Tumor chemoresistance caused by P-glycoprotein (P-gp) expression in cancer cells remains a significant challenge in cancer chemotherapy. Herein, a novel P-gp-inhibiting lopinavir derivative (LD) was synthesized via esterification of protease inhibitor lopinavir with 5-methyl-4-oxohexanoic acid. LD proved to be a potent P-gp inhibitor with EC50 similar to 1 mu M, capable of considerable sensitization of P-gp-expressing cancer cells to conventional cytostatic drugs in vitro. The oxo functional group introduced in LD allowed its covalent linkage with the N-(2-hydroxypropyl)methacrylamide copolymer carrier via a pH-sensitive hydrazone bond (P-LD). Polymer conjugation enhanced the pharmacological properties of LD in vivo, increasing its half-life in the bloodstream, protecting it from metabolic degradation, and promoting its accumulation in tumors via the enhanced permeability and retention effect. P-LD exhibited P-gp-inhibitory activity and sensitized cells to polymer-bound cytostatic drugs in vitro. Importantly, P-LD remarkably improved the antitumor efficacy of a polymer-bound doxorubicin in two P-gp-expressing mouse tumor models without exhibiting any systemic toxicity.

INTRODUCTION:The systemic administration of cytokines is constrained by their pleiotropic activity, dose-dependent toxicities, and short serum half-life, limiting their therapeutic window in cancer treatment. To overcome these challenges, strategies that restrict cytokine signaling to defined immune cell subsets within the tumor microenvironment have been developed to enhance efficacy while minimizing off-target effects. Among these, antibody-cytokine fusion proteins represent a rational design platform that enables selective and localized cytokine delivery to specific immune populations. AREAS COVERED:This report outlines the design principles underlying tumor-associated antigen-targeted and cis-delivered IL-2- and IL-15-based immunocytokine platforms, with particular emphasis on the PD-1-directed cis-signaling strategy. Preclinical data on SOT201 are summarized, highlighting how its affinity optimized IL-15 mutein promote selective proliferation and enhanced effector function of PD-1+ CD8+ T cells. EXPERT OPINION:Cis-acting immunocytokines represent a promising class of advanced therapeutics that selectively direct cytokine payload to tumor infiltrating lymphocytes. This strategy has been shown to induce durable antitumor immunity and, in some cases, promote immune memory formation while limiting systemic toxicity. Ongoing clinical evaluation and rational combination approaches will ultimately define its therapeutic positioning in precision cancer immunotherapy.

Natural killer (NK) cells contribute to tumor immunosurveillance, yet their heterogeneity across cancer types remains incompletely understood. Transcriptomic, spatial, and functional assays reveal that non-small cell lung carcinoma (NSCLC) is enriched in NK cells that mediate clinically relevant effector functions, whereas high-grade serous ovarian carcinoma (HGSOC) contains dysfunctional NK cells that express co-inhibitory receptors including NKG2A. Analysis of HGSOC patient samples and syngeneic mouse models indicates a crosstalk between NK cells and CD8⁺ T cells critical for effective antitumor immunity. Depletion of either population leads to phenotypic impairment of the reciprocal one. Blocking NKG2A restores NK cell cytotoxicity and promotes CD8⁺ T cell responses, significantly improving the efficacy of PD-1 blockade in murine HGSOC models. Thus, NK cells and CD8⁺ T cells engage in a functional interplay of immunological relevance. Moreover, the NKG2A-HLA-E axis represents a clinically actionable immunological checkpoint in tumors with impaired NK cell functions.

NACT-mediated adjuvanticity positively impacts clinically relevant TLS maturation in metastatic HGSOC. A, Representative image of immunofluorescence of CD4, CD8, CD20, CD21, CD23, DC-LAMP, and GZMB staining (immunofluorescence panel 1). Scale bars, 10, 100 and 500 µm. B and C, Distribution of early TLS (eTLS; B) and mature TLS (mTLS; C) across pTME and mTME HGSOC tumor samples with/without NACT. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. D, Supervised hierarchical clustering of TLS-relevant gene signature (CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, and CXCL13) across pTME and mTME HGSOC tumor samples with/without NACT. E and F, Overall survival (OS) of 60 (E) and 40 chemo-naïve and treated patients with mHGSOC (F), respectively (study cohort 1 and 2) based on median stratification of total mTLS. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. Number of patients at risk and P values are reported. G, Number of mTLS across patients with CALRLo and CALRHi mHGSOC with/without NACT as determined by median stratification. Mean and SEM are shown. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. H and I, OS of 60 and 40 patients with chemo-naïve and treated mHGSOC (study cohorts 1 and 2), upon stratification based on median frequency of mTLS and expression of CALR. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using the log-rank test.

