Combination of racotumomab immunotherapy with programmed death-1 blockade in a preclinical model of non-small cell lung cancer

Introduction

Lung cancer is one of the most commonly diagnosed malignancies and the leading cause of cancer-related deaths in both men and women (1). Notably, non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is typically diagnosed at an advanced stage (2). Therapeutic options for NSCLC include surgery for early-stage diagnoses, chemotherapy, radiotherapy, molecular targeted therapies, and more recently, immunotherapy utilizing immune checkpoint blockade (ICB) (3). Anti-programmed death-1 (PD-1) monoclonal antibodies (mAbs), pembrolizumab, and nivolumab, were initially approved as second-line treatments, achieving unprecedented results. The use of pembrolizumab was further extended as a first-line treatment for advanced NSCLC with expression of programmed death-ligand 1 (PD-L1) on at least 50% of tumor cells or in combination with chemotherapy in those patients whose tumors have PD-L1 less than 50% (4,5). Despite improvements in patient survival and quality of life since the advent of ICB, lung cancer prognosis remains poor. In this scenario, a combination of immunotherapeutic agents appears to be a promising approach for exploring new possibilities.

Anti-idiotype antibodies mimic the structure of the antigen of interest and can be used as vaccines to enhance specific immune responses. Racotumomab is an anti-idiotype mAb directed against N-glycolylneuraminic acid (NeuGc)-containing glycoconjugates (6), which has been approved in Latin American countries as switch maintenance immunotherapy for advanced NSCLC (7). Normal human cells cannot synthesize NeuGc because of a deletion of the gene encoding the key enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) (8). However, glycoconjugates such as ganglioside NeuGcGM3 have been identified as neoantigens in NSCLC in humans, and their presence is associated with poor prognosis (9-11). It is widely reported that aggressive human tumor cells can take up NeuGc from dietary sources and incorporate it into newly synthesized NeuGc-containing gangliosides (12-14). In contrast to humans, in normal murine tissues, NeuGc can be naturally synthesized by CMAH as a terminal glycan residue in cell membrane glycoconjugates, where it is also overexpressed as a tumor-associated antigen.

Preclinical studies have demonstrated that racotumomab increases CD8⁺ tumor-infiltrating T lymphocytes, induces tumor apoptosis (15), and reduces tumor blood vessels (16). Using a clinically relevant NSCLC mouse model with high NeuGc expression developed by our team, we demonstrated for the first time the feasibility of combining racotumomab with chemotherapy. We also observed the induction of NeuGcGM3-specific antibodies in the sera of tumor-bearing mice treated with racotumomab (17). In NSCLC patients treated with racotumomab, the presence of anti-NeuGcGM3 antibodies was confirmed, which can mediate the lysis of antigen-bearing cells via an oncosis-like mechanism (18), and patients who developed target-specific serum antibodies showed longer median survival times (19). Furthermore, serum samples from racotumomab-treated patients in a phase III trial revealed antigen-specific antibody-dependent cell-mediated cytotoxicity against NeuGcGM3-expressing cells (20).

The success of ICB therapies in treating various cancer types, including NSCLC, underscores the importance of reactivating the host immune response against tumors. Considering that the combination of ICB with other active immunotherapies against tumor-associated antigens is an interesting option (21-23), our study aimed to evaluate the antitumor effects of an anti-murine PD-1 mAb in combination with racotumomab immunotherapy in a preclinical mouse model of NSCLC. Additionally, we validated the antitumor activity of racotumomab in a humanized mouse model, while examining the potential impact of a NeuGc-rich diet. We present this article in accordance with the ARRIVE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-230/rc).

Methods

Racotumomab and anti-PD-1 mAb

Racotumomab (Vaxira^®) was obtained from ELEA Laboratories (Buenos Aires, Argentina). The final formulation was obtained by mixing racotumomab with aluminum hydroxide as an adjuvant at a final concentration of one mg/mL of purified anti-idiotype mAb. The anti-murine PD-1 mAb (clone RMP1-14) was obtained from Leinco Technologies, Inc. (USA) (Leinco CAT# P372, RRID: AB_1085306).

Tumor cells and culture conditions

We used the Lewis lung carcinoma (LLC) cell line, recently characterized as a lung adenocarcinoma with high mutational burden and metastatic potential, in syngeneic C57BL/6 mice (ATCC Cat# CRL-1642, RRID: CVCL_4358) (24). Tumor cells were grown in Dulbecco’s modified Eagle media (DMEM) culture medium containing 10% heat-inactivated fetal bovine serum (FBS) at 37 ℃ in a humidified atmosphere containing 5% CO₂. Cells were subcultured three times a week using a trypsin-ethylenediaminetetraacetic acid (EDTA) solution, cell viability was assessed using the trypan blue exclusion technique, and mycoplasma infection was routinely tested.

