Radioimmunotheranostic Pair Based on the Anti-HER2 Monoclonal Antibody: Influence of Chelating Agents and Radionuclides on Biological Properties

The oncogene HER2 is an important molecular target in oncology because it is associated with aggressive disease and the worst prognosis. The development of non-invasive imaging techniques and target therapies using monoclonal antibodies is a rapidly developing field. Thus, this work proposes the study of the radioimmunotheranostic pair, [111In]In-DTPA-trastuzumab and [177Lu]Lu-DOTA-trastuzumab, evaluating the influence of the chelating agents and radionuclides on the biological properties of the radioimmunoconjugates (RICs). The trastuzumab was immunoconjugated with the chelators DTPA and DOTA and radiolabeled with [111In]InCl3 and [177Lu]LuCl3, respectively. The stability of the RICs was evaluated in serum, and the immunoreactive and internalization fractions were determined in SK-BR-3 breast cancer cells. The in vivo pharmacokinetics and dosimetry quantification and the ex vivo biodistribution were performed in normal and SK-BR-3 tumor-bearing mice. The data showed that there was no influence of the chelating agents and radionuclides on the immunoreactive and internalization fractions of RICs. In contrast, they influenced the stability of RICs in serum, as well as the pharmacokinetics, dosimetry and biodistribution profiles. Therefore, the results showed that the nature of the chelating agent and radionuclide could influence the biological properties of the radioimmunotheranostic pair.


Introduction
Personalized oncology is based on evidence and offers more assertive decisions for each patient, leading to successful results and reduced healthcare costs. It involves genomic analysis, target-specific drugs, treatment and diagnosis by molecular imaging. It is a promising new approach with remarkable impact on personalized medicine [1]. HER2 is an important molecular target in oncology because it is related to more aggressive tumor development in patients [2][3][4]. Breast cancer is a tumor type that overexpresses the HER2 oncogene in approximately 20% to 30% of cases; this expression is associated with the worst prognosis [3,5,6]. Currently, the available diagnostic methods are invasive and may present inconsistent results due to intratumoral heterogeneity, and the sample size of the biopsied tumor may not represent the whole tumor expression, as well as the possibility of promoting metastatic lesions by repeated biopsies [2].
In this sense, innovative alternatives have been widely investigated and the use of radiolabeled monoclonal antibodies is a rapidly developing field, aiming to find new agents for radioimmunodiagnosis (RID) and radioimmunotherapy (RIT) [3,4]. The RID is a non-invasive imaging technique, which offers advantages in the diagnosis and staging of HER2-positive tumors, allowing the selection of patients who are responsive to a targeted therapy, as well as allowing the monitoring the therapeutic response and the identification of patients who become resistant to immunotherapy [2]. On the other hand, RIT allows for combining specific molecular target therapy with systemic treatment, in order to increase the cytotoxic effect of antibodies by means of their association with a radionuclide, providing the radiation deposition at the site of interest without compromising healthy tissues [7][8][9][10][11]. In addition, there is a great deal of interest in the development of radioimmnunoconjugates (RICs) for theranostic purposes [7].
The rapid growth in the number of molecular biomarkers and the development of drugs for targeted therapy in the treatment of breast cancer began after the discovery of tyrosine kinase receptors, which allowed the emergence of the first target therapy using the humanized anti-HER2 IgG1 monoclonal antibody, trastuzumab, approved by the Food and Drug Administration (FDA) in 1998 [12][13][14]. Due to its importance, trastuzumab has been radiolabeled and evaluated for the purposes of radioimmunotheranostics. In particular, the radioimmunotheranostic pair allows the diagnosis and staging of the disease, the confirmation of the target expression and, consequently, the choice of the patient for specific treatment, which is in agreement with personalized medicine [3,5,6].
In this context, the choice of the radionuclide depends on its physical properties, considering the slow blood clearance of trastuzumab, its local availability and economic viability for routine use [3,7]. On the other hand, the coordination chemistry of each metallic or lanthanide radionuclide requires a specific chelating agent, which can directly influence the biological properties of the RIC [12,[15][16][17][18][19][20]. Therefore, the study of the influence of different bifunctional chelating agents and radionuclides on the biological properties of RICs represents an important field of investigation for the development of new theranostic agents [21].
In this work, the trivalent metal ions indium-111 ( 111 In) and lutetium-177 ( 177 Lu) were chosen for radiolabeling the trastuzumab antibody. 111 In is produced in a cyclotron by the nuclear reaction 111 Cd (p, n) 111 In, and it has a half-life of 67.9 h (2.80 d). It decays 100% by electron capture with gamma (γ) ray emissions [171 KeV (91%) and 245 KeV (94%)]. It is used for diagnosis in single photon emission computed tomography (SPECT) imaging [17]. S-2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA) was conjugated to the antibody because it is an acyclic chelating agent that has the advantage of allowing high labeling efficiency with 111 In, leading to greater thermodynamic stability compared to other chelators [22]. On the other hand, 177 Lu can be produced in a reactor by irradiating the enriched 176 Lu ( 176 Lu (n, γ) 177 Lu) for a low-cost, high-yield and medium-specific activity. It decays to hafnium-177 ( 177 Hf) by beta (β − ) emissions [177 KeV (12%), 385 KeV (9%) and 498 KeV (79%)] and low-abundance γ ray emissions [113 KeV (7%) and 208 KeV (11%)], with a half-life of 160.4 h (6.7 d) [17]. Additionally, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) was conjugated to trastuzumab because it is a macrocyclic chelating agent that promotes greater kinetic and thermodynamic stabilities when radiolabeled with 177 Lu [22]. The γ emissions of 177 Lu allow it to be used for RID, particularly to track the uptake of lesions after the therapeutic procedure. Regarding RIT, among the particle-emitting radionuclides, 177 Lu has adequate physical properties that lead to a safer treatment, such as low-energy β − emission which has a short range (0.2-0.3 mm), promoting a lower radiation dose to the bone marrow compared, for example, to yttrium-90 ( 90 Y). Furthermore, the cross-fire effect of β − may be more effective than alpha (α) particles emitted by radium-223 ( 223 Ra), bismuth-213 ( 213 Bi) and astatine-211 ( 211 At) [7,8,23]. In cancer treatment, the antibody interacts with a limited number of cells in the tumor mass.
However, if a radiolabeled antibody is used, more cells will be killed due to the cross-fire of the emitted particles (β − , α and Auger electrons) [24].
Thus, this work aimed to study the potential of the [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab as a radioimmunotheranostic pair, evaluating the influence of different chelating agents and radionuclides on the biological properties of trastuzumab.

