Enhanced Tumor Targeting of Radiolabeled Mouse/Human Chimeric Anti-Tn Antibody in Losartan-Treated Mice Bearing Tn-Expressing Lung Tumors

Abstract

Aim:

ChiTn, a mouse/human chimeric anti-Tn monoclonal antibody, was radiolabeled with iodine-131 (¹³¹I) and technetium-99m (^99mTc) to assess its biodistribution and internalization in Tn-expressing (Tn+) and wild-type (Tn-) LL/2 lung cancer cells.

Results:

Selective accumulation and gradual internalization of ChiTn were observed in Tn+ cells. Biodistribution in mice with both Tn+ or Tn− lung tumors indicated that the uptake of radiolabeled ChiTn within tumors increased over time. Dual-labeling experiments with ^99mTc and ¹³¹I showed different biodistribution patterns, with ^99mTc exhibiting higher values in the liver, spleen, and kidneys, while ¹³¹I showed higher uptake in the thyroid and stomach. However, tumor uptake did not significantly differ between Tn+ and Tn− tumors. To improve tumor targeting, Losartan, an antihypertensive drug known to enhance tumor perfusion and drug delivery, was investigated. Biodistribution studies in Losartan-treated mice revealed significantly higher radiolabeled ChiTn uptake in Tn+ tumors. No significant changes were observed in the uptake of the control molecule IgG-HYNIC™^99mTc.

Conclusions:

These findings demonstrate the enhanced tumor targeting of radiolabeled ChiTn in Losartan-treated mice with Tn-expressing lung tumors. They highlight the potential of ChiTn as a theranostic agent for cancer treatment and emphasize the importance of Losartan as an adjunctive treatment to improve tumor perfusion and drug delivery.

Introduction

Monoclonal antibodies (mAbs) have been widely used in clinical practice for the diagnosis and treatment of various human pathologies, including cancer and infectious diseases, as well as for the modulation of the immune response.^1
–3 The development of therapeutic mAbs in cancer has been facilitated by the identification of cell surface structures. These structures, which are either present or overly expressed in abnormal cells compared with their normal counterparts, act as targets for the effective function of mAbs.⁴

Aberrant glycosylation is a common change in cancer cells, which causes incomplete elongation of the carbohydrate chains and unmasking of tumor-associated carbohydrate antigens on the cell surface.⁵ The Tn-antigen (CD175) is formed by an N-acetyl galactosamine (GalNAc) residue O-linked to the amino acids serine or threonine, present in the amino acid sequence of different glycoproteins, especially those of the mucin-type.⁶ This antigen is a cryptic determinant in cancer with high expressions in various epithelial cancers (ovarian, breast, prostate, colorectal, and lung cancers).^7,8 Tn is associated with metastasis and cancer aggressiveness.^9,10

Indeed, in Lewis lung cancer (LL/2), Tn antigen promotes tumor growth, immune evasion, and angiogenesis, resulting in larger and highly vascularized tumors.¹¹ This may be explained by the ability of LL/2 Tn+ cells and LL/2 Tn+ tumors to produce higher amounts of vascular endothelial growth factor (VEGF) compared with LL/2 wild type (Tn−).¹¹ VEGF is considered to be the primary angiogenesis factor and has been shown to play a role in inducing extracellular matrix (ECM) synthesis and the angio-fibrotic switch in fibrosis.^12
–14

Fibrosis, characterized by the excessive deposition of collagen, hyaluronan, and other ECM components, plays a critical role in tumor progression.¹⁴ The fibrotic ECM has been identified as a significant contributor to the elevated solid stress observed in tumors.¹⁵ Unlike fluid pressure, this physical force is specifically contained within and transmitted by the solid phase of the tumor, resulting in the compression of blood vessels. This compression, coupled with the structural abnormalities of tumor blood vessels, renders them highly susceptible to collapse under the influence of the increased compressive forces.

Consequently, the overall blood flow throughout the tumor mass is compromised.^15

–18 This reduction in perfusion has two major consequences: (1) compromised efficacy of treatment due to reduced drug delivery to tumors,¹⁹ and (2) increased tumor hypoxia, which promotes aggressive phenotypes, immunosuppression, and resistance to chemotherapy, radiation, and immunotherapy, all of which require oxygen to be effective.¹⁵

Therefore, strategies aimed at reducing solid stress and improving tumor perfusion are being explored to improve the delivery and efficacy of cancer therapeutics. One such strategy is the use of Losartan, an FDA-approved antihypertensive agent that has been shown to improve tumor perfusion and drug delivery in mouse models of breast and pancreatic cancer by reducing the collagen and hyaluronan content within tumors.^17,20,21

Several mAbs targeting the Tn-antigen with different specificities have been developed, some of which have demonstrated increased survival in mice inoculated with Jurkat,²² mammary,⁷ and lung carcinoma cell lines.²³ In addition to their inherent ability to inhibit tumor growth by inducing or stimulating the immune system,^24,25 the antitumor efficacy of mAbs can be significantly enhanced by conjugation with drugs,²⁶ oligonucleotides,²⁷ or radionuclides.^28

–31

The chimeric mouse/human IgG1 anti-Tn mAb, ChiTn, developed as an antibody-drug conjugate, effectively targets and internalizes in Tn+ tumor cells, showing selective accumulation in vivo.⁸ With the Tn-antigen present in both species and ChiTn's reduced immunogenicity in humans, this antibody is poised for a more efficient transition from preclinical studies to human trials.

Imaging and biodistribution studies utilizing radioactive tracers have emerged as a highly sensitive and versatile approach, making it one of the main approaches for in vitro and in vivo studies of molecular-level processes. It allows analysis of pharmacokinetics, heterogeneity, antigen expression and engagement, and uptake of radiolabeled mAbs in tumors and other organs.^31
–33 Iodine-131 (¹³¹I), a widely used radionuclide in nuclear medicine, serves as a dual β and γ emitter (8.02-d half-life).

Its gamma energy of 364.5 keV make it suitable for gamma spectrometry, whereas its beta emission has potential for cancer treatment.³⁴ In addition, technetium-99m (^99mTc), characterized by a 6-h half-life and a 140.5 keV γ-ray emission,³⁵ enables distinct gamma emission detection in gamma spectrometry, differentiating it from ¹³¹I for in vitro and biodistribution studies.

In this study, the authors aimed to explore the potential of a radiolabeled mouse/human IgG1 chimeric anti-Tn mAb (ChiTn) as a theranostic agent for cancer. The authors radiolabeled ChiTn with ¹³¹I and ^99mTc and evaluated its biodistribution and internalization in Tn+ and Tn− (wild-type) cells. Furthermore, they investigated the potential use of Losartan as an adjunctive treatment to enhance the delivery and targeting of radiolabeled ChiTn to Tn+ tumors in murine models of Tn+ and Tn− LL/2 lung cancer.