Animals

Pathogen-free standard C57BL/6 mice, 8–12 week-old (male and females), were obtained from the Animal Care Division of the National University of La Plata (La Plata, Argentina). CMAH-deficient (CMAH −/−) female mice were obtained from The Jackson Laboratory (USA) (RRID: IMSR_JAX:017588) and maintained under specific pathogen-free conditions at the Laboratory Animal Biotechnology Unit of Institut Pasteur de Montevideo, Uruguay. Briefly, they were generated by introducing an exon deletion similar to the human CMAH mutation into embryonic stem cells and were bred into C57BL/6 females, as previously described (25). CMAH deficiency results in the complete loss of NeuGc expression in normal murine tissues, making CMAH −/− mice a valuable humanized model for studying the effects of a NeuGc-rich diet.

Up to five to six mice were housed per cage under enriched and controlled environmental conditions at the animal facility of Quilmes National University (Quilmes, Argentina). They had access to food and water provided ad libitum, and were maintained on a regulated photoperiod of 12 hours of light followed by 12 hours of darkness. The general health condition was monitored daily and animal weight was assessed twice a week. Animals were excluded from the study if they presented body-weight reduction of 20%, or if they demonstrated any evidence of distress or discomfort. All experimental procedures were conducted at least one week after the animals had been habituated to the animal facility at Quilmes National University. The Animal Review Board of Quilmes National University approved all experimental protocols (Approval No. 010-15), and animal maintenance was conducted under accepted international standards for the care and use of animals. Protocols were prepared before the study without registration.

Flow cytometry analysis

Tumor cells were harvested using trypsin-EDTA solution and resuspended in serum-free DMEM. A total of 1×10⁶ cells per sample were incubated with an anti-PD-L1 mAb, clone 10F.9G2 (BioLegend Cat# 124302, RRID: AB_961228) or phosphate buffered saline (PBS) as a negative control for 30 minutes at 4 ℃. After washing, the cells were incubated with polyclonal goat anti-rat fluorescein-conjugated antibody (R and D Systems Cat# F0104B, RRID: AB_884223) for 30 min at 4 ℃. Acquisition was achieved using a Becton Dickinson FACSCalibur flow cytometer, and the data were analyzed using FlowJo^® software (RRID: SCR_008520).

Antitumor activity

The antitumor activity of racotumomab in combination with an anti-PD-1 mAb was evaluated in a preclinical NSCLC tumor model. LLC cells were preincubated with purified NeuGc and implanted into the lungs of C57BL/6 mice by intravenous (IV) injection, as previously described (17). We used two experimental approaches based on the concurrent or sequential administration of both treatments. In all experiments, a minimum of six to eight animals was used per experimental group, resulting in a total of 30 animals per experiment. Animals were randomly divided into the experimental groups.

Concurrent treatment (Figure 1A): mice were treated in the interscapular region with three subcutaneous (SC) doses of 200 µg of racotumomab on days −14, −7, and +7. We injected 1×10⁵ tumor cells per mouse (day 0), and at days +4 and +10, animals were treated with 200 µg of anti-PD-1 mAb in PBS via intraperitoneal (IP).

Figure 1 Schematic representation of concurrent (A) and sequential (B) treatments used for the preclinical evaluation of Raco in combination with an anti-PD-1 mAb. IP, intraperitoneal; IV, intravenous; LLC, Lewis lung carcinoma; mAb, monoclonal antibodie; NeuGc, N-glycolylneuraminic acid; PD-1, programmed cell death protein 1; Raco, racotumomab; SC, subcutaneous.

Sequential treatment (Figure 1B): Tumor cell injection was performed as described for concurrent treatment (day 0). Mice were injected with three SC doses of 200 µg of racotumomab on days +7, +14, and +21. On day +4, mice were treated with a single IP dose of 200 µg of anti-PD-1 mAb in PBS.

In both experimental approaches, control mice received 200 µL of PBS and an isotype control antibody (Leinco Technologies Cat# R1367, RRID: AB_2831721) under the same experimental conditions as those vaccinated with racotumomab or treated with anti-PD-1 mAb, respectively. All animals were euthanized on day 25 post-tumor cell inoculation. Lungs were subsequently harvested, fixed in Bouin’s solution, and examined under a dissecting microscope to enumerate and measure surface nodules for the evaluation of antitumor activity.