Cell Culture
The SK-BR-3 (ATCC HTB-30TM) cell line was grown in DMEM medium supplemented with 10% fetal bovine serum and 50 µg/mL gentamicin (Gibco, Life Technologies, Austin, TX, USA). The cells were kept in humidified air containing 5% CO 2 at 37 • C. The cells were grown to confluence and then harvested via trypsinization. After centrifugation (1200 rpm; 5 min), the cells were resuspended in supplemented DMEM medium for in vitro studies or in matrigel/supplemented DMEM medium (1:1) for in vivo tumor development.

Determination of the Immunoreactive Fraction
The determination of the immunoreactive fraction of each RIC was performed according to Lindmo and coworkers [26].
The SK-BR-3 cells suspension was serially diluted: 5, 2.5, 1.25, 0.625, 0.312 and 0.156 × 10 6 cells per vial. For specific binding (SB), 1% BSA in PBS and 2.2 pmol of the RIC were added to each vial (n = 3). For non-specific binding (NSB), 1% BSA in PBS, 2.2 nmol of trastuzumab and 2.2 pmol of the RIC were added to each vial (n = 3). After incubation for 1 h (4 • C; gentle agitation), the vials were centrifuged (2000 rpm; 5 min) and the supernatants were collected. The vials containing pellets and supernatants were quantified in an automatic gamma counter. Data were analyzed by linear regression using the GraphPad Prism v. 8.3.1 software (GraphPad Software Inc.-La Joya, CA, USA). The value in the Y-axis, when the value in the X-axis is equal to zero, corresponds to the immunoreactive fraction (r) expressed as 1/r. Radiochemical purity was assessed by ascending chromatography, using iTLC-SG (Agilent Technologies, Santa Clara, CA, USA) and 0.1 M sodium citrate buffer (pH 5.0) as eluent ( Figure 1B). When the radiochemical purity was <90%, the RIC was purified through a molecular exclusion column (Sephadex ® -G25 PD-10, GE Healthcare, Bronx, NY, USA), using 0.25 M ammonium acetate buffer (pH 6.5). High-purity-grade reagents were purchased from Merck Millipore (Burlington, MA, USA).

