Preclinical Combination Studies of an FGFR2 Targeted Thorium-227 Conjugate and the ATR Inhibitor BAY 1895344.

PURPOSE: Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle-emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344. METHODS AND MATERIALS: The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice. RESULTS: In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC-mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. CONCLUSIONS: The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.

Targeting angiogenesis for radioimmunotherapy with a 177Lu-labeled antibody.

PURPOSE: Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. METHODS: The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with 177Lu (t 1/2 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: 177Lu only, TRC105 only, 177Lu-DTPA-IgG (a nonspecific antibody), 177Lu-DTPA-TRC105 low-dose, and 177Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of 177Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose 177Lu-DTPA-TRC105. RESULTS: Biodistribution studies indicated steady uptake of 177Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p < 0.001, all groups). In most study groups (all except the nonspecific IgG group), the body weights of the mice did not decrease by more than 10%, indicating the safety of the injected agents. Serum alanine transaminase levels remained nearly constant indicating no damage to the liver (a primary clearance organ of the agent), and this was confirmed by ex vivo histological analyses. CONCLUSION: 177Lu-DTPA-TRC105, when administered at a sufficient dose, is able to curtail tumor growth and provide a significant survival benefit without off-target toxicity. Thus, this targeted agent could be used in combination with other treatment options to slow tumor growth allowing the other agents to be more effective.

Triple Therapy of HER2+ Cancer Using Radiolabeled Multifunctional Iron Oxide Nanoparticles and Alternating Magnetic Field.

By using radio-labeled multifunctional superparamagnetic iron oxide nanoparticles (SPIONs) and an alternating magnetic field (AMF), we carried out targeted hyperthermia, drug delivery, radio-immunotherapy (RIT), and controlled chemotherapy of cancer tumors. We synthesized and characterized Indium-111-labeled, Trastuzumab and Doxorubicin (DOX)-conjugated APTES-PEG-coated SPIONs in our previous work. Then, we evaluated their capability in SPECT/MRI (single photon emission computed tomography/magnetic resonance imaging) dual modal molecular imaging, targeting, and controlled release. In this research, AMF was introduced to evaluate therapeutic effects of magnetic hyperthermia on radionuclide-chemo therapy of HER2+ cells and tumor (HER2+)-bearing mice. In vitro and in vivo experiments using synthesized complex were repeated under an AMF (f: 100 KHz, H: 280 Gs). Instead of an intra-tumor injection in most hyperthermia experiments, SPIONs were injected to the tail vein, based on our delivery strategies. For magnetic delivery, we held a permanent Nd-B-Fe magnet near the tumor region. The results showed that simultaneous magnetic hyperthermia enhanced SKBR3 cancer cells, killing by 24%, 28%, 33%, and 80% at 48 hours post-treatment for treated cells with (1) bare SPIONs; (2) antibody-conjugated, DOX-free, surface-modified SPIONs; (3) 111In-labeled, antibody-conjugated surface-modified SPIONs; and (4) 111In-labeled, antibody- and DOX-conjugated surface-modified SPIONs, respectively. Moreover, tumor volume inhibitory rate was 85% after a 28 day period of treatment. By using this method, multimodal imaging-guided, targeted hyperthermia, RIT, and controlled chemotherapy could be achievable in the near future.

New insights into the pretargeting approach to image and treat tumours.