Racotumomab antitumor activity in humanized CMAH −/− mice

CMAH −/− mice were fed a NeuGc-rich diet consisting of beef fat and cattle bone powder (Premium chow, Argentina) for at least six weeks prior to tumor cell injection, and this diet was maintained throughout the entire protocol. The controls were fed standard rodent chow (Cooperación, Argentina). To evaluate the antitumor activity of racotumomab in this humanized mouse model, animals were injected with LLC cells and treated with racotumomab as described for sequential treatment. Mice were euthanized on day 25 post-tumor cell inoculation. Lungs were subsequently harvested, fixed in Bouin’s solution, and examined under a dissecting microscope for enumeration and measurement of surface nodules.

Biochemical analysis

To analyze blood toxicity in mice treated with the combination of anti-PD-1 therapy and racotumomab, at least five animals per group were anesthetized with a ketamine/xylazine combination, and blood was collected through cardiac puncture using sodium citrate as an anticoagulant. Immediately after blood samples were collected, the animals were euthanized by cervical dislocation. Whole blood was analyzed using an XS-1,000i Automated Hematology Analyzer through the service provided by Laboratorio Galizia (Don Bosco, Argentina).

Immunohistochemistry

Formalin-fixed lung specimens were processed using the standard paraffin technique. Briefly, after deparaffinization and hydration, the endogenous peroxidase activity was inhibited with a three percent volume of H₂O₂ solution. Antigen retrieval was performed according to the suggested protocol for each antibody. Lung sections were incubated with anti-CD8 mAb (Abcam Cat# ab217344, RRID: AB_2890649), anti-PD-L1 mAb (Cell Signaling Technology Cat# 64988, RRID: AB_2799672), or PBS as a negative control. The sections were incubated with biotinylated secondary antibodies, followed by peroxidase-conjugated avidin-biotin-horseradish peroxidase complex (Vector Laboratories, USA). Bound antibodies were detected by incubation with 3,3-diaminobenzidine as the chromogen, and the tumor sections were then counterstained with hematoxylin. The results were semi-quantitatively evaluated and scored by two independent observers, who compared specific staining intensity to that of the negative control.

Quantification of CD8⁺ tumor-infiltrating lymphocytes

Digital images of representative tumor areas, including regions with the highest density of infiltrating lymphocytes, were captured at 100× magnification using a light microscope equipped with a digital camera. Three to four representative high-power fields per experimental group were selected. Images were analyzed using the ImageJ/Fiji software (RRID: SCR_003070; RRID: SCR_002285). First, color deconvolution was applied to separate the diaminobenzidine chromogen signal, representing positive staining, from the hematoxylin counterstain. Thresholds were then set subjectively to distinguish positively stained cells from the background. Results were expressed as the percentage of the tumor area occupied by CD8⁺ T cells relative to the total tumor area.

Statistical analysis

Results were presented as mean values ± standard error of the mean (SEM) or standard deviation (SD) when indicated. Normal distribution of data was evaluated using D’Agostino and Pearson omnibus normality tests. To compare differences between two and multiple experimental groups, t-tests or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test were used. Differences were considered statistically significant at P<0.05. Data processing and statistical analyses were performed using the GraphPad Prism Software (RRID: SCR_002798).

Data availability

The data generated in this study are available upon request from the corresponding authors.

Results

Antitumor effect of anti-PD-1 therapy in a NSCLC mouse model with high expression of NeuGc-containing glycoconjugates

Although LLC cells express almost negligible amounts of NeuGc when maintained as monolayer cultures or injected subcutaneously, their expression increases significantly in lung lesions that spontaneously develop following the progression of SC heterotopic LLC tumors in syngeneic mice (26,27). As mentioned previously, we demonstrated racotumomab antitumor activity using an aggressive preclinical model with high expression of NeuGc-containing glycoconjugates developed by our group, in which LLC cells were pre-incubated with purified NeuGc (LLC-NeuGc) ex vivo before tumor cell injection. Despite the high variability in pulmonary nodule development after IV tumor cell inoculation and the inherently inefficient lung colonization process, this method establishes a reliable model for assessing immunotherapeutic strategies targeting the NeuGc antigen (17). Since combinatorial approaches involving long-term immunization against tumor-specific antigens together with ICB are attractive strategies for immunotherapy, the main objective of this study was to evaluate the antitumor activity of racotumomab in combination with ICB based on PD-1 inhibition. To this end, we first evaluated PD-L1 expression in LLC and LLC-NeuGc-cultured cells using flow cytometry. As shown in Figure 2, similar ligand expression was observed regardless of whether LLC cells were pre-incubated with NeuGc (Figure 2A,2B; P=0.43, ANOVA followed by Tukey’s multiple comparisons test). Considering that PD-L1 has already been reported in LLC tumors (28), we also assessed whether it was expressed in lung tumors that developed after injection of LLC-NeuGc cells using immunohistochemistry. Consistent with in vitro results, LLC-NeuGc tumors showed immunoreactivity to PD-L1 (Figure 2C), suggesting that the presence of NeuGc-containing glycoconjugates did not alter PD-L1 expression in LLC cells.