Methods

ChiTn and IgG linker formation

The mouse/human IgG1 chimeric anti-Tn mAb (Chi-Tn)³⁶ was obtained from Laboratorio de Glicobiología e Inmunología tumoral/Institut Pasteur Montevideo. A human IgG (Sigma-Aldrich) was used as a control molecule, which has no declared antigen-specificity. HYNIC conjugation to antibodies followed established protocols, typically yielding 3–5 chelators per molecule.³¹

The conjugation reaction was initiated by adding an N-succinimidyl 6-(trifluoroacetyl) hydrazinopyridine-3-carboxylic acid hydrazino-protected form of succinimidyl ester (NHS-HYNIC-Tfa), synthesized in the authors' laboratory,³⁷ and 100 nmol in 7 μL dimethylsulfoxide (DMSO; J.T. Baker) were added to purified ChiTn or IgG control (10 nmol) in, 0.15 M pH 8 NaC₂H₃O₂ buffer and incubated in darkness for 30 min at room temperature (RT). The ChiTn-HYNIC and IgG-HYNIC were subsequently purified by size-exclusion chromatography using NaCl 0.9% and a Sephadex G-25 (size exclusion chromatography [SEC]) column (PD-10; Amersham Biosciences).

ChiTn and IgG radiolabeling

Radiolabeling of ChiTn-HYNIC or IgG-HYNIC with ^99mTc was performed by adding 33.5 mol of tricine, 33 mol of SnCl₂.2H₂O, and 5 nmol of the antibody conjugate. Immediately, a Na^99mTcO₄ ⁻ solution was added, and the mixture was incubated at RT for 30 min. Radiolabeling of ChiTn or IgG with ¹³¹I was achieved using the Pierce precoated iodination tubes (PPCIT; Thermo Fisher Scientific, Waltham, MA), and the “Example Protocol I: The Chizzonite Indirect Method for Iodination” protocol according to Thermo Fisher Scientific manual 0016379.

In brief, the PPCIT was wetted with 1 mL of Tris iodination buffer and decanted. Then, 10 MBq of ¹³¹I were added to the PPCIT and activated for 6 min at RT. Subsequently, 50 μg of ChiTn or IgG were added and the reaction mixture was incubated for 10 min at RT.

ChiTn and IgG radiolabeling controls, purification, and stability

Both radiolabeling were evaluated for radiochemical purity by (1) SEC equilibrated and eluted with normal saline, at 1.0 mL/min flow rate, and (2) high-performance liquid chromatography with a Protein-Pak 300 SW 7.5 mm × 30 cm column (size exclusion-high-performance liquid chromatography [SEC-HPLC]; Waters) using isocratic mode with 0.05 M pH 7.0 phosphate buffer, 1 mL/min flow rate, equipped with ultraviolet and NaI(Tl) absorbance and scintillation detectors, respectively.

^99mTc radiolabeling was assessed using instant thin-layer chromatography (ITLC) chromatography with various systems: instant thin layer chromatography medium impregnated with a silica gel (ITLC-SG) (Pall Corporation) combined with 0.9% NaCl, ITLC-SG saturated with bovine serum albumin (BSA; Sigma-Aldrich) and EtOH:NH₃:H₂O (2:1:5), as well as Whatman 1 paper (Whatman International Ltd.) with methyl ethyl ketone (MEK). To ensure the integrity of the radiolabeled antibodies, the same SEC-HPLC system was employed. The radiolabeled antibodies were incubated in serum at 37°C for different time intervals, followed by high-performance liquid chromatography analysis to evaluate their stability.

Cell culture

Wild-type (Tn−) murine lung cancer cell line LL/2 (ATCC), and LL/2 cells expressing the Tn antigen (Tn+; Cells transfected by CRISPR guide targeted to Cosmc [c1galt1c1] and characterized at the Laboratorio de Inmunomodulacion y Desarrollo de Vacunas, Facultad de Medicina UdelaR¹¹), were grown in RPMI-1640 medium (Capricorn Scientific) supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific), 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Capricorn Scientific). All cultures were incubated at 37°C with 5% CO₂ and a 95% humidity atmosphere. Reconfirmation of Tn expression in LL/2 Tn+ cells was not deemed necessary due to the established stability and consistent characteristics of these cell lines.

Membrane-bound and cell internalization

Membrane-bound and cell internalization assay was performed by seeding 0.5 × 10⁶ Tn− or Tn+ cells with culture medium in 24-well plates and allowed to grow overnight. After washing once with culture medium, cells were incubated with ∼4 nM of ChiTn and/or IgG radioconjugated with ^99mTc and/or ¹³¹I for 1, 2, 3, 4 (quadruplicates), and 24 h (octuplicate) at 37°C.

Then, the reaction medium was aspirated, and cells were washed twice with 500 μL cold phosphate-buffered saline (PBS). Cells were washed with cold acid buffer solution (0.2 M pH 2 glycine buffer) for 5 min to remove membrane-bound radioconjugated antibodies. The remaining internalized radioconjugated antibodies were obtained by lysis with 1 M NaOH for 5 min. Membrane-bound and internalized radioactivities were measured, and the results were analyzed, according to Radioactivity Measurements and Statistical Analysis sections.

Animals and tumor induction

C57BL/6 female mice were purchased from URBE Laboratories. Mouse handling, care, and experiments were in accordance with ethical principles adopted by National Committee on Animal Research (CNEA; protocol approved by the Commission of Ethics in the Use of Animals, Facultad de Ciencias [CEUA] No. 240011-001327-18). Tn− or Tn+ LL/2 cells (2 × 10⁵/100 μL) were inoculated on the flank region of each mouse (8 weeks old, 19.8 g average weight) through subcutaneous (s.c.) or intradermal (i.d.) injection (5 mice/group). The animals were followed daily for at least 1 month, evaluating tumor growth. All procedures were confirmed to comply with ARRIVE guidelines.

Losartan treatment

Losartan pills (50 mg/pill; Losartan potassium) were ground using a mortar and pestle, and dissolved in PBS to obtain a concentration of 2.5 mg/mL. The solutions were then filtered and stored in a sterile container. Animals were treated daily with an intraperitoneal (i.p.) injection of Losartan (20 mg/kg) or saline (control) for 9 d.^17,20,21

Biodistribution studies

When tumors reached 500–1000 cm³ in size (mean and standard deviation [SD] mass (g): 0.35 ± 0.1 and 0.72 ± 0.2, Tn− and Tn+, respectively; 15–20 d), mice from the same cohort received 0.74–3 MBq of ChiTn-HYNIC-^99mTc or IgG-HYNIC-^99mTc (1–4 μg, in 100 μL PBS), and/or ∼185 kBq of ChiTn-¹³¹I or IgG-¹³¹I (1–4 μg, in 100 μL PBS) through the tail vein. The amount of radiolabeled antibody used falls within the range typical for biodistribution studies.⁶⁹

After 4, 24, and 48 h, the animals were euthanized with anesthetic drugs (xylazine—100 mg/kg and ketamin—300 mg/kg). Blood, urine, and feces were collected, tumor and selected organs were excised, rinsed of residual blood, weighed and their radioactivity was measured according to the Radioactivity measurements section. Results were expressed as percent injected dose (% ID) and per gram of tissue (% ID/g).