Tumour pretargeting is a promising strategy for cancer diagnosis and therapy allowing for the rational use of long circulating, highly specific monoclonal antibodies (mAbs) for both non-invasive cancer radioimmunodetection (RID) and radioimmunotherapy (RIT). In contrast to conventional RID/RIT where the radionuclides and oncotropic vector molecules are delivered as presynthesised radioimmunoconjugates, the pretargeting approach is a multistep procedure that temporarily separates targeting of certain tumour-associated antigens from delivery of diagnostic or therapeutic radionuclides. In principle, unlabelled, highly tumour antigen specific mAb conjugates are, in a first step, administered into a patient. After injection, sufficient time is allowed for blood circulation, accumulation at the tumour site and subsequent elimination of excess mAb conjugates from the body. The small fast-clearing radiolabelled effector molecules with a complementary functionality directed to the prelocalised mAb conjugates are then administered in a second step. Due to its fast pharmacokinetics, the small effector molecules reach the malignant tissue quickly and bind the local mAb conjugates. Thereby, corresponding radioimmunoconjugates are formed in vivo and, consequently, radiation doses are deposited mainly locally. This procedure results in a much higher tumour/non-tumour (T/NT) ratio and is favourable for cancer diagnosis and therapy as it substantially minimises the radiation damage to non-tumour cells of healthy tissues. The pretargeting approach utilises specific non-covalent interactions (e.g. strept(avidin)/biotin) or covalent bond formations (e.g. inverse electron demand Diels-Alder reaction) between the tumour bound antibody and radiolabelled small molecules. This tutorial review descriptively presents this complex strategy, addresses the historical as well as recent preclinical and clinical advances and discusses the advantages and disadvantages of different available variations.

Synthesis of Site-Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion.

Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope 64Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.

A pioneer experience in Malaysia on In-house Radio-labelling of (131)I-rituximab in the treatment of Non-Hodgkin's Lymphoma and a case report of high dose (131)I-rituximab-BEAM conditioning autologous transplant.

Radioimmunotherapy is an established treatment modality in Non-Hodgkin's lymphoma. The only two commercially available radioimmunotherapies - (90)Y-ibritumomab tiuxetan is expensive and (131)I-tositumomab has been discontinued from commercial production. In resource limited environment, self-labelling (131)I-rituximab might be the only viable practical option. We reported our pioneer experience in Malaysia on self-labelling (131)I-rituximab, substituting autologous haematopoietic stem cell transplantation (HSCT) and a patient, the first reported case, received high dose (131)I-rituximab (6000MBq/163mCi) combined with BEAM conditioning for autologous HSCT.

Radioimmunotherapy and Autologous Stem-Cell Transplantation in the Treatment of B-Cell Non-Hodgkin Lymphoma.

High-dose chemotherapy and autologous stem-cell transplantation (ASCT) is the standard therapy for patients with chemosensitive-relapsed or chemosensitive-refractory aggressive lymphoma. The use of rituximab, an anti-CD20 monoclonal antibody, has dramatically changed the outcome of patients with aggressive lymphoma, increasing both response and survival rates. However, despite this progress a significant proportion of patients are still refractory or relapse after frontline rituximab-containing therapy. Moreover, it is increasingly more difficult to rescue these patients with current salvage chemotherapy and ASCT approaches. Novel approaches are needed for these high-risk patients, especially in the rituximab era. Radioimmunotherapy (RIT) is a form of targeted therapy using the parent monoclonal antibody to deliver radiation emitted by a conjugated radioisotope, to the vicinity of antigen-positive tissues. Two radioimmunoconjugates--yttrium-90 ibritumomab tiuxetan (Zevalin) and iodine-131 tositumomab (Bexxar) have been in clinical use. There are multiple studies demonstrating the safety and efficacy of both agents in both indolent and aggressive lymphoma. Radiolabeled antibodies are ideal candidates to combine with high-dose chemotherapy and ASCT. RIT targets radiation to disease sites while limiting exposure of uninvolved critical organs, thus it can safely replace total-body irradiation during conditioning for ASCT. The major toxicity and limiting factor in RIT is myelotoxicity that is easily reversed by stem-cell rescue. RIT can be combined at standard doses with high-dose chemotherapy or can be given in escalated doses either alone or with high-dose chemotherapy before ASCT. Several phase II studies have shown the safety and potential efficacy of both agents using these approaches. A small randomized study comparing standard-dose Zevalin with combination of carmustine, etoposide, cytarabine, and melphalan (BEAM) high-dose chemotherapy and BEAM alone suggested a survival advantage of Zevalin-BEAM. However, a large randomized study comparing Bexxar-BEAM and rituximab-BEAM did not show any advantage. More studies are needed to establish the role and the dose of RIT given for ASCT.

