Safety and Efficacy of 188-Rhenium-Labeled Antibody to Melanin in Patients with Metastatic Melanoma

There is a need for effective “broad spectrum” therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of 188Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi 188Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of 188Re-6D2 was escalated to 54 mCi. SPECT/CT revealed 188Re-6D2 uptake in melanoma metastases. The mean effective half-life of 188Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that 188Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.


Introduction
e incidence of melanoma is increasing worldwide, with a concomitant rise in mortality from metastatic disease. Patients who progress to stage IV metastatic melanoma (MM) have a median survival of less than 1 year [1]. In the United States, about 9,180 people will die from melanoma in 2012 (American Cancer Society, 2012). Until recently, treatment options for patients with stage IV disease were limited and offered marginal, if any, improvement in overall survival. is situation changed with the newly approved by FDA ipilimumab (anti-CTLA4 monoclonal antibody), an immunomodulator which in a phase III trial was shown to improve overall survival [2]. In addition, vemurafenib that inhibits mutated B-RAF protein offers hope for 40-60% melanoma patients carrying this mutation [3,4]. However, the responses to the latter have been relatively short lasting followed by recurrences.
In our search for alternative therapeutic options for MM we turned to radioimmunotherapy (RIT). RIT takes advantage of the speci�city of the antigen-antibody interaction to deliver cytotoxic radiation to tumors [5,6]. e clinical success of FDA-approved drugs such as ibritumomab tiuxetan (anti-CD20 monoclonal antibody (mAb) labeled with 90 Yttrium) for treatment of primary, relapsed or refractory B-cell non-Hodgkin lymphoma demonstrates RIT potential as antineoplastic strategy. Unlike other therapies, RIT does not rely on speci�c genotypes, biochemical pathways, or the variability of an individual's immune response and thus can be offered to broad patient populations and is not a subject to multidrug resistance mechanisms which are already limiting the efficacy of vemurafenib.
Melanoma owes its name to melanin pigment with even "amelanotic" melanomas containing some melanin [7,8]. Historically, melanin was not considered a target for RIT because of its intracellular location in melanosomes beyond the reach of melanin-speci�c mAbs. However, in rapidly growing melanoma tumors cell necrosis releases melanin into the extracellular space where it can be targeted for delivery of radiation by radiolabeled melanin-binding mAbs. Several mAbs to fungal melanin were generated in our laboratories [9]. We established the feasibility of targeting melanin in melanoma xenogras with melanin-binding mAb 6D2 IgM labeled with beta-emitting radionuclide 188 Re ( max = 2.1 MeV, half-life 17.0 hrs) [10]. In spite of their fast clearance from the circulation which should result in favorable target to nontarget ratios, IgMs are oen overlooked in radioimmunoimaging and RIT. Importantly, experiments with melanin-binding mAbs conducted in C57Bl6 black mice demonstrated that melanin in normal pigmented tissues such as retina of the eye, pigmented skin, or hair follicles is not accessible to the mAb by virtue of its intracellular location [10]. Preclinical development of 188 Re-6D2 resulted in developing cGMP-compatible radiolabeling methodology while computer-simulated tumor dosimetry demonstrated that 188 Re-6D2 could deliver tumoricidal doses to tumors within the wide range of melanin concentrations (up to 100 less melanin than in primary tumors) [11,12]. Here we report the results of the recently completed consecutive Phase Ia and Ib trials of 188 Re-6D2 mAb in patients with MM.

Patients Eligibility and Screening.
Patients were eligible for enrollment if they had histologically or clinically con�rmed stage III (unresectable) or stage IV metastatic melanoma. e studies were conducted in accordance with International Conference on Harmonization guidelines and Good Clinical Practice guidelines. e studies were registered at clinicaltrials.gov and were numbered NCT00399113 and NCT00734188. e study protocols were approved by the Ethics Committees at Hadassah Medical Center, Jerusalem and Sheba Medical Center, Tel Hashomer, Israel where the study was conducted. All patients signed a written informed consent prior to participating in the study. Men and women (not pregnant or lactating, and following acceptable methods of birth control) were to be 18 years or older and have a life expectancy of at least 3 months. At least 4 weeks had to have elapsed since prior chemotherapy or radiation therapy and at least 1 week since IL-2 therapy. Patients had to have failed response to at least one previous therapy; adequate organ and marrow function and a negative human anti-mouse antibody (HAMA) result; no cerebral metastases by MRI or CT; no ocular diseases that may have led to an impaired blood-retinal brain barrier and no prior parenteral exposure to murine proteins.
2.2. Antibody, Radioisotope, and Radiolabeling. MAb 6D2, a murine IgM generated against fungal melanin, was previously described in references [8][9][10][11][12]. e clinical lot of 6D2 and all reagents used for manufacturing were produced in compliance with cGMPs by Goodwin Biotechnology Inc. (Plantation, FL). 188 Re was obtained from a 188 W/ 188 Re generator (Oak Ridge National Laboratory, Oak Ridge, TN, USA). e radiolabeling of 6D2 with 188 Re was performed as in [11]. e speci�c activity of 188 Re-6D2 in both Phase I studies was kept approximately at 1 mCi/mg to preserve 6D2 immunoreactivity towards melanin as was demonstrated in reference [11]. e highest level of activity achievable with 188 W/ 188 Re generators used in the study was 60 mCi/50 mg.