Radioactivity measurements

γ-ray measurements were acquired using a high-purity germanium detector (HPGe; Canberra) and appropriate low-background shielding. The HPGe has the following features: 20% counting efficiency (at 1332 keV) and full width at half maximum resolution <0.85 keV (at 122 keV), enabling simultaneous in vitro and/or biodistribution measurements of multiple γ-emitting radionuclide.^37,38 Results are reported for ^99mTc (140.5 keV, 89%) lower limit of detection (LLD) 110 Bq, and ¹³¹I (364.5 keV, 81.7%) LLD 1.0 kBq for 30-s measurements.

Statistical analysis

To evaluate the statistical significance of differences in medians, the authors used the Mann–Whitney U test with Bonferroni correction. The following annotation was used for statistical significance, ns: p ≤ 1e⁺⁰⁰; *: 1e⁻⁰² < p ≤ 5e⁻⁰²; **: 1e⁻⁰³ < p ≤ 1e⁻⁰²; ***: 1e⁻⁰⁴ < p ≤ 1e⁻⁰³; ****: p ≤ 1e⁻⁰⁴, also, a 95% confidence interval was applied. The authors performed all statistical analyses and data visualizations using multiple packages within Spyder v. 4.1.5,³⁹ an integrated development environment (IDE) for scientific programming in Python v. 3.8.5 (Python Software Foundation). The packages utilized include Pandas⁴⁰ for data manipulation, Matplotlib⁴¹ for data visualization, NumPy⁴² for numerical computations, Seaborn⁴³ for advanced data visualization, and Statannotations^44,45 for statistical annotations.

Results

Radiolabeling and in vitro quality controls

Both ¹³¹I and ^99mTc radiolabeled ChiTn or IgG antibodies were purified and their chemical identity was determined by SEC, SEC-HPLC, and ITLC. SEC analysis showed a retention volume of the radiolabeled antibodies between 3 and 4 mL, whereas free ^99mTc and free ¹³¹I showed a retention volume between 8 and 9 mL. SEC-HPLC profile showed retention times of 8.5, 8.9, and 9.0 min for ChiTn-HYNIC-^99mTc, ChiTn-¹³¹I, and unlabeled ChiTn, respectively (Fig. 1). Radiochemical purity exceeded 92% in all the experiments.

FIG. 1.

SEC-HPLC profile and the in vitro radiochemical stability of the radiolabeled ChiTn. The upper panel displays the retention profile of ChiTn when radiolabeled with either ^99mTc (red) or ¹³¹I (blue), as well as unlabeled ChiTn (gray and dashed lines) under UV at a wavelength of 280 nm. The lower panel illustrates the gamma signal of the radiolabeled ChiTn, measured in counts per second. In the bottom right box, the average and SD of the percentage radiochemical stability of the radiolabeled ChiTn over time in serum at 37°C. ¹³¹I, iodine-131; ^99mTc, technetium-99m; SD, standard deviation; UV, ultraviolet.

The analysis conducted by ITLC demonstrated that within the residual 8%, nearly two-thirds were attributed to ^99mTcO₂·H₂O, whereas the remaining one-third was associated with ^99mTc-tricine. The radiochemical stability exhibited a gradual decrease over time, with an average of 89% (SD 2.1) and 90% (SD 2.08) of antibodies retaining their radiolabeling with ^99mTc and ¹³¹I, respectively, after a 48 h incubation period in serum at 37°C (Fig. 1, lower right). Similar results of radiochemical purity and stability were obtained for IgG-HYNIC-^99mTc (data not shown).

Membrane-bound and cell internalization

Results for the membrane-bound internalization and uptake ratio between Tn+ and Tn− LL/2 cells for radioconjugated ChiTn and/or IgG are shown in Figure 2, and data are available at https://zenodo.org/record/8110446A gradual time-dependent increase in both membrane-bound and intracellular activities was observed for ChiTn-HYNIC-^99mTc during the 24 h experiment at 37°C (Fig. 2A). This increase was found to be significantly higher in Tn+ cells compared with Tn− cells (p ≤ 1e⁻⁰⁴ at 24 h).

FIG. 2.

Membrane-bound, internalization, and total cell association of radiolabeled antibodies in LL/2 Tn− or Tn+ cells, measured at different incubation times. The figure includes (A) boxplot and MW of ChiTn-HYNIC-^99mTc, (B) boxplot and MW analysis of co-incubated ChiTn-¹³¹I and IgG-HYNIC-^99mTc, and (C) average and 95% CI uptake ratio (%) between Tn+ and Tn− cells, for experiments A and B, where positive numbers indicate higher uptake in Tn+ cells. ns: p ≤ 1e⁺⁰⁰; *, 1e⁻⁰² < p ≤ 5e⁻⁰²; **, 1e⁻⁰³ < p ≤ 1e⁻⁰²; ***, 1e⁻⁰⁴ < p ≤ 1e⁻⁰³; ****, p ≤ 1e⁻⁰⁴. CI, confidence interval; MW, Mann–Whitney U test.

Similarly, when IgG-HYNIC-^99mTc and ChiTn-¹³¹I were co-incubated, a similar trend of gradual increase was observed for ChiTn-¹³¹I, with significantly higher activities observed in Tn+ cells (1e⁻⁰³ < p ≤ 1e⁻⁰² at 24 h). However, no specific trend or significant differences were observed between Tn+ and Tn− cells when using IgG-HYNIC-^99mTc (Fig. 2B). The total cell-associated uptake of ChiTn-HYNIC-^99mTc was ∼100% higher in Tn+ cells compared with Tn− cells, whereas for ChiTn-¹³¹I, the difference was ∼50% (Fig. 2C at 24 h). In contrast, no significant difference favoring Tn+ cells was observed for IgG-HYNIC-^99mTc (Fig. 2C).

Biodistribution studies

The results of the biodistribution of ChiTn-HYNIC-^99mTc in female C57BL/6 inoculated s.c. or i.d. with Tn− or Tn+ LL/2 cells obtained at 4, 24, 48 h postinjection, are shown in Figure 3 and Supplementary Figure S1, and data are available at https://zenodo.org/record/8110446 An increase in Tn+ tumor uptake occurred between 4 h (4.4% ± 1.6 ID/g) and 24 h (10.5% ± 2.3 ID/g) postinjection, remaining stable at 48 h (11.3% ± 2.5 ID/g). No significant differences in the uptake were found between Tn− and Tn+ or between s.c. or i.d. tumors.