Absorbed Doses and Risk Estimates of (211)At-MX35 F(ab')2 in Intraperitoneal Therapy of Ovarian Cancer Patients.

PURPOSE: Ovarian cancer is often diagnosed at an advanced stage with dissemination in the peritoneal cavity. Most patients achieve clinical remission after surgery and chemotherapy, but approximately 70% eventually experience recurrence, usually in the peritoneal cavity. To prevent recurrence, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation of (211)At in thyroid and other NIS-containing tissues. Mean absorbed doses to normal tissues were calculated from clinical data, including blood and i.p. fluid samples, urine, γ-camera images, and single-photon emission computed tomography/computed tomography images. Extrapolation of preclinical biodistribution data combined with clinical blood activity data allowed us to estimate absorbed doses in additional tissues. The equivalent dose was calculated using an RBE of 5 and the effective dose using the recommended weight factor of 20. All doses were normalized to the initial activity concentration of the infused therapy solution. RESULTS: The urinary bladder, thyroid, and kidneys (1.9, 1.8, and 1.7 mGy per MBq/L) received the 3 highest estimated absorbed doses. When the tissue-weighting factors were applied, the largest contributors to the effective dose were the lungs, stomach, and urinary bladder. Using 100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective dose potentially corresponds to a risk of treatment-induced carcinogenesis, optimization may still be valuable.

Preclinical testing of radiopharmaceuticals for novel applications in HIV, bacterial and fungal infectious diseases.

Antibiotics, antifungal and antiviral medications have traditionally been used in the management of infections. Due to widespread emergence of resistance to antimicrobial medications, and their side effects, there is a growing need for alternative approaches for management of such conditions. Antibiotic resistant bacterial pathogens are on the rise. A cure has not been achieved for viral infections like AIDS, while fungal and parasitic infections are constant threats to the health of general public. The incidence of opportunistic infections in immunocompromised individuals like HIV patients, patients receiving high dose steroids, chemotherapy patients, and organ transplant recipients is on the rise. Radioimmunotherapy (RIT) has the potential to be a suitable and viable therapeutic modality in the arena of infection management. Provided the target-associated antigen is expressed by the target cells and minimally or not expressed by other tissues, selective targeting of radiation to target sites can be theoretically accomplished with relative sparing normal tissues from radiation exposure. In our laboratory we successfully demonstrated the effectiveness of RIT for treating infectious diseases. We targeted murine cryptococcosis with a mAb to the Cryptococcus neoformans capsular glucuronoxylomannan labeled with Bismuth-213 ((213)Bi) or Rhenium-188 ((188)Re). We subsequently extended the applicability of RIT for treating bacterial and viral infections. One of the advantages of using RIT to treat infections as opposed to cancer is that, in contrast to tumor cells, cells expressing microbial antigens are antigenically very different from host tissues and thus provide the potential for exquisite specificity and low cross-reactivity. Ever increasing incidence of infectious pathologies, exhaustion of antimicrobial possibilities and rising drug resistance calls for use of alternative and novel therapeutic options and we believe RIT is the need of the hour to combat these infections.

Radioimmunoconjugates for the treatment of cancer.

Radioimmunotherapy (RIT) has been developed for more than 30 years. Two products targeting the CD20 antigen are approved in the treatment of non-Hodgkin B-cell lymphoma (NHBL): iodine 131-tositumomab and yttrium 90-ibritumomab tiuxetan. RIT can be integrated in clinical practice for the treatment of patients with relapsed or refractory follicular lymphoma (FL) or as consolidation after induction chemotherapy. High-dose treatment, RIT in first-line treatment, fractionated RIT, and use of new humanized monoclonal antibodies (MAbs), in particular targeting CD22, showed promising results in NHBL. In other hemopathies, such as multiple myeloma, efficacy has been demonstrated in preclinical studies. In solid tumors, more resistant to radiation and less accessible to large molecules such as MAbs, clinical efficacy remains limited. However, pretargeting methods have shown clinical efficacy. Finally, new beta emitters such as lutetium 177, with better physical properties will further improve the safety of RIT and alpha emitters, such as bismuth 213 or astatine 211, offer the theoretical possibility to eradicate the last microscopic clusters of tumor cells, in the consolidation setting. Personalized treatments, based on quantitative positron emission tomography (PET), pre-therapeutic imaging, and dosimetry procedures, also could be applied to adapt injected activity to each patient.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