Figure 2 Antitumor effect of anti-PD-1 therapy in a NSCLC mouse model with high expression of NeuGc-containing glycoconjugates. (A) PD-L1 expression in LLC and LLC-NeuGc. For each experimental condition, values were normalized to control cells and data are represented as mean ± SEM of gMFI. ANOVA followed by Tukey’s multiple comparisons test. (B) Representative histogram showing PD-L1 expression in LLC (dotted grey line) and NeuGc-LLC cells (continuous grey line). Isotype antibody was included as a negative control (continuous black line). (C) Immunohistochemical detection of PD-L1 in LLC-NeuGc tumors. Left panel: no primary antibody control. Scale bars: 200 μm. (D) Total, micro and macro lung nodules quantification in control and PD-1 treated mice. Results are shown as lung nodules per animal and mean values ± SEM for each experimental group. (E) Representative lung lobes from control (top panel) and anti-PD-1-treated mice (bottom panel) are depicted. Bouin dye. Arrowheads indicate representative lung nodules. ANOVA, analysis of variance; gMFI, geometric median fluorescent intensity; LLC, Lewis lung carcinoma; LLC-NeuGc, NeuGc-preincubated LLC; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; SEM, standard error of the mean.

Once the presence of PD-L1 was confirmed, we evaluated the effect of treatment with anti-PD-1 mAb on LLC cells using this tumor model. As shown in Figure 2D,2E, checkpoint blockade with an anti-murine PD-1 mAb injected IP at a dose of 200 µg/dose exerted a prominent antitumor effect. Total lung nodules, as well as micronodules—defined as tumors with diameters below 2 mm—were significantly reduced, showing decreases of approximately 60% and 65%, respectively, in anti-PD-1-treated animals compared to vehicle-treated controls (P=0.005; P=0.002 unpaired t-test). No significant differences were observed in macronodules (>2 mm) formation between control and treated animals. Tumor diameter measurements across both experimental groups are depicted in Figure S1.

Antitumor activity of concurrent treatment with racotumomab and anti-PD-1 therapy

After validating that anti-PD-1 treatment reduced tumor burden in the lungs, we evaluated the antitumor activity of racotumomab in combination with anti-PD-1 therapy using an experimental approach based on concurrent administration of both treatments (Figure 1A). As previously described, the mice received two doses of racotumomab prior to the injection of tumor cells, followed by two additional doses of anti-PD-1 mAb, with the final dose of racotumomab administered in between. We did not observe any signs of blood toxicity in this experimental setting (hematocrit: P=0.45; hemoglobin: P=0.53; erythrocytes: P=0.50; platelets: P=0.051; relative lymphocytes: P=0.61; ANOVA followed by Tukey’s multiple comparison test. Leucocytes: P=0.17; Kruskal-Wallis followed by Dunn’s multiple comparisons test). All hematological parameters were consistent with previously reported standard mouse values (29). Moreover, no differences in mouse weight or food and water consumption were detected among the experimental groups (Table 1; Figure S2).

The incidence of lung nodules was 100% in all the experimental groups, with mean values of 28 (control), 11 (anti-PD-1), 17 (racotumomab), and 15 (racotumomab + anti-PD-1) total nodules per experimental group. Upon analysis of lung nodule burden, despite the absence of disease-free animals, we observed a significant reduction in total lung tumor formation when comparing individual (P=0.001 vs. anti-PD-1; P=0.05 vs. racotumomab, ANOVA followed by Tukey’s multiple comparisons test) and combined treatments (P=0.01, ANOVA followed by Tukey’s multiple comparisons test) vs. the control group. Micronodule formation was also significantly reduced between control and treated animals (P<0.001 vs. anti-PD-1; P=0.03 vs. racotumomab; P=0.004 vs. racotumomab + anti-PD-1; ANOVA followed by Tukey’s multiple comparisons test). However, concurrent treatment did not improve the antitumor effect in comparison with monotherapy (Figure 3A,3B). No significant differences were observed in the progression of macronodules between experimental groups (P=0.86, ANOVA followed by Tukey’s multiple comparisons test) (Figure 3C). Tumor diameters for each experimental group are summarized in Figure S3A.