FIG. 3.

(A) Biodistribution profile of ChiTn-HYNIC-^99mTc in blood, muscle, and Tn− or Tn+ tumors inoculated in mice, by SC or ID injection, at 4, 24, and 48 h postinjection. The figure also shows results for noninoculated (normal) mice. (B) % ID eliminated through urine and feces. (C, D) T/B and T/M ratios, respectively. % ID, percent injected dose; ID, intradermal; SC, subcutaneous; T/B, tumor-to-blood; T/M, tumor-to-muscle.

Furthermore, there were no significant differences between Tn− and Tn+ or between s.c. or i.d. tumors concerning the ratios of tumor-to-blood (T/B) and tumor-to-muscle (T/M). The remaining organs showed a typical antibody biodistribution profile, with liver uptake and hepatobiliary elimination (Supplementary Fig. S1).

The biodistribution of double-labeled ChiTn with ^99mTc and ¹³¹I at 48h postinjection (Fig. 4 top panel) resulted in a profile with higher values for ^99mTc in the liver, spleen, and kidneys, and a tendency to show higher values in Tn− and Tn+ tumors, although this was not significant. The ¹³¹I shows a high preference for the thyroid and is also significantly higher in the stomach. No significant differences were found in the T/B and T/M ratios.

FIG. 4.

Biodistribution profile of double-labeled ChiTn (^99mTc and ¹³¹I; top panel) and co-injection of IgG-HYNIC-^99mTc and ChiTn-¹³¹I (bottom panel) at 48 h, in mice with SC Tn− or Tn+ tumors. The figure displays elimination (urine+feces), T/B, and T/M ratios for each tracer. ns: p ≤ 1e⁺⁰⁰; *, 1e⁻⁰² < p ≤ 5e⁻⁰²; **, 1e⁻⁰³ < p ≤ 1e⁻⁰²; ***, 1e⁻⁰⁴ < p ≤ 1e⁻⁰³; ****, p ≤ 1e⁻⁰⁴.

Also, the biodistribution of the co-injection of IgG-HYNIC-^99mTc and ChiTn-¹³¹I was performed at 48 h (Fig. 4 bottom panel). Most of the organs showed a higher % ID/g of IgG-HYNIC-^99mTc compared with ChiTn-¹³¹I, even in the Tn− tumor, but not in the Tn+. Increased uptake was also found in the thyroid by ChiTn-¹³¹I. No significant differences were found in the T/B and T/M ratios, for both biodistributions. All values, except for thyroid, are comparable with Figure 3 and Supplementary Figure S1 (Data available at https://zenodo.org/record/8110446).

Finally, the biodistribution of ChiTn-HYNIC-^99mTc and IgG-HYNIC-^99mTc at 48 h in mice pretreated with Losartan is shown (Fig. 5) (data available at https://zenodo.org/record/8110446). Although the profile was similar to Figures 3 and 4, interestingly a higher uptake was observed in Tn+ than in Tn− tumors for ChiTn-HYNIC-^99mTc. Also, higher T/B and T/M ratios were observed between Tn+ and Tn− tumors for ChiTn-HYNIC-^99mTc. No significant differences between Tn+ and Tn− tumors were observed for IgG-HYNIC-^99mTc. The average results and their SD are summarized in Table 1.

FIG. 5.

Biodistribution profile of ChiTn-HYNIC-^99mTc and IgG-HYNIC-^99mTc in mice treated with Losartan, at 48 h, with SC Tn− or Tn+ tumors. The figure displays elimination (urine+feces), T/B, and T/M ratios for both tracers. ns: p ≤ 1e⁺⁰⁰; *, 1e⁻⁰² < p ≤ 5e⁻⁰²; **, 1e⁻⁰³ < p ≤ 1e⁻⁰²; ***, 1e⁻⁰⁴ < p ≤ 1e⁻⁰³; ****, p ≤ 1e⁻⁰⁴.

Table 1.

Total 48 h Biodistribution Results for Tn+ and Tn− Tumors, Including % ID/g Averages, Standard Deviations, Tumor-to-Blood, and Tumor-to-Muscle Ratios

Molecule	Pretreatment	Tn− % ID/g (SD)	Tn+ % ID/g (SD)	Tn− T/B (SD)	Tn+ T/B (SD)	Tn− T/M (SD)	Tn+ T/M (SD)
ChiTn-HYNIC-^99mTc	Untreated	10.3 (±3.5)	11.3 (±2.5)	0.5 (±0.2)	0.8 (±0.5)	5.2 (±2.9)	7.7 (±3.3)
IgG-HYNIC-99mTc	Untreated	14.5 (±0.7)	14.7 (±2.6)	0.8 (±0.3)	0.6 (±0.1)	5.7 (±0.8)	8.4 (±3.6)
ChiTn-¹³¹I	Untreated	5.2 (±1.1)	9.1 (±2.2)	0.5 (±0.3)	0.5 (±0.2)	2.7 (±0.9)	5.3 (±2.6)
ChiTn-HYNIC-99mTc	Losartan	7.9 (±1.2)	14.9 (±2.1)	0.6 (±0.1)	1.2 (±0.2)	4.3 (±0.8)	10.4 (±3.3)
IgG-HYNIC-99mTc	Losartan	10.9 (±2.5)	8.4 (±1.9)	0.5 (±0.2)	0.4 (±0.1)	4.6 (±1.9)	3.4 (±1.6)

% ID/g, percent injected dose per gram of tissue; ¹³¹I, iodine-131; ^99mTc, technetium-99m; SD, standard deviation; T/B, tumor-to-blood; T/M, tumor-to-muscle.

Discussion

This study principally focused on the dynamics of the mAb ChiTn, radiolabeled for potential diagnostic and theranostic applications in cancer. Although various barriers such as interstitial resistance, hypoxia, and acidic conditions are known to impede the penetration of anticancer agents,^46

–50 the administration of Losartan can play an adjunctive role in modifying the tumor stroma and enhancing antibody delivery by alleviating solid stress.^17,20,21 The findings provide insights into the distribution and targeting efficiency of radiolabeled ChiTn to Tn+ tumors, contributing to a better understanding of antibody-based cancer diagnostics and therapy.

