Dosimetry of [212Pb]VMT01, a MC1R-Targeted Alpha Therapeutic Compound, and Effect of Free 208Tl on Tissue Absorbed Doses

[212Pb]VMT01 is a melanocortin 1 receptor (MC1R) targeted theranostic ligand in clinical development for alpha particle therapy for melanoma. 212Pb has an elementally matched gamma-emitting isotope 203Pb; thus, [203Pb]VMT01 can be used as an imaging surrogate for [212Pb]VMT01. [212Pb]VMT01 human serum stability studies have demonstrated retention of the 212Bi daughter within the chelator following beta emission of parent 212Pb. However, the subsequent alpha emission from the decay of 212Bi into 208Tl results in the generation of free 208Tl. Due to the 10.64-hour half-life of 212Pb, accumulation of free 208Tl in the injectate will occur. The goal of this work is to estimate the human dosimetry for [212Pb]VMT01 and the impact of free 208Tl in the injectate on human tissue absorbed doses. Human [212Pb]VMT01 tissue absorbed doses were estimated from murine [203Pb]VMT01 biodistribution data, and human biodistribution values for 201Tl chloride (a cardiac imaging agent) from published data were used to estimate the dosimetry of free 208Tl. Results indicate that the dose-limiting tissues for [212Pb]VMT01 are the red marrow and the kidneys, with estimated absorbed doses of 1.06 and 8.27 mGyRBE = 5/MBq. The estimated percent increase in absorbed doses from free 208Tl in the injectate is 0.03% and 0.09% to the red marrow and the kidneys, respectively. Absorbed doses from free 208Tl result in a percent increase of no more than 1.2% over [212Pb]VMT01 in any organ or tissue. This latter finding indicates that free 208Tl in the [212Pb]VMT01 injectate will not substantially impact estimated tissue absorbed doses in humans.


Introduction
Melanocortin 1 receptor (MC1R) is a G protein-coupled receptor that is expressed in melanocytes and is implicated in melanogenesis [1]. MC1R is overexpressed on many mouse and human melanoma cells [2,3]. Positron emission tomography (PET) imaging of an MC1R-targeted peptide 68 Ga-DOTA-GGNle-CycNSH hex in melanoma patients has established clinical proof-of-concept of MC1R as a target for imaging and therapy [4].
Targeted alpha-particle therapy (TAT) is a promising therapeutic strategy that is unique in its ability to deliver cytotoxicity circumventing cellular resistance [5] and has demonstrated significant responses in early clinical trials [6][7][8]. High linear energy transfer (LET) alpha emissions result in clustered DNA double strand breaks [9][10][11][12][13][14][15][16]. In cell culture, alpha emitters have been shown to be more effective in inducing cell death than gamma radiation [17]. Due to short tissue ranges (<100 µm in water, <40 µm in bone), it had previously been believed that TAT may be best suited for the treatment of micrometastases and other disseminated tumors. However, recent TAT studies have demonstrated efficacy in large tumors and there is a growing body of evidence that TAT can activate the immune system and impart both bystander and abscopal effects [18,19]. In the clinical setting, TAT has demonstrated patient benefit even in subjects refractive to beta particle therapy [6].
[ 203 Pb]VMT01 is an MC1R-targeted TAT ligand in clinical development (NCT04904120) with elementally matched gamma-emitting [ 203 Pb]VMT01 that can be used as an imaging surrogate. [ 212 Pb]VMT01 human serum stability and in vivo mouse biodistribution experiments demonstrate robust retention of the 212 Bi daughter within the chelator following beta emission of parent 212 Pb and no evidence of in vivo translocation (Li and collaborators, SNMMI-ACNM Mid-Winter Meeting 2022 Abstract) [20]. In addition, the decay physics for 212 Pb [21,22] (Figure 1) dictates that retention of 212 Bi within the chelator will subsequently lead to alpha decay via the 212 Po or 208 Tl branches at the site of localization due to the short half-lives of 212 Po (0.3 µs) and 208 Tl (3.05 m). Due to recoil energy, the 36% alpha emission from 212 Bi via the 208 Tl branch will result in the accumulation of free 208 Tl in the administered injectate. Here, we calculated [ 212 Pb]VMT01 human tissue absorbed doses from murine [ 203 Pb]VMT01 biodistribution data and the activity and effect of free 208 Tl in the injectate on tissue absorbed doses. demonstrated significant responses in early clinical trials [6][7][8]. High linear energy transfer (LET) alpha emissions result in clustered DNA double strand breaks [9][10][11][12][13][14][15][16]. In cell culture, alpha emitters have been shown to be more effective in inducing cell death than gamma radiation [17]. Due to short tissue ranges (<100 µm in water, <40 µm in bone), it had previously been believed that TAT may be best suited for the treatment of micrometastases and other disseminated tumors. However, recent TAT studies have demonstrated efficacy in large tumors and there is a growing body of evidence that TAT can activate the immune system and impart both bystander and abscopal effects [18,19]. In the clinical setting, TAT has demonstrated patient benefit even in subjects refractive to beta particle therapy [6].
[  [20]. In addition, the decay physics for 212 Pb [21,22] (Figure 1) dictates that retention of 212 Bi within the chelator will subsequently lead to alpha decay via the 212 Po or 208 Tl branches at the site of localization due to the short half-lives of 212 Po (0.3 µs) and 208 Tl (3.05 m). Due to recoil energy, the 36% alpha emission from 212 Bi via the 208 Tl branch will result in the accumulation of free 208 Tl in the administered injectate. Here, we calculated [ 212 Pb]VMT01 human tissue absorbed doses from murine [ 203 Pb]VMT01 biodistribution data and the activity and effect of free 208 Tl in the injectate on tissue absorbed doses.

