated 212Pb, an in vivo generator of alpha-emitting 212Bi and 212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, 212Pb-TCMC-YS5. We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 0.74 MBq (20 µCi) 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (0.37 MBq or 10 µCi 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment.

To date, inefficient delivery of therapeutic doses of radionuclides to solid tumors limits the clinical utility of radioimmunotherapy. We aim to test the therapeutic utility of Yttrium-90 ((90)Y)-radio-conjugates of a monoclonal antibody, which we showed previously to bind specifically to the abundant intracellular La ribonucleoprotein revealed in dead tumor cells after DNA-damaging treatment.

The aim of this study was to investigate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice using different fractionated treatment regimens. The study was performed using the monoclonal antibody MX35 F(ab')(2) labeled with the alpha-particle emitter (211)At. Methods. Nude mice were intraperitoneally inoculated with ~1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later 6 groups of animals were given 400 kBq (211)At-MX35 F(ab')(2) as a single or as a repeated treatment of up to 6 times (n = 18 in each group). The fractionated treatments were given every seventh day. Control animals were treated with unlabeled MX35 F(ab')(2) (n = 12). Eight weeks posttreatment the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined. Results. The tumor-free fractions (TFFs) of the animals, defined as the fraction of animals with no macro- and microtumors and no ascites, were 0.17, 0.11, 0.39, 0.44, 0.44, and 0.67 when treated with 400 kBq (211)At-MX35 F(ab')(2) once or 2, 3, 4, 5, or 6 times, respectively. Repeated treatment 3 times or more resulted in a significantly higher (P < .05) TFF than compared to treatment once or twice. The presence of ascites decreased from 15 out of 18 animals in the group given only one treatment to zero for the 2 groups given 5 or 6 fractions. Treatment with unlabeled MX35 F(ab')(2) resulted in a TFF of zero. Conclusion. Weekly repeated intraperitoneal injections of tolerable amounts of activity of (211)At-MX35 F(ab')(2) of up to 6 times produced increased therapeutic efficacy without observed toxicity, indicating a potential increase of the therapeutic index.

Osteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo.

Glioblastoma (GBM) is the most common primary brain tumor. Due to high resistance to treatment, local invasion, and a high risk of recurrence, GBM patient prognoses are often dismal, with median survival around 15 months. The current standard of care is threefold: surgery, radiation therapy, and chemotherapy with temozolomide (TMZ). However, patient survival has only marginally improved. Radioimmunotherapy (RIT) is a fourth modality under clinical trials and aims at combining immunotherapeutic agents with radiotherapy. Here, we develop in vitro assays for the rapid evaluation of RIT strategies. Using a standard cell irradiator and an Electric Cell Impedance Sensor, we quantify cell migration following the combination of radiotherapy and chemotherapy with TMZ and RIT with durvalumab, a PD-L1 immune checkpoint inhibitor. We measure cell survival using a cloud-based clonogenic assay. Irradiated T98G and U87 GBM cells migrate significantly (p < 0.05) more than untreated cells in the first 20−40 h post-treatment. Addition of TMZ increases migration rates for T98G at 20 Gy (p < 0.01). Neither TMZ nor durvalumab significantly change cell survival in 21 days post-treatment. Interestingly, durvalumab abolishes the enhanced migration effect, indicating possible potency against local invasion. These results provide parameters for the rapid supplementary evaluation of RIT against brain tumors.