Phase I Study Objectives and Design. Both open-label
Phase I studies had the following objectives: (1) characterization of pharmacokinetics and dosimetry to normal organs; (2) identi�cation of the dose-limiting organs; (3) evaluation of the HAMA response; (4) determination of safety and tolerability; (5) evaluation of tumor localization and antitumor activity of 188 Re-6D2. e �rst Phase I study (phase Ia) had an additional objective of determining the effect of preload with "cold" (unlabeled) mAb on the biodistribution and pharmacokinetics of 188 Re-6D2 while the second phase I (phase Ib) study evaluated possible toxicities associated with increasing radioactive doses of 188 Re-6D2. Phase Ia enrolled 13 patients into 4 cohorts with each cohort receiving 10 mCi/10 mg 188 Re-6D2 preceded by either 0, 10, 20, or 50 mg of unlabeled 6D2 depending upon cohort. Seven patients in the Phase Ib were enrolled into 2 cohorts: 20-30 mCi and 40-60 mCi. Patients returned for posttreatment followup at 2 and 6 weeks aer infusion (Phase Ia and Ib) and every 8 weeks thereaer until disease progression (Phase Ib).

Pharmacokinetics, Imaging, and Dosimetry for the Normal
Organs. MAb pharmacokinetics was determined from blood samples taken at speci�ed time intervals from predose to 48 h aer 188 Re-6D2 administration. Biodistribution of 188 Re-6D2 was evaluated by whole body planar imaging. SPECT/CT scans were performed for the regions of interest aer each planar imaging session as necessary. e percentages injected dose for the organs and whole body were calculated from regions of interest and �tted to an exponential kinetic model within the dosimetry code Organ Level Internal Dose Assessment (OLINDA) [13,14].

Tumor Response Assessment.
Tumor response (based upon evaluation of target, nontarget, and emergence of new lesions) was assessed utilizing RECIST version 1.0. Cutaneous melanoma lesions were measured and the longest diameter recorded in millimeters. Noncutaneous lesions were identi�ed by radiologic assessment and measured. Tumors were recorded at baseline and tumor response was monitored at 2 and 6 weeks aer infusion. In the Phase Ib, tumors In phase Ia all 13 patients received 10 mCi 188 Re-6D2 and an infusion of unlabeled 6D2 depending upon the cohort entered. ree patients entered cohort 1 and did not receive an infusion of unlabeled 6D2, 4 patients (cohort 2) received 10 mg 6D2, 3 patients (cohort 3) received 20 mg, and 3 patients (cohort 4)-50 mg. Two patients in cohort I received a second 10 mCi dose of 188 Re-6D2. In phase Ib, 4 patients (cohort 1) received 20-30 mCi 188 Re-6D2 and 3 patients (cohort 2)-41-54 mCi 188 Re-6D2.