Internalization
The internalization of RICs into SK-BR-3 cells was evaluated as described in previous studies, with some modifications [27,28]. For each RIC, [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab, 1% BSA in PBS and 2.2 pmol of the RIC were added to a vial containing 2 × 10 6 SK-BR-3 cells. After 1, 4 and 24 h of incubation at 37 • C, the vials were centrifuged (2000 rpm; 5 min), the supernatants were discarded, and the cell pellets were resuspended with 500 µL of 0.2 M acetic acid buffer (pH 2.8) and incubated at 4 • C for 10 min, removing the receptor-bound fraction (but not internalizing it). Then, the vials were centrifuged (2000 rpm; 5 min.), and the supernatants were collected. Pellets (the fraction internalized into the cells) and supernatants were quantified in the automatic gamma counter. The result was expressed as a percentage of total activity in each fraction as a function of time.

In Vivo Studies
Inbred female BALB/c and BALB/c nude mice (4-8 weeks; 20-30 g) were supplied by the animal facility of the Centro de Experimentação e Treinamento em Cirurgia (CETEC) of the Hospital Israelita Albert Einstein (Sao Paulo, SP, Brazil), certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The animals (3-5 mice per cage) were maintained under specific pathogen-free (SPF) conditions with ad libitum access to food and water. The mouse room was temperature-(22 ± 3 • C) and humidity (55 ± 10%)-controlled, with filtered air and a regulated light-dark cycle (12/12 h), with lights turned on at 07:00 a.m. Animals received nest material (paper) and rolls as environmental enrichment. All procedures involving mice were conducted in agreement with the National Council for Animal Experimentation Control (CONCEA) and were approved by the Ethics Committee on Animal Use of the Instituto de Pesquisas Energéticas e Nucleares (IPEN)-(protocol n • 170/16, 15 April 2016) and the Hospital Israelita Albert Einstein (HIAE)-(protocol n • 3463/18, 12 July 2018).

Pharmacokinetics
The pharmacokinetic study was performed in normal female BALB/c mice for both RICs, [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab. At 0, 4, 24, 48, 72, 96, 144 and 168 h after injection of each RIC, 60 µL of blood was collected from the animal's orbital plexus after anesthetic induction with 5% isoflurane. The radioactivity of the blood samples was measured in an automatic gamma counter and the pharmacokinetic parameters were quantified after adjustment for a two-compartment distribution model. Blood clearance was calculated by the ratio between the injected activity and the area under the curve (AUC), and the effective half-life was calculated considering the half-life of the slow phase as the biological half-life.

Xenographic Breast Tumor Animal Model
The xenographic breast tumor animal model was developed in female BALB/c nude mice. In each animal, an aliquot (100 µL) containing 5 × 10 6 SK-BR-3 breast cancer cells in matrigel:DMEM medium (1:1) was subcutaneously inoculated in the right lower flank. Tumor growth was assessed weekly using a caliper rule. The tumor volume was calculated as proposed by Faustino-Rocha and co-workers, considering the measurements obtained from the smallest and largest diameter of the tumor mass [29]. When the average volume of tumor masses reached approximately 196 mm 3 in diameter, the tumors were used for histopathological analysis or the animals were used in biodistribution studies.

Histopathological Analysis
Tumor tissue was obtained from the mice and fixed in formalin (10% v/v in PBS). The hematoxylin-and eosin-staining procedure was performed on paraffin-embedded sections (5 µm) mounted on glass slides. The images of histological sections were captured by a light Nikon Eclipse E600 microscope (Tokyo, Japan).