The experimental findings demonstrate that the radiochemical purity of both ^99mTc and ¹³¹I radiolabeled antibodies remained consistently >92% even after a 48 h incubation period in serum at 37°C (Fig. 1). These results align with previous studies that reported similar levels of radiochemical purity and stability over time using the same antibody labeling methodology with ^99mTc^30,31 and ¹³¹I.⁵¹

Different binding and internalization capacities of the ChiTn mAb to tumor cell lines expressing the Tn-antigen have been observed,^7,8,52 In addition, it is rapidly internalized by Tn+ tumor cells and primarily localizes in early endosomes.^7,8 The results show that membrane-bound and internalization of the labeled antibody in Tn+ and Tn− LL/2 cells exhibit slower kinetics than the unlabeled antibody, but are similar to those of other radiolabeled antibodies.^30,53

The ^99mTc labeling process involves the reaction of HYNIC with, for example, the ɛ-amino group of antibody lysines.^54,55 Similarly, for the chemical oxidation process used in ¹³¹I labeling, sodium iodide is converted into a reactive form that can be incorporated into the tyrosyl groups of the antibody.^56,57 Therefore, modification by radioactive labeling can alter the physical and chemical properties of the antibody, which can affect its interaction with cells.⁵⁸

ChiTn-HYNIC-^99mTc showed persistent membrane binding after 24 h, favoring Tn+ cells significantly (Fig. 2A). Notable Tn+ internalization was observed after 4 h (Fig. 2A). ChiTn-¹³¹I yielded similar results unlike the control IgG-HYNIC-^99mTc, which showed minimal binding and internalization with no significant Tn+ and Tn− differences (Fig. 2B). Figure 2C illustrates the disparities in Tn+/Tn− ratios.

Although intermediate affinities of radiolabeled antibodies may hinder binding and internalization, studies by Rudnick et al. have shown that low-affinity mAbs can penetrate solid tumors more efficiently.⁵⁹ To investigate the uptake and retention ability of radiolabeled ChiTn and IgG in Tn+ and Tn− tumors in vivo, the authors conducted various biodistribution studies.

The biodistribution results of ChiTn-HYNIC-^99mTc indicated a typical distribution pattern for ^99mTc radiolabeled antibodies through HYNIC, as reported by Camacho et al.³⁰ The radiotracer exhibited slow clearance in the bloodstream, liver, heart, and lungs, and sustained tumor uptake up to 48 h (11.3 ± 2.5% ID/g), with no significant differences between Tn+ and Tn− tumors or T/B and T/M ratios (Figs. 2 and 3 and Supplementary Fig. S1). Similar results were seen for i.d. and s.c. injections of LL/2 Tn+ and Tn− cells in mice. These results indicate that the uptake of ChiTn-HYNIC-^99mTc is not an artifact of the injection route of the tumor models. Tumors injected i.d. and s.c. may differ in their microenvironment due to increased immunogenicity in i.d. tumors because of a higher density of dendritic cells.⁶⁸

It should be clarified that no ChiTn excess biodistribution studies were conducted, relying on the specificity established in prior work.^8,11 Future experiments will consider this, contingent on increased ChiTn production.

To assess the impact of the radioactive labeling method on biodistribution,⁶⁰ the authors performed dual-labeling of the ChiTn antibody with ^99mTc and ¹³¹I. This enabled simultaneous measurement of biodistribution using gamma spectrometry,³⁹ thereby minimizing inter-mouse, inter-measurement, and handling variability.³⁸ Double-labeled ChiTn exhibited higher values for ^99mTc in the liver, spleen, and kidneys, with a slight tendency for higher values in both Tn− and Tn+ tumors (Fig. 4 top panel), but this was not significant. The ¹³¹I exhibited a high affinity for the thyroid and was also significantly higher in the stomach. The T/B and T/M ratios did not show any significant differences.

When radioiodinated antibodies are taken up by cells, they are quickly broken down in lysosomes, resulting in the release of monoiodotyrosine into the extracellular space. This metabolite is further broken down by deiodination enzymes, which release free radioiodide into the bloodstream.⁶¹ The radioiodide is then taken up by any tissues expressing the sodium-iodide (Na⁺/I⁻) symporter, which is present in the thyroid gland and stomach. The lysosomal degradation of radioiodinated antibodies leads to the rapid clearance of radioiodine from all tissues, except those that metabolize or process iodine.⁶² This occurrence leads to diminished activity concentrations within the tumor tissue in comparison with residualizing radiolabels that employ the radiometal ^99mTc.⁶³

The accumulation of the antibody within tumors ideally would solely rely on the specific target antigen. However, challenges arise when nonspecific factors contribute to overall tumor uptake. One example is the enhanced permeability and retention (EPR) effect, which occurs due to rapid and irregular angiogenesis, resulting in antibodies passively extravasating into the tumor tissue through the leaky vasculature.^64,65 These nonspecific contributions to tumor uptake can vary significantly between tumor models, within a single tumor (intratumoral heterogeneity), or because of different responses to treatment (intertumoral heterogeneity). Consequently, the sensitivity of these techniques may be reduced, increasing the likelihood of false discoveries.⁶⁶

The authors conducted an additional experiment to assess nonspecific uptake by comparing the biodistribution of Tn-specific ChiTn-¹³¹I with that of nonspecific IgG-HYNIC-^99mTc. Most of the organs showed a higher % ID/g of IgG-HYNIC-^99mTc compared with ChiTn-¹³¹I (Fig. 4 bottom panel). This is expected since the radioactive label with ¹³¹I tends to be more unstable in vivo, leading to increased uptake in the thyroid and stomach, as well as greater clearance from organs, as previously discussed.

Nevertheless, the Tn+ tumor demonstrated a comparable uptake of ChiTn-¹³¹I to that of IgG-HYNIC-^99mTc, whereas a reduced uptake was observed in Tn− tumors. Once again, no distinct advantage in uptake was noted for the Tn-specific ChiTn as compared with the nonspecific IgG. This observation is further corroborated by the similarities in the T/B and T/M ratios (Fig. 4 below).

The presence of solid stress may be a contributing factor hindering the efficient delivery of antibodies to tumors, particularly in highly vascularized tumors such as LL/2 Tn+. These tumors produce higher levels of VEGF compared with LL/2 wild type (Tn−), as demonstrated by da Costa et al.¹¹ VEGF is recognized as a key factor in angiogenesis, capable of inducing ECM synthesis and promoting the angio-fibrotic switch in fibrosis, as documented by Larsson-Callerfelt et al.,¹² Kuiper et al.,¹³ and Zhang and Chu.³⁴ The fibrotic ECM significantly contributes to elevated solid stress within tumors.¹⁵

To assess this phenomenon, biodistribution studies were conducted after pretreatment with Losartan, a compound known to alleviate solid stress in fibrotic tumors.^17,20 Previous research has indicated that Losartan treatment does not alter VEGF levels or microvessel density. However, it has been shown to significantly enhance the percentage of perfused blood vessels.²¹

The % ID/g values indicate that the biodistribution profiles of normal tissues in the Losartan pretreated mice (Fig. 5) were comparable with those of the untreated counterparts at 48 h postinjection for both IgG-HYNIC-^99mTc and ChiTn-HYNIC-^99mTc, which aligns with the findings of Chauhan et al.,¹⁷ where Losartan treatment does not affect accumulation in normal tissues. However, a slightly higher uptake in the kidneys and elimination through urine and feces were observed for ChiTn-HYNIC-^99mTc (Fig. 5).