Tumor Imaging and Absence of 188 Re-6D2 Localization in
Normal Melanized Tissues. Whole body planar scintigraphy showed no uptake of 188 Re-6D2 in the normal melanized tissues like the retina of the eye, skin, and melanized neurons in brain (Figure 1(a)). Tumor targeting was visible on both whole-body planar scintigraphy and SPECT/CT. SPECT/CT of 188 Re-6D2 demonstrated targeting of various lesions: mediastinal and lung (Figure 1(b)), pelvic (Figure 1(c)), cutaneous, muscular, and nodal metastases. e highest uptake in the tumors was observed at 2 and 8 h aer injection, with the tumor still visible 24 h aer injection (Figure 1(b)). 18 FDG Uptake aer Treatment. 18 FDG PET/CT scans were performed prior to treatment and between 2 and 6 weeks aer treatment for each patient. e maximum and the mean standardized uptake values (SUV max and SUV mean , resp.) were summed at baseline and at the last aer treatment assessment to determine the percentage change from baseline. e results are shown in Figure 2. SUV max was calculated for all 20 patients, whereas SUV mean was only calculated for 12 patients. e results from these analyses are in concordance with the changes seen in target lesions aer treatment per-RECIST. Based upon the SUV max percent change from baseline, 15 patients experienced stable metabolic disease, 2 patients had progressive metabolic disease, and 3 patients experienced a partial metabolic response. irteen patients had stable metabolic disease. One patient from phase Ia and one from phase Ib experienced stable metabolic disease or partial metabolic responses in 100% of identi�ed lesions. A complete metabolic response was observed in a single lesion in two additional patients, one from each study.  Table 2. e radiation doses of 188 Re-6D2 (up to 54 mCi) administered in these two studies were determined to be safe when compared to the tolerance of normal tissue to therapeutic irradiation causing 50% complications in 5 years [15]. e kidneys and bone marrow had the highest radiation absorbed doses relative to MTDs for normal organs. At the highest doses administered in phase 1b, the kidneys absorbed 13.2±1.9% of the maximum tolerated dose and the bone marrow absorbed 8.5 ± 0.9%. ese are the potential dose limiting organs. Calculation of the total dose which could be delivered to normal organs by 100 mCi 188 Re-6D2 demonstrated that doses to the critical organs would be 5 times below TD 50/5 and compare favorably to those from ibritumomab tiuxetan and tositumomab (Table 3). 188 Re-6D2 Administration and Survival. Although the efficacy was not the primary objective of these studies, antitumor activity of 188 Re-6D2 was evaluated within the con�nes of a phase I study (Supplementary Table  S2). e 10 mCi 188 Re-6D2 in Phase Ia was not considered a therapeutic dose but was sufficient for imaging and pharmacokinetic analysis. Aer receiving 10 mCi, 9 patients demonstrated stable disease in target lesions at the conclusion of the 6-week followup period. At the conclusion of the 6week followup period 3 patients met the RECIST criteria of stable disease and 8 patients had progressive disease. is is an encouraging observation as all patients had previously failed at least two standard therapies. During Phase Ib 4 patients met the criteria for stable disease through week 6. During continued followup one patient had an overall response of stable disease through followup week 22. Two patients continued to have an overall response of stable disease through week 14. Supplementary Figure S1 represents the data for the best percent change from baseline for the sum of the longest diameters (SLD) for the target lesions from each  patient. e target lesions for 15 patients were considered stable disease by RECIST, 2 patients had partial responses in their target lesions, and 2 patients had progressive disease. e nontarget lesions in 8 patients showed progression and 9 patients developed new lesions (5 patients had both new lesions and nontarget progression). Figure 3(a) shows 18 FDG PET/CT of a patient with massive lung and pleural involvement 10 days before the administration of 188 Re-6D2 mAb and 7 days aer the 10 mCi dose. ere was signi�cantly decreased uptake in the tumor 7 days aer 188 Re-6D2 mAb administration corresponding most likely to increased tumor necrosis seen on CT. e tumor necrosis could have been the result of radiation, considering the high uptake of the 188 Re-6D2 mAb in the lung mets, or a re�ection of the natural history of the disease. Figure 3(b) shows the tumor in a patient who received 30 mCi/16 mg 188 Re-6D2 mAb and whose tumor was stable at 24 weeks aer treatment. At the time of manuscript preparation, this patient was still alive with nonprogressive disease for 17 months. Figure 3(c) displays one tumor mass in the lung parenchyma of a patient with progressive disease who received 54 mCi/47 mg 188 Re-6D2 mAb.

Tumor Response to
A post hoc pooled analysis of overall survival (OS) performed in May 2011 established a median duration of OS at 13 months and mean OS-at 15.6 months. Ten out of 17 (59%) patients had an overall survival greater than 12 months, 3 patients (18%) greater than 24 months with one patient continued to be followed at month 42. Four patients still being followed as of May 2011 were at 22, 23, 25, and 42 weeks aer 188 Re-6D2 mAb administration.