Ex Vivo Biodistribution Studies
The biodistribution studies were conducted in normal female BALB/c mice and in SK-BR-3 tumor-bearing female BALB-c nude mice for both RICs, counter. The percentages of injected dose per gram of tissue (%ID/g) were calculated using a standard dose containing the same amount of the dose injected into the mice and defined as 100%. For tumor-bearing mice, the target-to-non-target ratio was calculated as tumor to contralateral muscle.

Dosimetric Study
The biodistribution data of RICs from six normal mice were used to estimate the absorbed dose in the period of 4, 24, 48, 72, 96 and 168 h, and then the extrapolation for the dose in humans was calculated using the Medical Internal Radiation Dose (MIRD) methodology [30] and the method described by Sparks and Aydogan [31]. The data from the International Commission on Radiological Protection (ICRP-60 and ICRP-89) [32,33] were also used, and the absorbed fractions were obtained from the OLINDA/EXT software [34,35]. The absorptions of the RICs were calculated using the residence times and the cumulative activity integral of the MIRD methodology.

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism v. 8.3.1 software (Graph-Pad Software Inc.-La Joya, CA, USA). Data were expressed as mean ± error. The means of two groups were compared using the Student's t-test. The means of three or more groups were compared by analysis of variance (ANOVA), followed by Tukey's multiple comparison test (one-way ANOVA) or the Bonferroni test (two-way ANOVA). p-values ≤ 0.05 were considered statistically significant different.

Immunoconjugation and Radioimmunoconjugation
The immuno-and radioimmunoconjugation were performed with no further modification from the previous proposed protocol [25], at a 1:20 (antibody:chelator) molar ratio. The RICs presented radiochemical purity >90%; however, for [ 177 Lu]Lu-DOTAtrastuzumab, the radiochemical yield was about 84% and, then it was necessary to perform the purification in a molecular exclusion column to achieve radiochemical purity >90%. For [ 111 In]In-DTPA-trastuzumab, this step was not necessary.

Evaluation of the Stability of the Radioimmunoconjugates in Serum
The stability of RICs in serum, assessed by ascending chromatography, is shown in . Data were expressed as mean ± error. The means of two groups were compared using the Student's t-test. The means of three or more groups were compared by analysis of variance (ANOVA), followed by Tukey's multiple comparison test (one-way ANOVA) or the Bonferroni test (two-way ANOVA). p-values ≤ 0.05 were considered statistically significant different.

Immunoconjugation and Radioimmunoconjugation
The immuno-and radioimmunoconjugation were performed with no further modification from the previous proposed protocol [25], at a 1:20 (antibody:chelator) molar ratio.
The RICs presented radiochemical purity >90%; however, for [ 177 Lu]Lu-DOTA-trastuzumab, the radiochemical yield was about 84% and, then it was necessary to perform the purification in a molecular exclusion column to achieve radiochemical purity >90%. For [ 111 In]In-DTPA-trastuzumab, this step was not necessary.

Evaluation of the Stability of the Radioimmunoconjugates in Serum
The stability of RICs in serum, assessed by ascending chromatography, is shown in Figure 2. [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab showed radiochemical purity >75% and 94%, respectively, up to 168 h.

Determination of the Immunoreactive Fraction
After immuno-and radioimmunoconjugation, both RICs were able to bind SK-BR-3 cells in a cellular-concentration-dependent manner ( Figure 3A,B). The binding was specific and the unlabeled monoclonal antibody was able to inhibit RICs binding to cells. In the described conditions, it was not possible to obtain a saturation of the receptors and therefore, the Kd and Bmax parameters were not calculated. The immunoreactive fractions of [ 111 In]In-DTPA-trastuzumab ( Figure 3A ,A") and [ 177 Lu]Lu-DOTA-trastuzumab ( Figure 3B ,B") were determined by plotting the double inverse plot of the applied radiolabeled antibody over the specific (Figure 3A ,B ) and non-specific ( Figure 3A",B") binding as a function of the inverse cell concentration. Theoretically, it was assumed that the unconjugated and unlabeled trastuzumab has an immunoreactivity of 100% [26].