Myeloablative anti-CD20 radioimmunotherapy +/- high-dose chemotherapy followed by autologous stem cell support for relapsed/refractory B-cell lymphoma results in excellent long-term survival.

BACKGROUND: Radioimmunotherapy (RIT) has been used to treat relapsed/refractory CD20+ Non-Hodgkin lymphoma (NHL). Myeloablative anti-CD20 RIT followed by autologous stem cell infusion (ASCT) enables high radiation doses to lymphoma sites. We performed a phase I/II trial to assess feasibility and survival. METHODS: Twenty-three patients with relapsed/refractory NHL without complete remission (CR) to salvage chemotherapy were enrolled to evaluate RIT with Iodine-131 labelled rituximab (131I-rituximab) in a myeloablative setting. Biodistribution and dosimetric studies were performed to determine 131I activity required to induce a total body dose of 21-27Gy to critical organs. In 6/23 patients RIT was combined with high-dose chemotherapy. 8/23 patients received a sequential high-dose chemotherapy with a second ASCT. The median follow-up is 9.5 years. RESULTS: 6.956-19.425GBq of 131I was delivered to achieve the limiting organ dose to lungs or kidneys. No grade III/IV non-hematologic toxicity was seen with RIT alone. Significant grade III/IV toxicity (mucositis, fever, infection, one therapy related death) was observed in patients treated with RIT combined with high-dose chemotherapy. The overall response rate was 87% (64% CR). The median progression-free (PFS) and overall survival (OS) is 47.5 and 101.5 months. An international prognostic index score >1 was predictive for OS. CONCLUSION: Myeloablative RIT with 131I-rituximab followed by ASCT is feasible, well-tolerated and effective in high risk CD20+ NHL. Combination of RIT and high-dose chemotherapy increased toxicity significantly. Long-term results for PFS and OS are encouraging.

Rhenium-188: availability from the (188)W/(188)Re generator and status of current applications.

Rhenium-188 is one of the most readily available generator derived and useful radionuclides for therapy emitting ß(-) particles (2.12 MeV, 71.1% and 1.965 MeV, 25.6%) and imageable gammas (155 keV, 15.1%). The (188)W/(188)Re generator is an ideal source for the long term (4-6 months) continuous availability of no carrier added (nca) (188)Re suitable for the preparation of radiopharmaceuticals for radionuclide therapy. The challenges associated with the double neutron capture route of production of the parent (188)W radionuclide have been a major impediment in the progress of application of (188)Re. Tungsten-188 of adequate specific activity can be prepared only in 2-3 of the high flux reactors operating in the World. Several useful technologies have been developed for the preparation of clinical grade (188)W/(188)Re generators. Since the specific activity of (188)W used in the generator is relatively low 185 GBq( < 5 Ci)/g], the eluted (188)ReO(4)(-) can have low radioactive concentration often insufficient for radiopharmaceutical preparation. However, several efficient post elution concentration techniques have been developed that yield clinically useful (188)ReO(4)(-) solutions. Rhenium-188 has been used for the preparation of therapeutic radiopharmaceuticals for the management of diseases such as bone metastasis, rheumatoid arthritis and primary cancers. Several early phase clinical studies using radiopharmaceuticals based on (188)Re-labeled phosphonates, antibodies, peptides, lipiodol and particulates have been reported. This article reviews the availability and use of (188)Re including a discussion of why broader use of (188)Re has not progressed as expected as a popular radionuclide for therapy.