Radioimmunotherapy (RIT) aims to deliver a high radiation dose to cancer cells, while minimizing the exposure of normal cells. Typically, monoclonal antibodies are used to target the radionuclides to cancer cell surface antigens. However, antibodies face limitations due to their poor tumor penetration and suboptimal pharmacokinetics, while the expression of their target on the cancer cell surface may be gradually lost. In addition, most antigens are expressed in a limited number of tumor types. To circumvent these problems, we developed a Nanobody (Nb)-based RIT against a prominent stromal cell (stromal-targeting radioimmunotherapy or STRIT) present in nearly all tumors, the tumor-associated macrophage (TAM). Macrophage Mannose Receptor (MMR) functions as a stable molecular target on TAM residing in hypoxic areas, further allowing the delivery of a high radiation dose to the more radioresistant hypoxic tumor regions. Since MMR expression is not restricted to TAM, we first optimized a strategy to block extra-tumoral MMR to prevent therapy-induced toxicity. A 100-fold molar excess of unlabeled bivalent Nb largely blocks extra-tumoral binding of 177Lu-labeled anti-MMR Nb and prevents toxicity, while still allowing the intra-tumoral binding of the monovalent Nb. Interestingly, three doses of 177Lu-labeled anti-MMR Nb resulted in a significantly retarded tumor growth, thereby outcompeting the effects of anti-PD1, anti-VEGFR2, doxorubicin and paclitaxel in the TS/A mammary carcinoma model. Together, these data propose anti-MMR STRIT as a valid new approach for cancer treatment.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer with limited meaningful treatment options. NEPC lesions uniquely express delta-like ligand 3 (DLL3) on their cell surface. Taking advantage of DLL3 overexpression, we developed and evaluated lutetium-177 (177Lu)-labeled DLL3-targeting antibody SC16 (177Lu-DTPA-SC16) as a treatment for NEPC. SC16 was functionalized with DTPA-CHX-A" chelator and radiolabeled with 177Lu to produce 177Lu-DTPA-SC16. Specificity and selectivity of 177Lu-DTPA-SC16 were evaluated in vitro and in vivo using NCI-H660 (NEPC, DLL3-positive) and DU145 (adenocarcinoma, DLL3-negative) cells and xenografts. Dose-dependent treatment efficacy and specificity of 177Lu-DTPA-SC16 radionuclide therapy were evaluated in H660 and DU145 xenograft-bearing mice. Safety of the agent was assessed by monitoring hematologic parameters. 177Lu-DTPA-SC16 showed high tumor uptake and specificity in H660 xenografts, with minimal uptake in DU145 xenografts. At all three tested doses of 177Lu-DTPA-SC16 (4.63, 9.25, and 27.75 MBq/mouse), complete responses were observed in H660-bearing mice; 9.25 and 27.75 MBq/mouse doses were curative. Even the lowest tested dose proved curative in five (63%) of eight mice, and recurring tumors could be successfully re-treated at the same dose to achieve complete responses. In DU145 xenografts, 177Lu-DTPA-SC16 therapy did not inhibit tumor growth. Platelets and hematocrit transiently dropped, reaching nadir at 2 to 3 wk. This was out of range only in the highest-dose cohort and quickly recovered to normal range by week 4. Weight loss was observed only in the highest-dose cohort. Therefore, our data demonstrate that 177Lu-DTPA-SC16 is a potent and safe radioimmunotherapeutic agent for testing in humans with NEPC.

Immunotherapy (IT) and radiotherapy (RT) can act synergistically, enhancing antitumor response beyond what either treatment can achieve separately. Anecdotal reports suggest that these results are in part due to the induction of an abscopal effect on non-irradiated lesions. Systematic data on incidence of the abscopal effect are scarce, while the existence and the identification of predictive signatures or this phenomenon are lacking. The purpose of this pre-clinical investigational work is to shed more light on the subject by identifying several imaging features and blood counts, which can be utilized to build a predictive binary logistic model.

To destroy both tumor blood vessels and adjacent tumor cells, an alpha-particle emitter, 213Bi, has been targeted with a monoclonal antibody (MAb) to vessels that feed lung tumors in mice. Animals, bearing approximately 100 EMT-6 carcinomas each of 50-400 cells in size in the lung, that were treated with 120 muCi of 213Bi-MAb 201B were all cured of their disease. Animals treated when tumors were larger (10(3)-10(4) cells) had extended life spans, but a small number of residual tumors eventually killed the animals. Significant extension of life span was also induced with another tumor model-rat tracheal carcinoma growing in the lungs of SCID mice that were then treated with 136 muCi 213Bi-MAb 201B. These studies indicate that attack of both blood vessels and tumor cells simultaneously is an effective mode of cancer treatment.

We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.