Figure 3 Antitumor activity of concurrent treatment with Raco and anti-PD-1 therapy. (A) Total, (B) micro, and (C) macro lung nodules quantification. Values were normalized to control and data are represented as mean ± SEM of each experimental group. (A) Total lung nodules: ***, P=0.001 vs. anti-PD-1; *, P=0.05 vs. Raco; **, P=0.01 vs. Raco + PD-1. (B) Micronodules: ***, P<0.001 vs. anti-PD-1; *, P=0.03 vs. Raco; **, P=0.004 vs. Raco + anti-PD-1; ANOVA followed by Tukey’s multiple comparisons test. ANOVA, analysis of variance; PD-1, programmed death-1; Raco, racotumomab; SEM, standard error of the mean.

Antitumor activity of sequential treatment with anti-PD-1 therapy followed by racotumomab

Given that the concurrent treatment scheme was less effective than two doses of anti-PD-1 monotherapy in reducing the lung tumor burden, we aimed to evaluate the efficacy of a sequential treatment approach. In addition, we also decrease the number of anti-PD-1 doses in order to further enhance the combined effects of both immunotherapies. To achieve this, mice were first treated with a single dose of anti-PD-1 mAb after tumor cell injection and then with three doses of racotumomab (Figure 1B). Consistent with the concurrent treatment, we observed no significant differences in mouse weight or food and water consumption among the experimental groups (Figure S2). The mean values of the total lung lesions obtained for each experimental group were 9 (control), 8 (anti-PD-1), 6 (racotumomab), and 4 (anti-PD-1 + racotumomab). There were no significant differences in total lung nodules or macronodules between the treated and control groups (P=0.14, ANOVA followed by Tukey’s multiple comparisons test). However, animals treated with anti-PD-1 mAb + racotumomab showed a reduced number of total nodules (Figure 4A). In particular, mice that received the sequential combination of therapies showed a significant reduction in the number of lung micronodules in comparison with control and anti-PD-1 treated groups (P=0.007; P=0.05 respectively, ANOVA followed by Tukey’s multiple comparisons test), showing a reduction in micronodule formation of 62% and 45% compared to anti-PD-1 or racotumomab-vaccinated groups, respectively (Figure 4B). Similar to what was obtained for concurrent treatment, no significant differences in macronodules were observed between the control and treated mice, regardless of whether they received monotherapies or a combination of both treatments (P=0.94, ANOVA followed by Tukey’s multiple comparisons test) (Figure 4C). Figure S3B presents the tumor diameter distribution data for each experimental group, highlighting the significant reduction in micronodule formation.

Figure 4 Antitumor activity of sequential treatment with anti-PD-1 therapy followed by Raco. (A) Total, (B) micro and (C) macro lung nodules quantification. Values were normalized to control, and data are represented as mean ± SEM of each experimental group. ANOVA followed by Tukey’s multiple comparisons test. ANOVA, analysis of variance; PD-1, programmed death-1; Raco, racotumomab; SEM, standard error of the mean.

To further our analysis, we evaluated lung disease incidence and observed a significant reduction in those mice treated with the combination of active immunotherapies in comparison to control mice (P=0.04, Chi-square test). All animals in the control group developed lung tumors, while for those treated with anti-PD-1 and racotumomab, tumor incidence was 84.6% and 76.1%, respectively. Conversely, 54.5% of mice treated with anti-PD-1 mAb + racotumomab developed lung tumors, leaving almost half of the animals free of disease (Table 2).

Recognizing that the effectiveness of immunotherapy depends on activating pre-existing immunity to enable the immune system to identify and eliminate tumor cells, we subsequently evaluated CD8⁺ tumor-infiltrating lymphocytes in the lung nodules using immunohistochemistry. Formalin-fixed, paraffin-embedded lungs were histologically analyzed, revealing a significant increase in CD8⁺ T cells infiltration in lung nodules of animals treated with anti-PD-1 mAb combined with racotumomab compared to controls (P=0.02, ANOVA followed by Tukey’s multiple comparisons test). In contrast, animals treated with monotherapies exhibited weaker immunoreactivity, showing no significant differences from the control group (Figure 5A). Representative immunostaining images from all experimental groups are shown in Figure 5B-5G.