Murine [ 203 Pb]VMT01 Biodistribution
Murine biodistribution results following intravenous administration of [ 203 Pb]VMT01 in female and male CD-1 IGS naïve mice are provided in Supplemental Table S1 and S2 (supplementary material). [ 203 Pb]VMT01 cleared rapidly through the kidneys with an accumulation of 6.24 ± 0.35%ID/g and 8.30 ± 1.90% ID/g in females and males, respectively at 0.5 h. Kidney activity decreased to 1.09 ± 0.12% ID/g and 0.55 ± 0.10% ID/g in females and males, respectively at 55 h. Accumulation and retention in other organs were minimal.

Murine [ 203 Pb]VMT01 Biodistribution
Murine biodistribution results following intravenous administration of [ 203 Pb]VMT01 in female and male CD-1 IGS naïve mice are provided in Supplemental Tables S1 and S2 (Supplementary material). [ 203 Pb]VMT01 cleared rapidly through the kidneys with an accumulation of 6.24 ± 0.35% ID/g and 8.30 ± 1.90% ID/g in females and males, respectively at 0.5 h. Kidney activity decreased to 1.09 ± 0.12% ID/g and 0.55 ± 0.10% ID/g in females and males, respectively at 55 h. Accumulation and retention in other organs were minimal.    [23,24].

Dosimetry
Human biodistribution of 201 Tl chloride published in the literature [25,26] and the calculated activity fraction of free 208 Tl in the injectate at a shelf-life of 6 h was used to estimate human tissue absorbed doses of administered free 208 Tl. The activity fraction of free 208 Tl in the injectate was calculated at a shelf-life of 6 h to be 0.44 MBq 208 Tl per MBq 212 Pb (Table 3).  Table 4. The estimated percent increase in absorbed tissue doses from free 208 Tl in the injectate was 0.03% and 0.09% in the red marrow and kidneys, respectively. In addition, absorbed doses from free 208 Tl result in a percent increase of less than 1.2% over [ 212 Pb]VMT01 in any organ or tissue, and were within the values that would be expected to be the uncertainty in absorbed dose estimates for [ 212 Pb]VMT01 alone.