Accumulating evidence indicates that cancer stem cells (CSCs) are the cause of tumor drug/radio-resistance or distant metastasis; therefore, it is essential to eliminate CSCs to cure cancer completely. The purpose of this study was to utilize radioimmunotherapy (RIT) to target CD133(+) colonic CSCs and observe whether this prevented tumor development, by assessing the maximum tolerated dose (MTD) of HCT116 tumor-bearing nude mice with escalating doses of 131I-AC133.1 monoclonal antibody (mAb), and determining the therapeutic efficacy of RIT with 131I-AC133.1 mAb. For RIT trials, animals were randomly divided into 4 groups of 6 per group, and injected with 131I-AC133.1 mAb (16.65 MBq/100 μl), AC133.1 mAb (173.1 μg/100 μl), saline (100 μl), or unrelated IgG1 as an isotype control. Iodine-131 was radiolabeled to AC133.1 mAb by conjugation with N-succinimidyl 3-(tri-n-butylstannyl) benzoate. The MTD of HCT116 tumor-bearing nude mice was 16.65 MBq. Both of the tumor volume doubling time and the survival time of the 131I-AC133.1 mAb group were significant longer than other groups (P < 0.001). CD133 expression was assessed by flow cytometry. Protein levels of cancer stem-like biomarkers (CD133, ALDH1, Lgr5, Vimentin, Snail1), and the proliferative rate of 131I-AC133.1 mAb group were lower than other groups (P<0.001); while its protein level of E-cadherin was higher than other groups. Furthermore, a large proportion of tumor necrosis was also observed in the 131I-AC133.1 mAb group, suggesting that RIT can destroy CSCs and effectively inhibit tumor development.

Immunotherapy has changed the oncology landscape during the last decade and become standard of care for several cancers. The combinations of immunotherapy with other treatment modalities are also being investigated. One of the challenges to investigate such combinations is to identify suitable mouse models for the pre-clinical experiments. In the past, we and other researchers showed that murine B16-F10 melanoma in C57Bl6 mice is refractory to treatment with immune checkpoint inhibitors. In this work we studied the suitability of an alternative syngeneic model, Cloudman S91 murine melanoma in DBA/2 mouse (DBA/2NCrl), to study the combination of immunotherapy targeting PD-1 and radioimmunotherapy targeting melanin. DBA/2 male and female mice were injected subcutaneously with 3-6 million Cloudman S91 cells. When the tumors reached ~150 mm3 volume, the animals were treated intraperitoneally with PBS (sham), h8C3 unlabeled (cold) antibody to melanin, immunotherapy with anti-PD-1 antibody, radioimmunotherapy with 213Bismuth (213Bi)-labeled h8C3 antibody, or several combinations of immunotherapy and radioimmunotherapy. Treatments with immunotherapy alone produced very modest effect on the tumor size, while combination therapy resulted in significant slowing down of the tumor growth, increased animal survival, and no decrease in animal body weight. We conclude that Cloudman S91 murine melanoma in DBA/2 mouse is a suitable model to evaluate combination of immunotherapy of melanoma with tangentially targeted treatments.

Prostate cancer ranks as the second most lethal malignancy in the Western world. Previous targeting of prostate-specific antigen and human kallikrein-related peptidase 2, two related enzymes abundantly expressed in prostatic malignancies, with radioimmunoconjugates intended for diagnostic purposes, have proven successful in rodent prostate cancer (PCa) models. In this study, we investigated the uptake and therapeutic efficacy of (177)Lu-m11B6, a human kallikrein-related peptidase 2 (hK2)-targeting radioimmunoconjugate in a pre-clinical setting.

Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (64Cu, t1/2 = 12.7 h) and beta particle-emitting copper-67 (67Cu, t1/2 = 61.8 h). This strategy is predicated on the in vivo ligation between a trans-cyclooctene (TCO)-bearing antibody and a tetrazine (Tz)-based radioligand via the rapid and bioorthogonal inverse electron-demand Diels-Alder reaction. Longitudinal therapy studies were conducted in a murine model of human colorectal carcinoma using an immunoconjugate of the huA33 antibody modified with TCO (huA33-TCO) and a 67Cu-labeled Tz radioligand ([67Cu]Cu-MeCOSar-Tz). The injection of huA33-TCO followed 72 h later by the administration of 18.5, 37.0, or 55.5 MBq of [67Cu]Cu-MeCOSar-Tz produced a dose-dependent therapeutic response, with the median survival time increasing from 68 d for the lowest dose to >200 d for the highest. Furthermore, we observed that mice that received the highest dose of [67Cu]Cu-MeCOSar-Tz in a fractionated manner exhibited improved hematological values without sacrificing therapeutic efficacy. Dual radionuclide experiments in which a single administration of huA33-TCO was followed by separate injections of [64Cu]Cu-MeCOSar-Tz and [67Cu]Cu-MeCOSar-Tz revealed that the positron emission tomography images produced by the former accurately predicted the efficacy of the latter. In these experiments, a correlation was observed between the tumoral uptake of [64Cu]Cu-MeCOSar-Tz and the subsequent therapeutic response to [67Cu]Cu-MeCOSar-Tz.