Figure 5 CD8⁺ tumor-infiltrating lymphocytes in lung nodules. (A) CD8⁺ T cell quantification was performed using ImageJ. Results are expressed as the percentage of tumor area occupied by CD8⁺ T cells relative to the total tumor area, and data are presented as mean ± SEM for each experimental group. ANOVA followed by Tukey’s multiple comparisons test. (B-E) Representative microphotographs of immunohistochemical detection of total CD8⁺ T cells in lung sections from mice receiving (B) saline solution, (C) anti-PD-1 therapy, (D) Raco, and (E) sequential combination of both immunotherapies. (F) No primary antibody and (G) CD8 staining in the mouse spleen were included as negative and positive controls, respectively. Scale bars: 100 μm. ANOVA, analysis of variance; PD-1, programmed death-1; Raco, racotumomab; SEM, standard error of the mean.

Exploratory analysis of racotumomab antitumor activity and the role of a NeuGc-rich diet using a humanized CMAH −/− mouse model of NSCLC

As outlined before, in human tumors, NeuGc is regarded as a neoantigen derived from dietary sources, with no expression in normal tissues owing to the lack of the key enzyme CMAH (8). In contrast, in normal murine tissues, NeuGc is naturally expressed and NeuGc glycoconjugates are described as tumor-associated antigens. Considering this, we used CMAH −/− mice to confirm the antitumor activity of racotumomab and to examine the role of a NeuGc-rich diet in the context of a humanized model that more closely resembles the human glycan phenotype. When CMAH −/− mice were fed with standard rodent chow containing very low NeuGc, injected with LLC cells, and treated with three doses of racotumomab as described for the sequential treatment, no antitumor effect was observed (Figure 6A-6D). However, in mice fed with a NeuGc-rich diet, total lung nodules and micronodules were significantly reduced in racotumomab-treated animals (P=0.02, P=0.03, respectively; unpaired t-test) (Figures 6E,6F). In addition, a decrease in macronodules was found, with marginal statistical significance (P=0.052; unpaired t-test). In particular, while 71% (5/7) of control animals developed lung macronodules, only 25% (2/8) of racotumomab-treated animals developed them (Figure 6G). Figure 6H shows representative lung lobes from control and racotumomab-treated mice. Tumor diameter measurements across experimental groups for both the standard diet with low NeuGc content and the rich-NeuGc diet are depicted in Figure S4.

Figure 6 Role of a NeuGc-rich diet in Raco antitumor activity using a humanized CMAH −/− mouse model of NSCLC. (A) Total, (B) micro and (C) macro lung nodules quantification in CMAH −/− female mice fed with a standard diet with low NeuGc content and treated or not with Raco. (E) Total, (F) micro, and (G) macro lung nodules quantification in CMAH −/− mice fed with a NeuGc-rich diet and treated or not with Raco. Results are shown as lung nodules per animal and mean values ± SEM for each experimental group. * Total lung nodules, P=0.02; * lung micronodules, P=0.03; unpaired t-test. (D,H) Representative lung lobes from control (top panel) and Raco treated mice (bottom panel) fed with NeuGc-low and rich diets, respectively, are depicted. Bouin dye. Arrowheads indicate representative lung nodules. CMAH −/−, CMAH-deficient mice; NeuGc, N-Glycolylneuraminic acid; NSCLC, non-small cell lung cancer; Raco, racotumomab; SEM, standard error of the mean.

Discussion

With the advent of ICB-based immunotherapy, the therapeutic landscape of a broad spectrum of cancers, including NSCLC, has profoundly changed. Although patients’ clinical outcomes have improved, with improvements in quality of life and overall and progression-free survival (PFS), only a subset of patients respond favorably to these therapies or show transient responses (30). Moreover, many of them require interruption or even have to withdraw treatment because of immune-related adverse events (31,32). For some patients, ICB alone may be insufficient to overcome immune tolerance induced by both tumor cells and the surrounding immunosuppressive microenvironment, in which cancer-associated fibroblasts, abnormal angiogenesis, and suppressive immune cells play central roles (33-35). In this scenario, a combination of multiple strategies, including multiple immunotherapies, is considered a rational approach to enhance antitumor immunity. Standard chemotherapy and radiotherapy, as well as other checkpoint blockers, angiogenesis inhibitors, targeted therapy, stimulator of an interferon gene (STING) agonists, and cell therapy, among others, have been shown to synergize with PD-1/PD-L1 blockade (36).

Therapeutic cancer vaccines are targeted active immunotherapies aimed at tumor-associated antigens or neoantigens; however, they have yielded disappointing results in early clinical studies. As evaluated in patients with established tumors, immunosuppressive mechanisms induced by cancer cells and the microenvironment prevent T cell activation after antigen stimulation (37). However, ICB success has reinvigorated interest in therapeutic cancer vaccines and the possibility of achieving long-lasting tumor-specific memory T cells (38). To potentiate the immune response against cancer cells, proper combination strategies require temporally designed immunotherapy treatment schedules (39). Consequently, in this study, we evaluated the antitumor activity of a specific active immunotherapy with racotumomab in combination with ICB based on PD-1 blockade in a clinically relevant NSCLC mouse model using two different schedules. No signs of adverse effects were observed in either phasing dose scheme. The combination of both immunotherapies did not alter animal weight or blood parameters in the treated mice, indicating that this treatment approach was safe.