Development of 177Lu-nanobodies for radioimmunotherapy of HER2-positive breast cancer: evaluation of different bifunctional chelators.

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor-to-background-ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2-overexpressing minimal residual disease. The therapeutic radionuclide Lutetium-177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators - p-SCN-Bn-DOTA, DOTA-NHS-ester, CHX-A"-DTPA or 1B4M-DTPA - in order to select the optimal chemical link between Lutetium-177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator-conjugation. High stability in time was observed, together with nanomolar affinities on HER2-expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro-CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M-DTPA-coupled conjugate was further evaluated in a high HER2-expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor-to-background ratios. No specific binding was observed in a HER2-negative model. In conclusion, the in-house developed anti-HER2 nanobody 2Rs15dHIS can be successfully labeled with (177) Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M-DTPA-based conjugate.

Pretargeted radioimmunotherapy with α-particle emitting radionuclides.

Alpha-particle emitting radionuclides are attractive for targeted cancer therapies due to their physicochemical properties. Their high linear energy transfer (LET) and short particle range makes them particularly toxic at a microscopic level, which is ideal for treating disseminated micrometastases. However, their cytotoxic properties also place special demands on the pharmacokinetics of the tumor specific carrier vector, where high tumor-to-normal-tissue ratios are a prerequisite. Tumor specific antibodies are perhaps the most common vector for targeted therapy, but due to pharmacokinetics considerations antibodies will generally not meet the standard for α-particle radioimmunotherapy. However, the tumor specificity of monoclonal antibodies may be used in pretargeting techniques, strategies used to increase the selectivity of the radioactivity. The basic concept of pretargeting relies on a separate administration of a modified antibody and a radioactive ligand. The modified antibody is first injected and allowed to localize on the tumor. Then, the radiolabeled ligand is injected, which is a small molecule that rapidly localizes the modified antibody on tumor cells while non-localized ligand rapidly clears from the circulation, preferably through renal filtration. Several pretargeting strategies have been developed, in particular the avidin-biotin system and bispecific antibodies. Approaches under evaluation are the use of complementary DNA, morpholinos, and the use of infinite antigen binding. Preclinical and clinical studies of pretargeting have shown that favorable distribution of the radioactivity can be achieved, which may increase dose to the tumor as compared with the dose from directly labeled antibodies, and most important decrease the dose to normal tissues. This survey describes different pretargeting strategies, and includes a review of pretargeting with α emitting radionuclides.

(124)I-huA33 antibody PET of colorectal cancer.

UNLABELLED: Humanized A33 (huA33) is a promising monoclonal antibody that recognizes A33 antigen, which is present in more than 95% of colorectal cancers and in normal bowel. In this study, we took advantage of quantitative PET to evaluate (124)I huA33 targeting, biodistribution, and safety in patients with colorectal cancer. We also determined the biodistribution of (124)I-huA33 when a large dose of human intravenous IgG (IVIG) was administered to manipulate the Fc receptor or when (124)I-huA33 was given via hepatic arterial infusion (HAI). METHODS: We studied 25 patients with primary or metastatic colorectal cancer; 19 patients had surgical exploration or resection. Patients received a median of 343 MBq (44.4-396 MBq) and 10 mg of (124)I-huA33. Nineteen patients received the antibody intravenously and 6 patients via HAI, and 5 patients also received IVIG. RESULTS: Ten of 12 primary tumors were visualized in 11 patients. The median concentration in primary colon tumors was 0.016% injected dose per gram, compared with 0.004% in normal colon. The PET-based median ratio of hepatic tumor uptake to normal-liver uptake was 3.9 (range, 1.8-22.2). Quantitation using PET, compared with well counting of serum and tissue, showed little difference. Prominent uptake in bowel hindered tumor identification in some patients. Pharmacokinetics showed that patients receiving IVIG had a significantly shorter serum half-time (41.6 ± 14.0 h) than those without (65.2 ± 9.8 h). There were no differences in clearance rates among the intravenous group, IVIG group, and HAI group, nor was there any difference in serum area under the curve, maximum serum concentration, or volume of distribution. Weak titers of human-antihuman antibodies were observed in 6 of 25 patients. No acute side effects or significant toxicities were associated with huA33. CONCLUSION: Good localization of (124)I-huA33 in colorectal cancer with no significant toxicity has been observed. PET-derived (124)I concentrations agreed well with those obtained by well counting of surgically resected tissue and blood, confirming the quantitative accuracy of (124)I-huA33 PET. The HAI route had no advantage over the intravenous route. No clinically significant changes in blood clearance were induced by IVIG.

