Reconfiguration of tumor cells with LCOR transcription factor mRNA nanotherapy to enhance immunotherapy efficacy

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   Jul 27, 2025

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   Jul 27, 2025

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   Referee #2

   Evidence, reproducibility and clarity

   SUMMARY OF THE PRESENTED FINDINGS

   Abstract

   1. LCOR (Ligand-dependent corepressor), which suppresses tumor growth by inducing the antigen presentation machinery (APM) of the tumor cells and constrains cellular plasticity.
   2. poly β-(amino esters) (pBAE) nanoparticles (NPs).. Our results show optimal endosomal escape, which results in high transfection efficiency in vitro and in vivo
   3. the combination of Lcor mRNA-loaded NPs with anti-PDL1 or anti-CTLA4 immunotherapies eradicated most of the tumors in our preclinical TNBC model.

   Introduction

   a. These structures facilitate endosomal escape due to protonation of tertiary amines at lower pH7.

   Resu…

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #2

   Evidence, reproducibility and clarity

   SUMMARY OF THE PRESENTED FINDINGS

   Abstract

   1. LCOR (Ligand-dependent corepressor), which suppresses tumor growth by inducing the antigen presentation machinery (APM) of the tumor cells and constrains cellular plasticity.
   2. poly β-(amino esters) (pBAE) nanoparticles (NPs).. Our results show optimal endosomal escape, which results in high transfection efficiency in vitro and in vivo
   3. the combination of Lcor mRNA-loaded NPs with anti-PDL1 or anti-CTLA4 immunotherapies eradicated most of the tumors in our preclinical TNBC model.

   Introduction

   a. These structures facilitate endosomal escape due to protonation of tertiary amines at lower pH7.

   Results

   b. In human models MDAMB-231 and MCF7 cells, the NPs also showed high eGFP mRNA transfection efficiency

   c. The efficiency of eGFP mRNA-loaded pBAE-NPs to transfect mRNA into different mouse breast cancer cells (AT3, 4T07, EO771, EMT6, 66cl4, EpRAS, and 4T1) was tested using NPs encapsulating eGFP mRNA,

   d. Synthetic Lcor mRNA contained a Cap1, 5' and 3' untranslated regions (UTR) and a standard polyA tail (Fig. S2A), and all uracil were replaced for 5-methoxyuracil (5-moU) to avoid immunogenic reactions27,28. First, we measured and detected high levels of Lcor mRNA by qRT-PCR

   e. NPs were stable at 25ºC for 24 h (Fig. S2C). In contrast, under conditions simulating the physiological environment (37ºC), a decrease in FRET signaling was detected ... indicating disassembly of the NPs after 2 h (Fig. S2C).

   f. Lcor mRNA NPs, induces the expression of APM genes in AT3 and 4T07 cell lines

   g. AT3 cells that constitutively overexpress ovalbumin (OVA). In these cells, OVA is cleaved, generating the SIINFEKL antigen peptide presented in the H-2Kb context. This can be used to measure APM activity using the anti-SIINFEKL antibody via flow cytometry.

   h. We also observed a time- and dose-dependent effect regarding APM induction.

   i. When tumors reached 0.5 x 0.5 cm2, we treated them intratumorally with pBAE-NPs loaded with 5 ug of synthetic FLuc or eGFP mRNA. We detected BLI at 3 h, meaning that tumor cells had taken up the mRNA-loaded NPs and translated a luciferase active protein within 3 h. In both models, expression peaked around 6 to 10 hours after administration

   j. After local administration of 5 μg of Lcor mRNA-loaded NPs, we observed a rapid increase in Lcor mRNA in the tumor tissue, followed by a decrease, reaching baseline levels after 24 h (Fig. 3C). ..To unravel the protein dynamics, we used ... LCOR-HA protein and uniquely detect the ectopic protein using anti-HA by IF. As expected, LCOR-HA protein expression was delayed, peaking 3 h after administration (Fig. 3D). Linked to protein expression, at 3 h and 6 h after administration, we detected an increase in APM genes by RT-qPCR (Fig. 3E and S3D).

   k. the combination of Lcor mRNA-loaded NPs with anti-PDL1 therapy not only reduced tumor growth but also led to tumor eradication in 5 out of 7 mice.

   l. The combination of Lcor mRNA-loaded NPs with different ICIs showed high efficiency in preclinical models, thus supporting the feasibility of starting clinical studies and thus bringing the treatment closer to patients.

   Major points

   L. 277: "NPs were stable at 25ºC for 24 h (Fig. S2C). In contrast, under conditions simulating the physiological environment (37ºC), a decrease in FRET signaling was detected ... indicating disassembly of the NPs after 2 h (Fig. S2C)."

   • The disassembly of the NPs after 2 h is key to the performance of the chosen approach.

   L. 296: "The results showed an increased number of cells with higher OVA-SIINFEKL presentation, indicating the enhanced activity of the APM induced by the Lcor mRNA-loaded pBAE-NPs... demonstrate the efficiency of this mRNA nanotechnology to rescue the function of the LCOR TF in inducing tumor cell immunogenicity and thus modulating tumor phenotypes."