Toxicity, Adverse Effects, and HAMA.
During the phase Ib no toxicities were observed for the administered doses including the maximum dose of 54 mCi 188 Re-6D2 manufactured during the study, suggesting that the doses of 188 Re-6D2 were well below MTD. All patients in both studies were negative for HAMA at baseline. Supplementary Table S3 displays the dynamics of the HAMA response in patients aer receiving 188 Re-6D2 mAb in both studies. In the phase 1a study, 5 of 13 patients developed a positive HAMA response at 2 weeks aer administration with this number decreasing to 3 patients at 6 weeks. ree patients who were HAMA positive at week 2 became negative by week 6. In the second trial, all 7 patients were negative for HAMA at 2 weeks aer

Discussion
Even in the era of B-RAF inhibition, there continues to be an enormous need for effective "broad spectrum" therapies for MM which would be suitable for all patients diagnosed with the disease. In 1981 DeNardo et al. reported a curative therapy of murine melanoma with 131 I-labeled mAbs against P-51 murine melanoma [16]. RIT of melanoma moved into the clinical trial in 1985 when 50% tumor reduction was observed in a patient treated with 131 I-labeled Fab' fragments of a mAb against high molecular weight melanoma-associated antigen [17]. Despite early successes during the 80s, RIT of melanoma did not develop into a clinical modality for a variety of reasons that included disappointing results in clinical trials of different solid tumors during that time. e availability of novel mAbs and radionuclides with optimal emission characteristics encouraged us to revisit the RIT for MM. We targeted melanin which is a novel antigen for radioimmunotherapy with an IgM mAb 6D2. IgMs are oen overlooked in RIT despite fast blood clearance, ability to trigger ADCC and CDC immune responses, and early encouraging data in patients [18]. ough the doses of melanin-binding mAb 188 Re-6D2 were limited by the activity of the 188 W/ 188 Re generators at the time of the study, the combined survival data for the two studies demonstrated that the median survival of patients receiving 188 Re-6D2 was approximately 13 months which is longer than the 8.5 months for the MM patients receiving standard care. Although this result must be interpreted cautiously, the �nding is encouraging. Importantly, no uptake in the healthy melanized tissues was observed which con�rmed our prior observations in the melanoma animal models [10]. e treatment was not accompanied by the severe toxic effects. On the contrary, toxicity was very mild with no hematological AEs observed even in patients receiving the highest doses of 188 Re-6D2. Several factors contribute to the nontoxic nature of 188 Re-6D2: (1) fast clearance of 188 Re-6D2 from the blood which prevents harmful irradiation of bone marrow; (2) relatively short physical half-life of 188 Re (17.0 hrs) in comparison with 2.8 and 8 days for 90 Y and 131 I used in ibritumomab tiuxetan and tositumomab, respectively; (3) 188 Re nonresidualizing nature which results in its fast excretion through the kidneys; (4) the absence of 6D2 cross-reactivity with normal tissues leading to very low uptake of 6D2 in nontarget tissues. In this regard, the dosimetry calculations showed that up to 100 mCi 188 Re-6D2 could be safely administered to the patients which should further improve the therapeutic results.
HAMA in the majority of patients was transient and not dependent on the unlabeled mAb dose. In this regard it is important to emphasize that the mAb used in ibritumomab tiuxetan and tositumomab for treatment of NHL is also murine. One can suggest that the radiolabeled murine mAb to melanin can be administered safely to melanoma patients for up to two doses. e work on the conversion of the murine mAb into the human-mouse chimera which would allow for multiple administrations of the radiolabeled mAb to MM patients is currently ongoing in our laboratories.
Our study has some limitations with major ones being relatively small number of patients which precludes statistical analyses of the data and not achieving the maximum tolerated dose for the 188 Re-6D2 due to the technical restrictions of the 188 W/ 188 Re generators available to us at the time of the trial. e high activity/high speci�c activity 188 W/ 188 Re generators have become available commercially aer the completion of this study which will ensure that higher doses could be administered to patients in the follow-up trials.
In conclusion, Phase I trials of 188 Re-6D2 mAb to melanin in patients with MM demonstrated tumor targeting and safety of the drug, as well as prolongation in survival. ese results are encouraging and suggest the need for further investigation of this reagent by itself or in combination with other therapies.

Con�ict of �nterests
e authors declare no con�ict of interests and no �nancial relationship with Goodwin Biotechnology.

Authors' Contribution
M. Klein and M. Lotem contributed equally to this paper.