Disease control and ototoxicity using intensity-modulated radiation therapy tumor-bed boost for medulloblastoma.

PURPOSE: We previously reported excellent local control for treating medulloblastoma with a limited boost to the tumor bed. In order to decrease ototoxicity, we subsequently implemented a tumor-bed boost using intensity-modulated radiation therapy (IMRT), the clinical results of which we report here. PATIENTS AND METHODS: A total of 33 patients with newly diagnosed medulloblastoma, 25 with standard risk, and 8 with high risk, were treated on an IMRT tumor-bed boost following craniospinal irradiation (CSI). Six standard-risk patients were treated with an institutional protocol with 18 Gy CSI in conjunction with intrathecal iodine-131-labeled monoclonal antibody. The majority of patients received concurrent vincristine and standard adjuvant chemotherapy. Pure-tone audiograms were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. RESULTS: Median age was 9 years old (range, 4-46 years old). Median follow-up was 63 months. Kaplan-Meier estimates of progression-free survival (PFS) and overall survival (OS) rates for standard-risk patients who received 23.4 or 36 Gy CSI (not including those who received 18 Gy CSI with radioimmunotherapy) were 81.4% and 88.4%, respectively, at 5 years; 5-year PFS and OS rates for high-risk patients were both 87.5%. There were no isolated posterior fossa failures outside of the boost volume. Posttreatment audiograms were available for 31 patients, of whom 6%, at a median follow-up of 19 months, had developed Grade 3 hearing loss. CONCLUSION: An IMRT tumor-bed boost results in excellent local control while delivering a low mean dose to the cochlea, resulting in a low rate of ototoxicity.

Tandem high-dose therapy in relapsed and refractory B-cell lymphoma: results of a prospective phase II trial of myeloablative chemotherapy, followed by escalated radioimmunotherapy with (131)I-anti-CD20 antibody and stem cell rescue.

A phase II trial evaluated safety, feasibility and efficacy of a sequential tandem approach combining myeloablative BEAM chemotherapy and autologous stem cell transplantation (ASCT) with myeloablative radioimmunotherapy (HD-RIT), with (131)I-anti-CD20 antibody ((131)I-rituximab), followed by a second ASCT in patients with relapsed or refractory CD20+ B-cell lymphoma. According to protocol, 16 patients with relapsed (n = 14) and refractory (n = 2) CD20+ B-cell lymphoma received salvage therapy with rituximab and Dexa-BEAM, followed by BEAM (HD chemotherapy) and high-dose myeloablative radioimmunotherapy 2-6 months after BEAM. Nine of 16 patients received HD-RIT; seven patients were excluded before HD-RIT because of toxicity or progressive disease. Disease histologies were follicular lymphoma (FL) grades 1 and 2 (n = 4), transformed follicular (FL 3b; n = 6), diffuse large B-cell (DLBCL; n = 4), mantle cell (n = 1) and marginal zone lymphoma (n = 1). After a median follow-up of 50.4 months for OS and 39.7 months for progression-free survival (PFS), estimated 4-year OS and PFS were 67% and 64%, respectively. The estimated 4-year OS and PFS for patients with FL were 80% and 78%, respectively. Toxicity was significant, including one fatal outcome due to pneumonitis. Tandem transplants consisting of HD chemotherapy followed by HD-RIT with (131)I-coupled anti-CD20 are manageable and effective but toxic treatment modalities for relapsed poor prognosis CD20+ B-NHL.