Determination of the Immunoreactive Fraction
After immuno-and radioimmunoconjugation, both RICs were able to bind SK-BR-3 cells in a cellular-concentration-dependent manner ( Figure 3A,B). The binding was specific and the unlabeled monoclonal antibody was able to inhibit RICs binding to cells. In the described conditions, it was not possible to obtain a saturation of the receptors and therefore, the Kd and Bmax parameters were not calculated. The immunoreactive fractions of [ 111 In]In-DTPA-trastuzumab ( Figure 3A′,A″) and [ 177 Lu]Lu-DOTA-trastuzumab ( Figure 3B′,B″) were determined by plotting the double inverse plot of the applied radiolabeled antibody over the specific (Figure 3A′,B′) and non-specific ( Figure 3A″,B″) binding as a function of the inverse cell concentration. Theoretically, it was assumed that the unconjugated and unlabeled trastuzumab has an immunoreactivity of 100% [26].    19.0% (4 h),and 32.4% (24 h) of internalization. However, no significant differences between RICs were observed when comparing the same evaluated times (p > 0.05).

Internalization
The internalization of the RICs into SK-BR-3 cells is illustrated in Figure 4. For both RICs, the percentage of internalization increased with time.  19.0% (4 h),and 32.4% (24 h) of internalization. However, no significant differences between RICs were observed when comparing the same evaluated times (p > 0.05).

Pharmacokinetics
The pharmacokinetic parameters, by two-compartment mathematical models, were obtained from a plasma concentration curve (% of RIC's activity in total blood) as a function of time ( Figure 5), simulating the processes of absorption, distribution, metabolism and excretion (ADME) of the RICs (Table 1).

Pharmacokinetics
The pharmacokinetic parameters, by two-compartment mathematical models, were obtained from a plasma concentration curve (% of RIC's activity in total blood) as a function of time ( Figure 5), simulating the processes of absorption, distribution, metabolism and excretion (ADME) of the RICs (Table 1).

Ex Vivo Biodistribution Studies
The ex vivo biodistribution of RICs, [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab, in normal female BALB/c mice are shown in Figure 7A,B, and in SK-BR-3 breast-tumor-bearing female BALC/c nude mice in Figure 7A ,B.
The data showed that both RICs presented slow blood clearance, associated with significative uptake by the liver, spleen and kidneys. The RICs also accumulated in the heart, lungs, intestines and bones. The uptake of [ 177 Lu]Lu-DOTA-trastuzumab by bone was lower than that of [ 111 In]In-DTPA-trastuzumab at 4 h and 168 h (p < 0.05). On the other hand, the brain, pancreas, stomach and muscle presented low accumulation of RICs.
Ex vivo biodistribution data that were obtained in xenograft breast-tumor-bearing mice showed that tumor uptake was significant and increased over the evaluated period for both RICs (Figure 7A",B")

Ex Vivo Biodistribution Studies
The ex vivo biodistribution of RICs, [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab, in normal female BALB/c mice are shown in Figure 7A,B, and in SK-BR-3 breast-tumor-bearing female BALC/c nude mice in Figures 7A′,B. The data showed that both RICs presented slow blood clearance, associated with significative uptake by the liver, spleen and kidneys. The RICs also accumulated in the heart, lungs, intestines and bones. The uptake of [ 177 Lu]Lu-DOTA-trastuzumab by bone was lower than that of [ 111 In]In-DTPA-trastuzumab at 4 h and 168 h (p < 0.05). On the other hand, the brain, pancreas, stomach and muscle presented low accumulation of RICs.
Ex vivo biodistribution data that were obtained in xenograft breast-tumor-bearing mice showed that tumor uptake was significant and increased over the evaluated period for both RICs ( Figure 7A″,B″) Table 2.