In addition, ChiTn-HYNIC-^99mTc demonstrated a higher blood clearance compared with IgG-HYNIC-^99mTc, as evident in Figure 5, consistent with the outcomes of the previous biodistribution studies where ChiTn-HYNIC-^99mTc values ranged from 15% to 20% ID/g and IgG-HYNIC-^99mTc values ranged from 22% to 30% ID/g at 48 h in bloodstream (Figs. 3–5).

In mice pretreated with Losartan, a significantly higher uptake of ChiTn-HYNIC-^99mTc was observed in Tn+ tumors (% ID/g 14.9 ± 2.1) compared with Tn− tumors (% ID/g 7.9 ± 1.2; p ≤ 0.05), as indicated in Table 1 and Figure 5. However, there was no significant change in the uptake of IgG-HYNIC-^99mTc in either tumor type.

These findings are further supported by the T/B and T/M ratios (Table 1 and Fig. 5). ChiTn-HYNIC-^99mTc demonstrated significantly higher ratios in favor of Tn+ over Tn− tumors (p ≤ 0.01 and p ≤ 0.05, respectively), whereas IgG-HYNIC-^99mTc did not show the same pattern. Specifically, the T/B ratio in Tn+ tumors increased from 0.8 ± 0.5 in untreated mice to 1.2 ± 0.2 in mice pretreated with Losartan for ChiTn-HYNIC-^99mTc. The T/M ratio also exhibited a moderate increase from 7.7 ± 3.3 to 10.4 ± 3.3.

These values indicate a substantial increase in Tn+ uptake compared with Tn− in mice pretreated with Losartan, with an average of 88% higher uptake in Tn+ tumors. In addition, there was a significant enhancement in the T/B ratio, with a 50% increase. Similarly, the T/M ratio exhibited a 35% increase in Tn+ tumors. All of these values closely align with the findings reported by Chauhan et al.,¹⁷ where Losartan treatment resulted in a 74% increase in the accumulation of the small-molecule chemotherapeutic 5-FU in AK4.4 pancreatic tumors.

The findings indicate that LL/2 Tn+ tumors exhibit elevated solid stress compared with their wild-type (Tn−) counterparts. As a result, Losartan is expected to be more effective in alleviating this stress specifically in Tn+ tumors. In addition, the radiolabeled ChiTn mAb demonstrates enhanced penetration into this tumor type, allowing for increased interaction with the Tn antigen. In contrast, nonspecific IgG lacks the capability to undergo specific binding and subsequent internalization into tumor cells, leading to a lack of this effect. Its uptake primarily occurs through nonspecific binding or due to its higher concentration in the bloodstream.

Although Losartan may increase the blood flow to the tumor by alleviating solid stress, and this could lead to an increase in tumor size, the use of Losartan should be understood as an adjuvant treatment in conjunction with a therapeutic drug. Even so, Kumar et al.⁶⁷ found that the administration of 5FU, Doxil, or Losartan alone did not affect the growth of pancreatic tumors, but tumors were significantly smaller in mice treated with Losartan combined with either Doxil or 5FU.

The authors' data suggest a differential modulation by Losartan when used as an adjuvant therapy on Tn− versus Tn+ tumors, with an observed augmentation in ChiTn antibody uptake specifically in Tn+ tumors. This phenomenon implies that Losartan may selectively enhance the delivery and therapeutic efficacy of ChiTn in Tn+ tumor environments. It is essential, therefore, that the authors' observations serve as a catalyst for subsequent targeted investigations dedicated to elucidating the distinct mechanisms by which Losartan interacts with and potentially alters the tumor microenvironment in both Tn− and Tn+ malignancies. Such focused studies are critical to fully understand the role of Losartan in modifying ChiTn uptake and to determine the translational potential of these findings in the context of therapeutic interventions.

In conclusion, this study provides insights into the potential use of radiolabeled ChiTn in combination with Losartan for improved delivery and targeting of Tn+ tumors, which may have clinical implications for the treatment of epithelial cancers expressing the Tn-antigen. Further studies are warranted to explore the theranostic efficacy of this approach and optimize the radiolabeling and targeting strategies.

Data Availability Statement

Comprehensive data sets for in vitro and biodistribution assays can be accessed through this link: https://zenodo.org/record/8110446

Footnotes

Acknowledgments

This study is part of the Master's and Doctoral Program in Medical Sciences (Proinbio) from the Facultad de Medicina at the Universidad de la Republica, Uruguay, where M.T., MSc, is pursuing his doctorate in medical science. The authors would like to extend special thanks to Roger Chammas, MD, PhD, professor of oncology and deputy dean at the faculty of medicine of the University of São Paulo and the Cancer Institute of the State of São Paulo, for his insightful advice on the use of Losartan for tumor delivery.

Authors' Contributions

M.T., X.C., C.P., M.C., M.F.G., M. F., J.P.G., and P.C. participated in the antibody radiolabeling and in vitro/in vivo studies in both models. T.F., V.C., and E.O. developed cell lines, tumor models, and the ChiTn antibody. M.T. drafted the article. All authors participated in the editing and review of the article.

Disclosure Statement

The authors declare no competing interests.

Funding Information

This work was funded by “Comisión Sectorial de Investigación Científica CSIC I+D 2018 - Imagenología Molecular en Oncología”, Uruguay.

Supplementary Material

Supplementary Figure S1

References

Buss

, Henderson

, McFarlane

, et al. Monoclonal antibody therapeutics: History and future. Curr Opin Pharmacol, 2012; 12:615–622.

Hafeez

, Gan

, Scott

. Monoclonal antibodies as immunomodulatory therapy against cancer and autoimmune diseases. Curr Opin Pharmacol, 2018; 41:114–121.

Kaplon

, Reichert

. Antibodies to watch in 2021. MAbs, 2021; 13:1860476.

Martineau

, Watier

, Pèlegrin

, et al. Targets for MAbs: Innovative approaches for their discovery & validation. MAbs. 2019; 11(5):812–825. doi: 10.1080/19420862.2019.1612691

Hakomori

. Glycosylation defining cancer malignancy: New wine in an old bottle. Proc Natl Acad Sci U S A, 2002; 99:10231–10233.