Discussion
212 Pb is a promising alpha-emitting isotope with an elementally matched gammaemitting isotope 203 Pb that can be used as an imaging surrogate via single photon emission computed tomography (SPECT). 212 Pb physical half-life (10.64 h) is attractive from a clinical translation perspective with regard to patient care and waste management. A recently published phase 1 dose escalation trial of targeted alpha therapy with 212 Pb-DOTAMTATE demonstrated patient safety and promising preliminary efficacy in patients with somatostatin receptor-positive neuroendocrine tumors [27].
From a toxicity standpoint, recoil energy from the emission of an alpha particle decouples the daughter nuclide from any chelator or other chemical bond, and untargeted daughter nuclides are known to accumulate in normal tissues, such as in bone or kidneys [28]. In the work presented here, we calculated estimated human tissue absorbed doses for [ 212 Pb]VMT01 from preclinical murine biodistribution data. In addition, we calculated estimated human tissue absorbed doses for free 208 Tl (that will accumulate in the injectate prior to administration).
One limitation in the dosimetry of alpha radiotherapeutics is the unknown RBE value. Here, according to the method published by dos Santos and collaborators [21], an RBE value of 5 was used for 212 Pb alpha emissions and a value of 1 was used for beta and gamma radiation. Recent studies performed in mammary carcinoma NT2.5 cells treated with 212 Pb-labeled anti-HER2 antibody reported an RBE of 8.3 at 37% survival [29]. Notably, the dose contribution of extracellular unbound 212 Pb-labeled antibody to the absorbed dose was about 2 orders of magnitude smaller compared to the bound and internalized 212 Pb, suggesting that extracellular 212 Pb delivers minimal radiation to cells. The authors conclude that these findings suggest that the actual lesion to dose-limiting tissue absorbed dose could be an order of magnitude greater than that predicted by the calculated absorbed dose.
The analysis presented here demonstrates that accumulated 208 [20]. Retention of 212 Pb daughter 212 Bi within the chelator will result in decay of subsequent daughters 212 Po and 208 Tl at the site of localization due to their short half-lives. Prior to administration, accumulation of unchelated 208 Tl will occur in the formulated product due to the recoil energy of the alpha decay from 212 Bi. Accumulation of unchelated 212 Po may also occur prior to administration as a result of beta decay from 212 Bi; this decay has not yet been characterized. However, due to the extremely short half-life of the 212 Po daughter (0.3 µs), decay from any free 212 Po in the intravenously administered product can be assumed to occur in the plasma with negligible radiation to blood cells [29].

Ex Vivo Gamma Counting
The activity of each collected tissue was measured on a Wizard 1480 (Perkin Elmer Life and Analytical Sciences, Bridgeport, CT, USA) or Wizard 2470 (Perkin Elmer Life and Analytical Sciences, Bridgeport, CT, USA) with a 279 keV peak position and 68% window coverage in units of counts per minute (CPM). Triplicate aliquots of the radiotracer, pulled from the dose-calibrated bulk injectate prepared fresh on each day of injections, were weighed, and assayed via gamma counting to convert CPM to units of grams of injected material. The uptake (percent of the injected dose, % ID) and concentration (% ID per gram, % ID/g) were calculated for each sample count using the known injected dose mass, corrected for tail uptake. Concentration estimates used the sample weight of the gamma-counted tissue in grams (g).