Discussion
The nature of the chelating agent and radionuclide influences the biological properties of RICs. In this sense, in the development of a monoclonal antibody-based radioimmunotheranostic pair, it is important to evaluate such influence. In the present work, the anti-HER2 monoclonal antibody trastuzumab was immuno-and radioimmunoconjugated with [ 111 In]In-DTPA and [ 177 Lu]Lu-DOTA. The radioimmunotheranostic pair, [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab, were compared concerning their immunoreactivity and biological properties in order to assess the influence of the chelating agents and radionuclides.
The number of chelators per antibody molecule may influence the radiochemical stability of the radioimmunoconjugate. Previous MALDI-TOF studies revealed an average number of 6-7 molecules of p-SCN-Bn-DTPA and 8-9 molecules of DOTA-NHS-ester coupled to trastuzumab for a 1:20 M ratio [25]. The [ 177 Lu]Lu-DOTA immunoconjugate showed greater stability in serum than [ 111 In]In-DTPA, especially in the first 4 h of incubation. Indium atoms present similar coordination chemistry and biological properties compared to Fe 3+ . Then, in vivo, a slight transchelation process of 111 In may occur in the presence of specific protein-binding sites, such as transferrin, lactoferrin and ferritin [22,36]. Lub and coworkers observed a reduction of 7% per day in the stability of [ 111 In]In-DTPAtrastuzumab after incubation in serum at 37 • C, related to the transchelation of 111 In to transferrin [37]. In the same direction, Blend and coworkers reported a slight decrease in stability in human plasma after 96 h of incubation [38]. In biodistribution studies, the bone uptake of [ 111 In]In-DTPA-trastuzumab increased with time and this uptake is probably related to the free 111 In. On the other hand, [ 177 Lu]Lu-DOTA-trastuzumab showed no stability reduction in serum within time (p > 0.05), which is consistent with the study of Rasaneh and coworkers that showed stability for up to 96 h [8]. Incubation with an excess of unlabeled trastuzumab reduced the binding of RICs to SK-BR-3 cells, indicating the binding specificity. The percentages of the immunoreactive fraction obtained for both RICs in this study were higher than those previously reported [8,23,27,37]. The immunoreactivity of a RIC is related to the antibody:chelator molar ratio employed in the immunoconjugation, the possible binding of the chelator to the antigen binding site and the type of chelator [39][40][41][42]. In this study, there was no influence of the different chelating agents (DTPA and DOTA) and radionuclides ( 111 In and 177 Lu) on the immunoreactivity of the monoclonal antibody trastuzumab.
The incorporation of [ 111 In]In-DTPA and [ 177 Lu]Lu-DOTA also did not influence the internalization of the trastuzumab into the SK-BR-3 cells. Both RICs showed an increment of internalization within the same time-frame, and no significant differences were observed between them. The internalization process is slow and probably related to the binding of RICs to the HER2 receptors expressed on the surface of SK-BR-3 cells. The slow internalization is possibly justified by the molecular weight of trastuzumab (≈150 kDa). After internalization, the antibody is metabolized intracellularly to smaller peptides and amino acids [43]. The percentage of internalization obtained in this study, for both RICs, was higher than was previously published [27,37]. These differences are probably due to the different molar ratios (antibody:chelator) and cell lines used by each author.
The pharmacokinetic study showed that both RICs presented slow clearance, influencing the slow phase half-life (elimination)-T 1 2 Ke.  [43,46]. This clearance pattern is due to the high molecular weight of trastuzumab.
The biodistribution data showed high uptake of RICs by liver, spleen and kidneys. This associated uptake indicates that RICs are metabolized by the liver and spleen, and that the metabolic elimination occurs through the kidneys [47][48][49]. Furthermore, biodistribution showed slow blood clearance, which is in agreement with the pharmacokinetic data and with the previously determined partition coefficients, [ 111 In]In-DTPA-trastuzumab (log p ≈ 3.0) and [ 177 Lu]Lu-DOTA-trastuzumab (log p ≈ 1.8), that showed lipophilic features of both RICs [25]. Cooper and coworkers obtained similar results for the rituximab antibody immunoconjugated to different chelators, including DTPA and macrocyclic chelators [50].