   • There is a key difference between activating antigen-presenting machinary and inducing immunogenicity, i.e. recognition by the immune system and activation of effector cells. There is no indication on how effective endogenous immune responses (e.g. antibody titers, TIL infiltration, cytokine release) are to the administration of Lcor mRNA-loaded NPs.

   L. 325: "Based on these results, we estimated an optimal therapeutic regimen of Lcor-mRNA-loaded pBAE-NPs administration in our preclinical experimental models would be every 3 days."

   • It is highly unclear how the authors came to this conclusion, as it should be based on the time frame of optimal immune responses.

   L. 332: "Lcor mRNA-loaded NPs were administered at a dose of 250 μg/kg by intratumoral (i.t.) injection twice a week"

   • This possibly is the strongest limitation of this study. Intratumor injections of largely unfeasible/unrealistic in clinical setting. Even more, the management of metastatic disease appears out of question.

   L. 337: "the results revealed that Lcor mRNA monotherapy was enough to reduce 4T07 tumor 338 growth."

   • These effects appear rather limited (Fig. 4A,B) and are not statistically significant in Fig. S4B and Fig. S5A.

   L. 338: "the combination of Lcor mRNA-loaded NPs with anti-PDL1 therapy not only reduced tumor growth but also led to tumor eradication in 5 out of 7 mice"

   • Fig. 4A bottom left panel. Three of the tumor growth curves abruptly stop at below 200 mm3. Typically, this is mouse death. This reduces the tumor pool to four xenografts. Among these, we notice two complete responses and two tumor progressions. Two tumor progressions are seen also in the combination Lcor mRNA+ α-PD-L1 group. We are unsure about the statistics of this experiment.

   L. 350: "The combination of Lcor mRNA-loaded NPs with different ICIs showed high efficiency in preclinical models, thus supporting the feasibility of starting clinical studies and thus bringing the treatment closer to patients."

   • Please see comment on L. 332. It appears unrealistic to consider clinical studies in patients unless a systemic administration of Lcor mRNA-loaded NPs is tackled and corresponding therapeutic efficacy is shown.

   Significance

   General assessment:strengths and limitations.

   The identification of a candidate therapeutic means, by supplying Lcor mRNA for induction of antigen-presenting molecules is of potential interest. As this is not a basic science study, but aims at developing feasible therapeutics, it falls short in this respect, as most likely unfeasible in patients. The combined effect with anti-immune blockade agents is of interest. However, if one assumes that effective immunostimulation was indeed induced by Lcor mRNA, its overall impact on tumor growth is per se weak, if any. Maybe only antigen presentation is induced, but this is in the absence of costimulatory signals? This needs to be investigated.

   Advance

   This article is based on good papers that were published years ago. The science novelty is limited. As the idea is to develop a novel therapeutic approach, the lack of realistic feasibility severely limits merits.

   Audience

   Scientists involved in preclinical studies.

   Reviewer expertise

   This reviewer and his research group have cloned the genes and biochemically characterized novel tumor drivers. He identified their function as stimulators of tumor cell growth and of metastatic spreading, together with roles in cell-cell adhesion, signal transduction and local cancer invasion. This led to the discovery of their prognostic / predictive relevance in human cancer. Two murine models of rare genetic diseases were generated by ablating the corresponding murine genes. He then pioneered the development of software for the identification of fusion oncogenes and of transcription factor-DNA binding sites. This reviewer fostered novel anti-cancer immunotherapies. He generated anti-cancer cytotoxic T lymphocytes, by the use of in vitro engineered antigen presenting cells. Using proprietary discovery platforms, this reviewer developed novel anti-cancer monoclonal antibodies, that selectively target cancer cells. This led to the engineering of humanized antibody-drug conjugates, bispecific anti-CD3/activated Trop-2 antibodies and innovative CAR-T designs. ADCs are now being tested in clinical trials in cancer patients.

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3. 

   Review Commons

   Jul 27, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   In this manuscript, Serra-Mir et al investigate the therapeutic potential of delivering the mRNA of LCOR transcription factor via nanoparticles to enhance the efficacy of immune checkpoint inhibitors. The authors show that the mRNA delivery mediated by H and R-nanoparticles was efficient in multiple breast cancer cell lines in vitro. Moreover, using mouse models, they show that LCOR mRNA delivery may improve the efficacy of the treatment with anti-PDL1 or anti-CTLA4 checkpoint inhibitors against tumors. Although this proof-of-concept study has promising aspects, there are significant weaknesses that should be addressed. Details below.

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   In this manuscript, Serra-Mir et al investigate the therapeutic potential of delivering the mRNA of LCOR transcription factor via nanoparticles to enhance the efficacy of immune checkpoint inhibitors. The authors show that the mRNA delivery mediated by H and R-nanoparticles was efficient in multiple breast cancer cell lines in vitro. Moreover, using mouse models, they show that LCOR mRNA delivery may improve the efficacy of the treatment with anti-PDL1 or anti-CTLA4 checkpoint inhibitors against tumors. Although this proof-of-concept study has promising aspects, there are significant weaknesses that should be addressed. Details below.