Programmed cell death 1 ligand 1(PD-L1) is overexpressed in many tumors. The radionuclide-labeled anti-PD-L1 monoclonal antibody can be used for imaging and therapy of PD-L1 overexpressing cancer. Here, we described 131I-labeled Atezolizumab (131I-Atezolizumab, targeting PD-L1) as a therapeutic agent for colorectal cancer with PD-L1 overexpression.

Ionizing radiation induces direct and indirect killing of cancer cells and for long has been considered as immunosuppressive. However, this concept has evolved over the past few years with the demonstration that irradiation can increase tumor immunogenicity and can actually favor the implementation of an immune response against tumor cells. Adoptive T-cell transfer (ACT) is also used to treat cancer and several studies have shown that the efficacy of this immunotherapy was enhanced when combined with radiation therapy. α-Radioimmunotherapy (α-RIT) is a type of internal radiotherapy which is currently under development to treat disseminated tumors. α-particles are indeed highly efficient to destroy small cluster of cancer cells with minimal impact on surrounding healthy tissues. We thus hypothesized that, in the setting of α-RIT, an immunotherapy like ACT, could benefit from the immune context induced by irradiation. Hence, we decided to further investigate the possibilities to promote an efficient and long-lasting anti-tumor response by combining α-RIT and ACT. To perform such study we set up a multiple myeloma murine model which express the tumor antigen CD138 and ovalbumine (OVA). Then we evaluated the therapeutic efficacy in the mice treated with α-RIT, using an anti-CD138 antibody coupled to bismuth-213, followed by an adoptive transfer of OVA-specific CD8+ T cells (OT-I CD8+ T cells). We observed a significant tumor growth control and an improved survival in the animals treated with the combined treatment. These results demonstrate the efficacy of combining α-RIT and ACT in the MM model we established.

The aim of the present study was to explore the biodistribution, normal tissue toxicity, and therapeutic efficacy of the internalizing low-dose rate alpha-particle-emitting radioimmunoconjugate 227Th-trastuzumab in mice with HER2-expressing breast cancer xenografts.

Glioblastoma multiforme (GBM) is the most common and lethal type of adult brain cancer. Current GBM standard of care, including radiotherapy, often ends up with cancer recurrence, resulting in limited long-term survival benefits for GBM patients. Immunotherapy, such as immune checkpoint blockade (ICB), has thus far shown limited clinical benefit for GBM patients. Therapeutic vaccines hold great potential to elicit anti-cancer adaptive immunity, which can be synergistically combined with ICB and radiotherapy. Peptide vaccines are attractive for their ease of manufacturing and stability, but their therapeutic efficacy has been limited due to poor vaccine co-delivery and the limited ability of monovalent antigen vaccines to prevent tumor immune evasion. To address these challenges, here, we report GBM radioimmunotherapy that combines radiotherapy, ICB, and multivalent lymph-node-targeting adjuvant/antigen-codelivering albumin-binding vaccines (AAco-AlbiVax). Specifically, to codeliver peptide neoantigens and adjuvant CpG to lymph nodes (LNs), we developed AAco-AlbiVax based on a Y-shaped DNA scaffold that was site-specifically conjugated with CpG, peptide neoantigens, and albumin-binding maleimide-modified Evans blue derivative (MEB). As a result, these vaccines elicited antitumor immunity including neoantigen-specific CD8+ T cell responses in mice. In orthotopic GBM mice, the combination of AAco-AlbiVax, ICB, and fractionated radiation enhanced GBM therapeutic efficacy. However, radioimmunotherapy only trended more efficacious over radiotherapy alone. Taken together, these studies underscore the great potential of radioimmunotherapy for GBM, and future optimization of treatment dosing and scheduling would improve the therapeutic efficacy.