In the concurrent treatment setting, we assessed the antitumor activity on lung nodule development when animals were first treated with two doses of racotumomab, followed by two doses of anti-PD-1 mAb interspersed with the last dose of racotumomab. The rationale behind this strategy was to enhance the infiltration of NeuGc-specific effector T-cells into the tumors after two doses of racotumomab and potentiate T-cell activation in the lungs with anti-PD-1 treatment. The last dose of racotumomab consolidates the tumor-specific cellular response (40). Results showed that a single treatment with two doses of ICB was the most effective therapy against lung colonization by tumor cells, showing no treatment improvement when racotumomab was combined with ICB.

In the sequential approach, mice were administered a single, suboptimal dose of anti-PD-1 mAb, followed by repeated immunizations with racotumomab. In this setting, our initial objective was to minimize the inactivation of lung-infiltrating T cells, thereby promoting a NeuGc-specific response to racotumomab. Furthermore, by limiting anti-PD-1 administration to a single dose, we aimed to evaluate a dosing regimen that could potentially enhance the combined therapeutic effects of both agents, as previously reported in both preclinical and clinical studies (41,42). This sequential scheme better reflects the clinical setting for advanced NSCLC treatment, since ICB with pembrolizumab can be administered as a first-line treatment (43) and racotumomab has been approved as switch maintenance immunotherapy (44). Although we have demonstrated that three doses of racotumomab showed therapeutic activity in the same murine model (17), shorter dosing frequencies may also represent a suboptimal therapeutic scheme for racotumomab. Nonetheless, sequential administration of both immunotherapies was highly effective against lung nodules, showing an improved antitumor activity, since combination treatment significantly reduced the incidence of total lung nodules, as well as micronodule progression.

As mentioned previously, cancer vaccines can prime antigen-specific T cells, which play an important role in the activation of the antitumor immune response. The remarkable decrease in lung micronodules smaller than 2 mm suggests that racotumomab induced NeuGc-specific T cells that were able to infiltrate early tumors, in which immunosuppression was not as relevant as in larger tumors (37). Moreover, an increase in total CD8⁺ tumor-infiltrating T cells was observed in mice treated with a combination of both immunotherapies, as the percentage of the tumor area occupied by stained CD8⁺ T cells was significantly higher compared to vehicle-treated animals. Although we did not evaluate different subpopulations of CD8⁺ T cells within the tumors, among which may be an exhausted population, there is much evidence supporting an association between total CD8⁺ T cells and treatment outcomes in NSCLC, as well as other tumors (45,46).

Combination strategies based on ICB therapies and cancer vaccines may have different sequencing times, showing evidence that seems controversial. While some preclinical and clinical studies have reported that ICB was most effective when administered after vaccination (47,48), others have demonstrated that initiating treatment with a checkpoint inhibitor and administering a vaccine was the most effective combination (49,50). In our experimental design, sequential administration of anti-PD-1 mAb followed by racotumomab not only improved its antitumor activity but also suggested that a single dose of the immune checkpoint inhibitor was sufficient to reduce lung nodule formation in a combination approach. These preclinical findings demonstrate that the sequential treatment scheme is an effective combinatorial approach, consistent with clinical settings for NSCLC patients, where PD-1 blockade therapy is administered as first-line treatment and racotumomab is utilized as switch maintenance immunotherapy. Unlike therapies based on cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade, which have demonstrated a direct correlation between dose and toxicity, anti-PD-1 inhibitors do not increase the frequency or severity of adverse events when higher doses are used (51). However, since large amounts of data suggest that there is no difference in patient outcomes between higher and lower doses of anti-PD-1 mAb and reports survival benefits at doses much lower than those already approved, there is an opportunity to optimize the anti-PD-1 treatment regimen (52-54). Indeed, current dosing regimens may be derived from overtreatment, with consequent implications in cost and quality of life (55,56). In this regard, as treatment opportunities based on ICB are available for a limited number of patients in low- and middle-income countries, combination therapies that reduce the doses of ICB and demonstrate similar clinical benefits are worthy of evaluation in preclinical studies and clinical trials (57). Supporting this approach, recent data by Ozasa et al. in a preclinical murine model of colorectal cancer validated a combinatorial strategy employing immunomodulatory agents to reduce the anti-PD-1 mAb dosage by modulating the tumor microenvironment, without compromising antitumor efficacy (41).