, Otto

, Cummings

, et al. The Tn antigen-structural simplicity and biological complexity. Angew Chem Int Ed Engl, 2011; 50:1770–1791; doi: 10.1002/anie.201002313

Hubert

, Heitzmann

, Viel

, et al. Antibody-dependent cell cytotoxicity synapses form in mice during tumor-specific antibody immunotherapy. Cancer Res, 2011; 71(15):5134–5143; doi: 10.1158/0008-5472

Sedlik

, Heitzmann

, Viel

, et al. Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen. Oncoimmunology, 2016; 5(7):e1171434; doi: 10.1080/2162402X.2016.1171434

, Zhao

, Wang

, et al. Tumor-associated antigens: Tn antigen, sTn antigen, and T antigen. HLA, 2016; 88(6):275–286; doi: 10.1111/tan.12900

10.

Liu

, Liu

, Dong

, et al. Tn antigen promotes human colorectal cancer metastasis via H-Ras mediated epithelial-mesenchymal transition activation. J Cell Mol Med, 2019; 23(3):2083–2092; doi: 10.1111/jcmm.14117

11.

da Costa

, van Vliet

, Carasi

, et al. The Tn antigen promotes lung tumor growth by fostering immunosuppression and angiogenesis. Cancer Lett, 2021; 518:72–81; doi: 10.1016/j.canlet.2021.06.012

12.

Larsson-Callerfelt

, Andersson Sjöland

, Hallgren

, et al. VEGF induces ECM synthesis and fibroblast activity in human lung fibroblasts. Eur Respir J, 2017; 50(Suppl 61):PA1045; doi: 10.1183/1393003.congress-2017.PA1045

13.

Kuiper

, Van Nieuwenhoven

, de Smet

, et al. The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy. PLoS One, 2008; 3(7):e2675; doi: 10.1371/journal.pone.0002675

14.

Zhang

, Chu

. Differential roles of VEGF: Relevance to tissue fibrosis. J Cell Biochem, 2019; 120:10945–10951.

15.

Jain

. Antiangiogenesis strategies revisited: From starving tumors to alleviating hypoxia. Cancer Cell, 2014; 26:605–622.

16.

Liu

, Liao

, Diop-Frimpong

, et al. TGF-β blockade improves the distribution and efficacy of therapeutics in breast carcinoma by normalizing the tumor stroma. Proc Natl Acad Sci U S A, 2012; 109:16618–16623.

17.

Chauhan

, Martin

, Liu

, et al. Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat Commun, 2013; 4:2516–2527.

18.

Nia

, Liu

, Seano

, et al. Solid stress and elastic energy as measures of tumour mechanopathology. Nat Biomed Eng, 2016; 1:4–29.

19.

Stylianopoulos

, Munn

, Jain

. Reengineering the physical microenvironment of tumors to improve drug delivery and efficacy. Trends Cancer, 2018; 4:292–319.

20.

Diop-Frimpong

, Chauhan

, Krane

, et al. Losartan inhibits collagen I synthesis and improves the distribution and efficacy of nanotherapeutics in tumors. Proc Natl Acad Sci U S A, 2011; 108:2909–2914.

21.

Zhao

, Cao

, Melamed

, et al. Losartan treatment enhances chemotherapy efficacy and reduces ascites in ovarian cancer models. Proc Natl Acad Sci U S A, 2019; 116(6):2210–2219; doi: 10.1073/pnas.1818357116

22.

Ando

, Matsushita

, Wakitani

, et al. Mouse-human chimeric anti-Tn IgG1 induced anti-tumor activity against Jurkat cells in vitro and in vivo. Biol Pharm Bull, 2008; 31:1739–1744.

23.

Welinder

, Baldetorp

, Borrebaeck

, et al. A new murine IgG1 anti-Tn monoclonal antibody with in vivo antitumor activity. Glycobiology, 2011; 21:1097–1107; doi: 10.1093/glycob/cwr048

24.

Scott

, Wolchok

, Old

, et al. Antibody therapy of cancer. Nat Rev Cancer, 2012; 12:278–287; doi: 10.1038/nrc3236

25.

Vacchelli

, Galluzzi

, Eggermont

, et al. Trial watch: Monoclonal antibodies in cancer therapy. Oncoimmunology, 2013; 2:e22789; doi: 10.4161/onci.22789

26.

Chau

, Steeg

, Figg

, et al. Antibody-drug conjugates for cancer. Lancet, 2019; 394:793–804.

27.

Dovgan

, Kolodych

, Koniev

, et al. Antibody-drug conjugates and other nanomedicines: The frontier of gynaecological cancer treatment. Bioconjug Chem, 2019; 30:2483–2501.

28.

Steiner

, Neri

. Antibody-radionuclide conjugates for cancer therapy: Historical considerations and new trends. Clin Cancer Res, 2011; 17:6406–6416.

29.

Audicio

, Castellano

, Tassano

, et al. [177Lu] DOTA-anti-CD20: Labeling and pre-clinical studies. Appl Radiat Isot, 2011; 69(7):924–928; doi: 10.1016/j.apradiso.2011.01.026

30.

Camacho

, García

, Calzada

, et al. ^99mTc-labeled bevacizumab via HYNIC for imaging of melanoma. J Anal Oncol, 2014; 3(1):53–64; doi: 10.6000/1927-7229.2014.03.01.9

31.

Camacho

, Machado

, García

, et al. Technetium-99m- or Cy7-labeled rituximab as an imaging agent for non-Hodgkin lymphoma. Oncology, 2017; 92(4):229–242; doi: 10.1159/000452419

32.

Harsini

, Rezaei

. Cancer Imaging with Radiolabeled Monoclonal Antibodies. In: Cancer Immunology. ( Rezaei

. ed.) Springer: Cham, 2020; pp. 739–760; doi: 10.1007/978-3-030-30845-2_32

33.

Lin

, Paolillo

, Le

DB et al

. Monoclonal antibody based radiopharmaceuticals for imaging and therapy. Curr Probl Cancer, 2021; 45(5):100796; doi: 10.1016/j.currproblcancer.2021.100796

34.

Zhang

, Zhao

, Jiang

et al. Radioimmunotherapy study of ¹³¹I-labeled Atezolizumab in preclinical models of colorectal cancer. Eur J Nucl Med Mol Imaging, 2022; 12:70; doi: 10.1186/s13550-022-00939-2

35.

Duatti

. Review on ^99mTc radiopharmaceuticals with emphasis on new advancements. Nucl Med Biol, 2021; 92:202–216; doi: 10.1016/j.nucmedbio.2020.05.005

36.

Osinaga

, Bay

, Tello

, et al. Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy. FEBS Lett, 2000; 469(1):24–28; doi: 10.1016/s0014-5793(00)01248-5

37.

García

, Calzada

, Camacho

, et al. Microwave-assisted synthesis of HYNIC protected analogue for ^99mTc labeled antibody. Curr Radiopharm, 2014; 7(2):84–90.