203 Pb Dosimetry Analysis
The radioactivity concentration of [ 203 Pb]VMT01 in each organ (fraction of injected activity per gram) over time was used to compute time-integrated activity coefficients (TIAC) [31] for each organ. For all organs except the total body and blood, uptake at time zero was assumed to be 0% ID. Total body and blood were assumed to be 100% ID at time zero. Human TIAC values were defined by multiplying individual mouse concentration values by animal body weight and by the human phantom organ weight to body weight ratio. This method is equivalent to the percent kilogram per gram method [32]. The human phantom organ weight to body weight ratios were determined from the ICRP 89 adult male and adult female phantom organ and total body weights from OLINDA/EXM 2.0 (Hermes Medical Solutions, Stockholm, Sweden). Each time point value was computed from the group average of the data.
TIAC through the last experimental time point was generated using trapezoidal integration of the seven data points. The contribution to the TIAC following the last experimental time point was estimated by fitting decay-corrected data to a single or a bi-exponential model to estimate biological clearance or assuming physical decay only following the last time point. The combination of physical decay and biological clearance was then analytically integrated. Human TIAC values were then adjusted for radioactivity leaving the body via the renal and gastrointestinal (GI) systems using the dynamic voiding bladder [33] (2 h void) and human alimentary tract model [34]. Excreted urine activity at each time point was defined as 100%-total body % ID-feces % ID. The fraction of excreted urine activity and the voiding half-life were determined by fitting the data to an exponential function. These coefficients were used with a 2 h human voiding time to calculate the urinary bladder TIAC. The ICRP 100 human alimentary tract (HAT) model [34] was utilized with the assumption that radioactivity enters the GI tract via the small intestine. For all animals in each sex group, the radioactivity (% ID, decay corrected) within the small and large intestine, cecum, and all contents were summed at each time point. The peak sum across time for each sex were then determined and used as input into the HAT model in OLINDA/EXM 2.0 to calculate the small intestines, left colon, right colon, and rectum TIACs. Total body radioactivity was calculated as the sum of all measured tissues except for bladder wall, urine, GI, and feces. Total body % ID human was assumed to be equivalent to total body % ID in mouse. The remainder of body TIAC was calculated by subtracting source organ TIACs except for excreta and those derived from the voiding and HAT models. Cortical and trabecular bone TIACs were calculated based on relative surface densities assuming radioactivity distributed to the bone surface. TIAC values were used to compute tissue absorbed dose values for the human adult male and female using OLINDA/EXM 2.0 with ICRP 89 adult male and female phantoms.

212 Pb Dosimetry Analysis
[ 203 Pb]VMT01 data were extrapolated to [ 212 Pb]VMT01 by adjusting the radioactive decay half-life. Assuming transient equilibrium between 212 Pb and its daughters ( 212 Bi, 212 Po, and 208 Tl), the same residence times as for 212 Pb were applied to the daughter nuclides as described by dos Santos and collaborators [21] OLINDA/EXM 2.0 calculations were performed for all nuclides manually. For 208 Tl and 212 Po, the relevant branching fraction was applied. A relative biological effectiveness (RBE) value of 5 was used for the alpha emissions from 212 Bi and 212 Po (while an RBE of 1 was used for beta and gamma emissions); absorbed doses are presented in units of Gray (Gy RBE=5 ).

208 Tl Dosimetry Analysis
Human biodistribution of 201 Tl chloride (a cardiac imaging agent) via scintigraphy imaging published in the literature [25,26] was used to estimate the dosimetry of free 208 Tl. Thallous ion behaves as a potassium analog and tissue uptake is essentially intracellular. Biodistribution of thallium at early times in organs is thus related to regional blood flow. Human % ID values for heart, brain, kidney, liver, intestine, spleen, testes, and the remainder of body were determined from scintigraphy imaging as reported by Svensson and collaborators [26] and Krahwinkel and collaborators [25] for 201 Tl chloride (using the earliest imaging time point from a combination of at rest and after exercise) and conservatively assuming no biological clearance and 100% ID in the total body (Table 5). Radioactive decay of 208 Tl (3.05 m half-life) and resulting TIAC values were used to determine tissue-absorbed doses in the ICRP 89 human adult male using OLINDA/EXM 2.2. The activity fraction of free 208 Tl in the injectate at a shelf-life of 6 h was calculated using the 212 Pb decay scheme and branching fraction of 35.94% for 208 Tl. The 208 Tl activity fraction was used to calculate the 208 Tl mGy RBE=5 /MBq administered 212 Pb activity. a Activity was split equally between the small intestine, upper large intestine wall, lower large intestine wall, and rectum wall based on ICRP 89 target wall organ masses.

Conclusions
The critical tissues for [ 212 Pb]VMT01 based on human dosimetry estimates from murine [ 203 Pb]VMT01 biodistribution data and tissue threshold doses from external beam irradiation data are anticipated to be red marrow and kidneys. Dosimetry analysis indicates that free 208 Tl that will accumulate in the [ 212 Pb]VMT01 injectate prior to administration will not substantially impact estimated tissue absorbed doses in humans. The dosimetry estimations support the clinical evaluation of [ 212 Pb]VMT01.