177Lu labeling of Herceptin and preclinical validation as a new radiopharmaceutical for radioimmunotherapy of breast cancer.

INTRODUCTION: In the present study, Herceptin was labeled with lutetium-177 via DOTA, and the necessary preclinical quality control tests (in vitro and in vivo) were performed to evaluate its use as a radioimmunotherapy agent. MATERIAL AND METHODS: Herceptin was conjugated to DOTA as a chelator in three different conjugation buffers (ammonium acetate, carbonate and HEPES buffer); each of the resulting conjugates was compared with respect to in vitro characteristics such as number of chelates per antibody, incorporated activity, immunoreactivity and in vitro stability in PBS buffer and blood serum. The biodistribution study and gamma camera imaging were performed in mice bearing breast tumors. To assess the therapeutic effects of (177)Lu-Herceptin, cytotoxicity was investigated for 7 days in a SKBr3 breast cancer cell line. RESULTS: Carbonate buffer was the best conjugation buffer (number of chelates per antibody: 6; incorporated activity: 81%; immunoreactivity: 87%; buffer stability: 86%; serum stability: 81%, after 4 days). The efficient tumor uptake observed in the biodistribution studies was consistent with the gamma camera image results. At a concentration of 4 µg ml(-1), (177)Lu-Herceptin (surviving cells: 5 ± 0.6% of the total cells) of the total cells corresponded to an approximately eightfold increase in cytotoxicity in comparison to unmodified Herceptin (surviving cells: 43 ± 3.9%). CONCLUSION: The new complex described herein could be considered for further evaluation in animals and potentially in humans as a radiopharmaceutical for use in the radioimmunotherapy of breast cancer. These results may be important for patients who cannot tolerate the therapeutic dosage of Herceptin currently used because of heart problems.

A Phase II study of anti-epidermal growth factor receptor radioimmunotherapy in the treatment of glioblastoma multiforme.

OBJECT: This single-institution Phase II study tests the efficacy of adjuvant radioimmunotherapy with (125)I-labeled anti-epidermal growth factor receptor 425 murine monoclonal antibody ((125)I-mAb 425) in patients with newly diagnosed glioblastoma multiforme (GBM). METHODS: A total of 192 patients with GBM were treated with (125)I-mAb 425 over a course of 3 weekly intravenous injections of 1.8 GBq following surgery and radiation therapy. The primary end point was overall survival, and the secondary end point was toxicity. Additional subgroup analyses were performed comparing treatment with (125)I-mAb 425 (RIT, 132 patients), (125)I-mAb 425 and temozolomide (TMZ+RIT, 60 patients), and a historical control group (CTL, 81 patients). RESULTS: The median age was 53 years (range 19-78 years), and the median Karnofsky Performance Scale score was 80 (range 60-100). The percentage of patients who underwent debulking surgery was 77.6% and that of those receiving temozolomide was 31.3%. The overall median survival was 15.7 months (95% CI 13.6-17.8 months). The 1- and 2-year survivals were 62.5 and 25.5%, respectively. For subgroups RIT and TMZ+RIT, the median survivals were 14.5 and 20.2 months, respectively. No Grade 3 or 4 toxicity was seen with the administration of (125)I-mAb 425. The CTL patients lacked Karnofsky Performance Scale scores, had poorer survival, were older, and were less likely to receive radiation therapy. On multivariate analysis, the hazard ratios for RIT versus CTL, TMZ+RIT versus CTL, and TMZ+RIT versus RIT were 0.49 (p < 0.001), 0.30 (p < 0.001), and 0.62 (p = 0.008), respectively. CONCLUSIONS: In this large Phase II study of 192 patients with GBM treated with anti-epidermal growth factor receptor (125)I-mAb 425 radioimmunotherapy, survival was 15.7 months, and treatment was safe and well tolerated.