38.

Knight

, Mosley

, Kersemans

, et al. Dual-isotope imaging allows in vivo immunohistochemistry using radiolabelled antibodies in tumours. Nucl Med Biol, 2019; 70:14–22; doi: 10.1016/j.nucmedbio.2019.01.010

39.

Tassano

, Cabrera

, Camacho

, et al. Simultaneous biodistribution studies of Tc-99m/I-131 labeled antibody with HPGe gamma spectrometry. Eur J Nucl Med Mol Imaging, 2021; 48:423–424.

40.

Raybaut

. Spyder-Documentation. 2009. Available from: Pythonhosted.org [Last accessed: October 1, 2023].

41.

McKinney

. Data Structures for Statistical Computing in Python. In: Proceedings of the 9th Python in Science Conference, Austin, Texas; 2010; pp. 51–56.

42.

Hunter

. Matplotlib: A 2D graphics environment. Comput Sci Eng, 2007; 9:90–95. doi: 10.1109/MCSE.2007.55

43.

Harris

, Millman

, van der Walt

, et al. Array programming with NumPy. Nature, 2020; 585:357–362; doi: 10.1038/s41586-020-2649-2

44.

Waskom

. seaborn: Statistical data visualization. J Open Source Softw, 2021; 6(60):3021; doi: 10.2110/5/joss.03021

45.

Charlier

, Weber

, Izak

, et al. Statannotations (v0.5). Zenodo: Geneva, Switzerland;, 2022; doi: 10.5281/zenodo.7213391

46.

Curti

. Physical barriers to drug delivery in tumors. Crit Rev Oncol Hematol, 1993; 14:29–39; doi: 10.1016/1040-8428(93)90004-N

47.

Sriraman

, Aryasomayajula

, Torchilin

. Barriers to drug delivery in solid tumors. Tissue Barriers, 2014; 2:e29528; doi: 10.4161/tisb.29528

48.

Libutti

, Tamarkin

, Nilubol

. Targeting the invincible barrier for drug delivery in solid cancers: Interstitial fluid pressure. Oncotarget, 2018; 9:35723–35725.

49.

Jain

. Barriers to drug delivery in solid tumors. Sci Am, 1994; 271(1):58–65.

50.

Anchordoquy

, Barenholz

, Boraschi

, et al. Mechanisms and barriers in cancer nanomedicine: Addressing challenges, looking for solutions. ACS Nano, 2017; 11(1):12–18; doi: 10.1021/acsnano.6b08244

51.

Vinod

, Kim

, Choi

, et al. Combination of ¹³¹I-trastuzumab and lanatoside C enhanced therapeutic efficacy in HER2 positive tumor model. Sci Rep, 2021; 11:12871; doi: 10.1038/s41598-021-92460-0

52.

Castro

, Berois

, Malanga

, et al. Docetaxel in chitosan-based nanocapsules conjugated with an anti-Tn antigen mouse/human chimeric antibody as a promising targeting strategy of lung tumors. Int J Biol Macromol, 2021; 182:806–814; doi: 10.1016/j.ijbiomac.2021.04.054

53.

Kuo

, Lin

, Hsu

, et al. Noninvasive assessment of characteristics of novel anti-HER2 antibodies by molecular imaging in a human gastric cancer xenograft-bearing mouse model. Sci Rep, 2018; 8:13735; doi: 10.1038/s41598-018-32094-x

54.

García

, Zhang

, Shah

, et al. (99m)Tc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging. Bioorg Med Chem, 2016; 24(6):1209–1215; doi: 10.1016/j.bmc.2016.01.046

55.

Meszaros

, Dose

, Biagini

, et al. Hydrazinonicotinic acid (HYNIC)—Coordination chemistry and applications in radiopharmaceutical chemistry. Inorg Chim Acta, 2010; 363:1059–1069.

56.

Gupta

, Batra

, Jain

. Antibody labeling with radioiodine and radiometals. Methods Mol Biol, 2014; 1141:147–157; doi: 10.1007/978-1-4939-0363-4_9

57.

Feng

, Meshaw

, McDougald

, et al. Evaluation of an ¹³¹I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers. Sci Rep, 2022; 12:3020; doi: 10.1038/s41598-022-07006-9

58.

Tolmachev

, Orlova

, Andersson

. Methods for radiolabelling of monoclonal antibodies. Methods Mol Biol, 2014; 1060:309–330.

59.

Rudnick

, Lou

, Shaller

, et al. Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors. Cancer Res, 2011; 71:2250–2259; doi: 10.1158/0008-5472.CAN-10-2277

60.

Williams

. Comparison of iodine-labeled and indium-labeled antibody biodistributions. Med Phys, 2014; 41:133; doi: 10.1118/1.4887962

61.

Press

, Shan

, Howell-Clark

, et al. Comparative metabolism and retention of Iidine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res, 1996; 56(9):2123–2129.

62.

Vivier

, Sharma

, Zeglis

. Understanding the in vivo fate of radioimmunoconjugates for nuclear imaging. J Labelled Comp Radiopharm, 2018; 61(9):672–692; doi: 10.1002/jlcr.3628

63.

Deyev

, Vorobyeva

, Schulga

, et al. Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1. Int J Biol Macromol, 2020; 145:216–225.

64.

Heneweer

, Holland

, Divilov

, et al. Magnitude of enhanced permeability and retention effect in tumors with different phenotypes: 89Zr-albumin as a model system. J Nucl Med, 2011; 52:625–633.

65.

Fang

, Nakamura

, Maeda

. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev, 2011; 63:136–151.

66.

Börjesson

PKE

, Jauw

YWS

, Boellaard

, et al. Performance of immuno–positron emission tomography with zirconium-89-labeled chimeric monoclonal antibody U36 in the detection of lymph node metastases in head and neck cancer patients. Clin Cancer Res, 2006; 12:2133.

67.

Kumar

, Boucher

, Liu

, et al. Noninvasive assessment of Losartan-induced increase in functional microvasculature and drug delivery in pancreatic ductal adenocarcinoma. Transl Oncol, 2016; 9(5):431–437; doi: 10.1016/j.tranon.2016.07.004

68.

Bonnotte

, Gough

, Phan

, et al. Intradermal injection, as opposed to subcutaneous injection, enhances immunogenicity and suppresses tumorigenicity of tumor cells. Cancer Res, 2003; 63(9):2145–2149.

69.

Cicone

, Denoël

, Gnesin

, et al. Preclinical evaluation and dosimetry of [111In]CHX-DTPA-scFv78-Fc targeting endosialin/tumor endothelial marker 1 (TEM1). Mol Imaging Biol, 2020; 22:979–991; doi: 10.1007/s11307-020-01479-8

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.13 MB