The development of a successful cancer vaccine requires the proper validation of tumor-associated antigens or neoantigens, against which direct specific antitumor immunity occurs. Although healthy human tissues contain negligible levels, tumor cells can incorporate large amounts of NeuGc from dietary sources (12-14), resulting in NeuGc-containing glycoconjugate neonates in many tumor types, including NSCLC. Conversely, normal murine cells have a functional CMAH; therefore, the presence of NeuGc has previously been described (58,59), while some cell lines derived from different mouse tumors showed limited expression of CMAH and NeuGc (26,27,60-62). We previously reported that LLC cells can take exogenous NeuGc when cultured in vitro as described for human tumor cells (17,60). To our knowledge, this is the first report that uses CMAH −/− mice to explore not only racotumomab antitumor activity but also any antitumor therapy in a humanized CMAH −/− mouse model. Furthermore, we report, for the first time, the role of a NeuGc-rich diet in the racotumomab effect. Our data suggest that tumors that developed in CMAH −/− mice after direct implantation of LLC cells into the lungs could incorporate NeuGc from dietary sources, similar to human tumors. This result is in accordance with previous findings suggesting that murine tumor cell lines can accumulate NeuGc in vivo (26,62). Moreover, our findings in this humanized mouse model of NSCLC are aligned with previous data that demonstrated the importance of tumor antigen expression in the effectiveness of racotumomab immunotherapy (63), highlighting the role of NeuGc intake from dietary sources.

One of the main limitations of our study is the lack of a comprehensive evaluation of changes in the immunogenicity profile of LLC cells following preincubation with purified NeuGc, as we assessed only PD-L1 expression. Although we did not directly investigate whether cell-surface NeuGc modulates MHC class I expression, previous studies indicate that certain gangliosides, such as NeuGcGM3, exert immunosuppressive effects within the tumor microenvironment, particularly when shed by tumor cells. This suppression mainly impairs dendritic cell and cytotoxic T cell functions (64-66). In our model, pre-treatment with purified NeuGc increased membrane-bound NeuGcGM3 levels, which correlated with enhanced tumor progression and metastatic potential.

Secondly, the absence of a CD8⁺ T cell depletion experiment limits the mechanistic validation of the improved antitumor effects observed with the combined immunotherapies and warrants further investigation. However, recent data demonstrate that patients treated with racotumomab who exhibited an increased ratio of CD8⁺ T cells to CD4⁺ T regulatory cells and a higher frequency of EMRA CD8⁺ T cells at baseline and after six to eight months of treatment are long-term survivors (67). Moreover, favorable responses to ICB have been primarily linked to immune activation of certain lymphocyte populations, including CD8⁺ T cells, which show increased activity in interferon and cytokine signaling pathways (68).

Conclusions

Our preclinical data support the combination of PD-1 checkpoint blockade therapy with the active vaccine immunotherapy racotumomab in advanced NSCLC. The sequential administration of both immunotherapies appears to be an appropriate treatment regimen to further elucidate the mechanisms underlying enhanced antitumor activity.

Acknowledgments

The authors gratefully acknowledge BSc Gabriel Fernández-Graña and MSc Geraldine Schlapp from the Laboratory Animal Biotechnology Unit of Institut Pasteur de Montevideo, Uruguay, for their valuable contributions to CMAH-deficient mice. The authors also express their gratitude to Mr. Nicolás Mairaú for his valuable assistance in preparing the figures.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-230/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-230/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-230/prf

Funding: This work was supported by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Argentina (grant No. PICT 2020-01277) to V.I.S.; Consejo Nacional de Investigaciones Científicas y Técnicas (grant No. PIBAA 2022-2023-28720210100406CO) to V.I.S.; and Universidad Nacional de Quilmes (program grant No. 2283/22).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-230/coif). All authors disclose that Laboratorio Elea-Phoenix provided study materials. V.I.S. reports that this study was supported by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Argentina (grant No. PICT 2020-01277); Consejo Nacional de Investigaciones Científicas y Técnicas (grant No. PIBAA 2022-2023-28720210100406CO). V.I.S., M.R.G., and D.F.A. have served in a consultant/advisory role for Laboratorio Elea-Phoenix, Argentina. I.A.D. and E.S. are full-time employees of mAbxience, Argentina and Laboratorio Elea-Phoenix, Argentina, respectively. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The Animal Review Board of Quilmes National University approved all experimental protocols (Approval No. 010-15), and animal maintenance was conducted under accepted international standards for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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