   Major points:

   1. In vitro delivery of LCOR appears to be effective in both AT3 and 4T07 cell lines when continuously exposed to the mRNA loaded nanoparticles. However, the impact of LCOR on antigen presentation machinery (APM) is rather mixed and not very convincing. The expression pattern and kinetics of several APM genes are inconsistent with LCOR kinetics and at several timepoints the expression in LCOR samples is essentially the same as in mutant LCOR negative controls (Figure 2D). Moreover, the APM reporter assay experiments show that APM in LCOR transduced 4T07 cells is induced rather modestly at best (Figure 2E). The APM effect needs to be demonstrated more rigorously to be convincing.
   2. Considering that previous studies by the authors suggest a role for LCOR in regulating stem cell properties in normal and malignant mammary cells (Celia-Terrassa et al Nat Cell Bio 2017; Perez-Nunez et al Nat Can 2022), it is important to address whether transduced LCOR mRNA impacts these properties. Moreover, other autocrine cell functions such as proliferation and apoptosis are also relevant and should be analyzed.
   3. The impact of LCOR delivery on immune responses in mouse models could be more rigorous. Analysis of APM genes shows rather modest difference in these gene after LCOR transduction (Figure 3E). Is this sufficient to induce effective anti-tumor immune response? What is the status of T cell activity or exhaustion? Furthermore, LCOR may regulate cytokines and chemokines that are critical for modulation of the immune environment. Did the authors measure any immune-modulating cytokines in the tumor microenvironment, following LCOR expression? Finally, whereas the study focuses on APM and its function, LCOR may directly modulate expression of checkpoint activators on cancer cells. The impact of LCOR transduction on PD-L1, PD-L2 and CTLA-4 expression in cancer cells should be determined.
   4. In line with point nr 2, it would be important to analyze the impact of delivered LCOR mRNA on cell functions such as proliferation and apoptosis in the mouse tumors. Even if LCOR delivery sensitizes tumors to checkpoint inhibitors, it cannot be assumed that the impact of LCOR is primarily due to induction of the APM.
   5. The experiments analyzing treatment efficacy in the 4T07 model in mice show lack of consistency and a substantial variation between mice that are treated in the same manner. Even the group treated with PBS and Ctr-mRNA contains mice with tumors that regress (Figure 5A). This inconsistency suggests that more mice are required to generate a convincing pattern. Furthermore, the inclusion of a second model would provide a stronger case for a broad applicability of the LCOR treatment with checkpoint inhibitors. Indeed, it is surprising that the authors did not use the AT3 model in vivo considering that mRNA delivery and LCOR expression is substantially more efficient in AT3 compared to 4T07.
   6. Following the injection of LCOR nanoparticles to the tumor, the proportion and spatial distribution of LCOR expressing cells should be determined. This is particularly relevant in light of the almost complete elimination of the tumors treated with combination therapy (Figures 4 and 5). Is this striking impact on tumors in spite of mRNA being delivered only to a small portion of cells within the tumor?
   7. The in vivo results indicate that expression levels of Fluc mRNA decline rapidly post-treatment, returning to baseline within 24 hours after peaking at 10 hours (Supplementary Figure 3). Although the investigators treat mice every 3rd day with LCOR nanoparticles in their therapeutic experiments, the analysis of durability of immune responses after single injection should be done and can provide important practical insights to guide therapeutic design.

   Minor points:

   1. The authors mention that LCOR mRNA delivery synergizes with checkpoint inhibitor treatment. However, synergy has a specific meaning when drug interaction is analyzed. This was not really addressed or calculated.
   2. There seems to be a mistake in the text (lines 261-263). Based on Figure 1C the mRNA delivery efficiency is higher in AT3 cells compared to 4T07 cells (very difficult to determine anything from Figure 3D, since the cell density is not visible).
   3. It is surprising how little expression of luciferase is observed in the 4T07 model (Figure S3), even if almost 60% of cancer cells and 40% of stromal cells are positive (Figure 3A). What could explain this discrepancy?
   4. Representative FACS plots from Figure 3 should be shown.
   5. There are issues with the figure legends of Figure 3 (from 3C onwards) and Figure S2 (from 2D onwards) that need to be fixed.

   Significance

   The study is a proof-of-concept investigation addressing whether LCOR mRNA can be delivered by nanoparticles to sensitize tumors to immunotherapy. This approach aims to overcome the limitations and difficulties of targeting transcription factors for therapeutic purposes. However, although the delivery of LCOR mRNA appears to be sufficient, further characterization of the resulting impact needs to be done. This includes both impact on immune responses as well as cell-autonomous impact on cancer cell proliferation and apoptosis.

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4. 

   Version published to 10.1101/2025.04.02.646434 on bioRxiv

   Apr 3, 2025