The uptake of RICs by the lungs and intestines was also observed, which may be associated with the physiological presence of the HER2 oncogene in these organs. [27,37] On the other hand, low uptake by the brain, pancreas, stomach and muscle was observed. The uptake of [ 177 Lu]Lu-DOTA-trastuzumab by bone was lower than that of [ 111 In]In-DTPAtrastuzumab. The metal complexes of the DOTA conjugates exhibit greater in vivo stability compared to the complexes of the chelating agent DTPA [51]. This profile is in agreement with Lub de Hooge and coworkers, who also demonstrated low bone uptake resulting from the possible decomplexation of the 111 In radionuclide from the RIC molecule [37]. Beyond that, it is worth mentioning that the chelating agent influences the biodistribution profile of a RIC. Labile chelators can be cleaved from the antibody molecule by enzymes present in the serum and liver, leading to low-molecular-weight radioactive metabolites that will be eliminated by the kidneys [52].
For the RID and RIT of cancer to be successfully performed, the immunoreactivity of RIC must be preserved, as shown by our immunoreactivity data. Consequently, the specificity to the tumor must also be maintained, which was evidenced by the biodistribution data that showed high accumulation in the SK-BR-3 tumor, as well as great target-to-non-target ratios (tumor/muscle).
However, it is important to mention that high immunoreactivity does not guarantee effective uptake of RICs by tumors. Therefore, there are other issues that can influence this uptake and must be evaluated in its development process, such as the determination of the best molar activity and molar ratio (antibody:chelator) and the lipophilic features [25,53].
Considering the activities of the RICs administered in humans, from 148 to 185 MBq for [ 111 In]In-DTPA-trastuzumab and 7400 MBq for [ 177 Lu]Lu-DOTA-trastuzumab, the dosimetric data showed that the doses absorbed in the organs for [ 177 Lu]Lu-DOTA-trastuzumab are higher than those for [ 111 In]In-DTPA-trastuzumab. Absorption is different for the two RICs, since 177 Lu presented the highest dose in the kidneys, followed by the liver, and 111 In showed highest dose for the liver, followed by the spleen. Dosimetrically, there is a relative difference in the injected activity between the two radionuclides and also between the energy of both, which can lead to this difference in dose absorption. The fact that the RICs present in some different organs for absorption is due to the affinity of interaction between the organs and the RICs.
Although a greater permanence in the organism is desirable when considering applications in therapy, such permanence must be directly related to the target tissue, in this case, the tumor. In the case of [ 177 Lu]Lu-DOTA-trastuzumab, the longer circulation time may represent higher irradiation of non-target organs, especially those that are more vascularized. However, the data presented in this work for both RICs are in accordance with previously published data [54,55].
Taking into account these data, the chelating agents and radionuclides may impair the biological properties of the monoclonal antibodies, such as trastuzumab. However, our data suggest that [ 111 In]In-DTPA-trastuzumab and [ 177 Lu]Lu-DOTA-trastuzumab are a good theranostic pair for HER2-overexpressing breast tumors. Our data also indicate that SPECT imaging with [ 111 In]In-DTPA-trastuzumab should be performed from 72 h to 168 h after RIC administration, and our results are in accordance with previous data [55][56][57].
Finally, this comparative study is important in the development of radioimmunotheranostic pairs, which are used for radioimmunodiagnostic and radioimmunotherapy. The diagnostic agent, [ 111 In]In-DTPA-trastuzumab, may be used to assess by SPECT imaging the presence of HER2-positive breast tumors in the patient. If positive, [ 177 Lu]Lu-DOTAtrastuzumab may be used for target-specific radionuclide therapy. If negative, other treatment options are indicated ( Figure 8). Furthermore, in the first case, [ 111 In]In-DTPAtrastuzumab may also be used to evaluate the patient's response to radioimmunotherapy. Further studies should be conducted in patients to confirm the potential of these radioimmunoconjugates as a radioimmunotheranostic pair.

Conclusions
In conclusion, our results demonstrated that the nature of the chelating agent and radionuclide influences the biological properties of the trastuzumab-based radioimmunoconjugates, highlighting the importance of this evaluation in the development of a theranostic pair. In this work, data showed that the immuno-and radioimmunoconjuga-

Conclusions
In conclusion, our results demonstrated that the nature of the chelating agent and radionuclide influences the biological properties of the trastuzumab-based radioimmunoconjugates, highlighting the importance of this evaluation in the development of a theranostic pair. In this work, data showed that the immuno-and radioimmunoconjugation of [ 111 In]In-DTPA and [ 177 Lu]Lu-DOTA preserved the immunoreactivity of the trastuzumab molecule. Although, different pharmacokinetic, dosimetric and biodistribution behaviors were observed between both RICs, our results suggest that they are suitable for radioimmunotheranostics of HER2 overexpressed-tumors.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.