Interleukin-2 (IL-2) is a multifunctional cytokine that plays a critical role in the differentiation, proliferation, activation, and survival of immune cells and has been used therapeutically for over 30 years as a cancer treatment. Unfortunately, the cytokine’s narrow therapeutic window and its short circulation half-life have severely limited its clinical application. Strategies designed to bias the activities of IL-2 represent emergent and promising avenues in the treatment of cancer, as they afford the opportunity to selectively target and stimulate certain immune cell subtypes. Recently, our lab has developed one such therapy, termed an immunocytokine (IC), which fuses IL-2 to an anti-IL-2 antibody that biases the cytokine towards immunostimulatory activities while also extending its persistence in the bloodstream. Additionally, we further established that the IC was able to synergize with immune checkpoint inhibitors (ICIs) to effectively clear tumors through expansion of CD8+ T cells and stimulation of their effector functions. Interestingly, it was observed that the therapeutic effects of this combination treatment were highly sensitive to dosing regimen; specifically, robust antitumor activity was observed when IC was delivered after ICI, but not when the order was reversed. Building on this observation of time-dependent anti-cancer activity, here we expand upon this work through the development of a biomaterial system that will enable further exploration of the kinetics surrounding the combinatorial delivery landscape using IC and ICIs. Our biomaterial system involves formulation of nanoparticles that encapsulate IC and ICIs using a flash nano complexation and flash nanoprecipitation system. These particles are further enclosed within a hydrogel system for protection and sustained release within the tumor environment, controlling both the temporal and spatial distribution of the encapsulated immunotherapies. We have successfully designed and characterized the delivery platform and established time-dependent release of the therapeutic cargo. In vivo immune stimulation and tumor clearance studies are ongoing to further evaluate our approach. Overall, our work introduces an innovative and highly versatile bioengineering approach that can be tuned to optimize immunotherapy strategies in a wide range of cancer types. Pilar O'Neal, Julia Lu, Yicheng Zhang, Marek Kovar, Hai-Quan Mao, Jamie Spangler. Novel delivery platform for temporally-controlled release of immunocytokines and immune checkpoint inhibitors to enhance cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3462.

Immunomodulation by NACT in metastatic HGSOC. A, Representative images of CALR immunostaining in CALRLo and CALRHi patients. Scale bars, 10 and 100 µm. B, CALR expression levels determined by immunostaining and (C) ER stress signature level [expression level of DNA damage inducible transcript 3 (DDIT3, best known as CHOP), heat shock protein family A (Hsp70) member 5 (HSPA5, best known as BIP), and heat shock protein 90 beta family member 1 (HSP90B1)] as determined by RNA-seq in pTME and mTME HGSOC with/without NACT. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. D, Supervised hierarchical clustering of gene signatures related to immune populations (orange), immune functions (blue) and immune phenotype (purple) as determined by RNA-seq data from pTME and mTME HGSOC tumor samples with/without NACT. IS, immunosuppression; mDCs, myeloid dendritic cells; NK cells, natural killer cells; TLS, tertiary lymphoid structures. E, Gene expression signature associated with CD8+ T cells, B cells, cytotoxicity, mDCs, TLS, and immunosuppression as determined on RNA-seq data from pTME and mTME HGSOC with/without NACT. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F, Representative image of CD20/DC-LAMP double immunostaining. Scale bars, 500 and 100 µm. G, Density of CD8+ T cells, CD20+ B cells, and DC-LAMP+ cells as determined by immunostaining in pTME and mTME HGSOC samples with/without NACT. Mean and SEM are shown. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. ns, not significant.

AbstractPurpose: Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICI) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. Experimental Design: We harnessed a variety of transcriptomic, spatial, and functional assays to characterize the differential impact of neoadjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to neoadjuvant chemotherapy (NACT)-naïve HGSOC samples from five independent patient cohorts. Results: We found NACT-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD1+ CD8+ T cells over their ICI-insensitive TIM-3+PD1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. Conclusions: Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC. See related commentary by Bravo Melgar and Laoui, p. 10

Univariate Cox proportional hazard analyses.

NACT-mediated adjuvanticity positively impacts the density of follicular T cells (TFH) and in situ activation of intratumoral B cells in mHGSOC. A–C, Representative image (A) and box plots showing the density of CD4+ cells (B) and CXCR5+PD1+FoxP3−CD4+ TFH cells (C) in the pTME and mTME of chemo-naïve and treated HGSOC (study cohort 1 and 2). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by the Mann–Whitney test. P values are indicated. D and E, Dot plot (D) and box plot (E) showing expression profile of gene signatures of B-cell subtypes, e.g., plasma cells (PC), germinal center (GC), and memory B cells within pTME and mTME HGSOC tumor samples with/without NACT. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F and G, Representative image (F) and density of CD68+CD163+ TAMs in pTME and mTME of chemo naïve and treated HGSOC (G). Mean and SEM are shown. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated.

Supplemental material, figures and tables

BACKGROUND:SOT201 and its murine surrogate mSOT201 are novel cis-acting immunocytokines consisting of a humanized/murinized/, Fc-silenced anti-programmed cell death protein 1 (PD-1) monoclonal antibody (mAb) fused to an attenuated human interleukin (IL)-15 and the IL-15Rα sushi+ domain. Murine mPD1-IL2v is a conjugate of a murinized, Fc silenced anti-PD-1 mAb bearing human IL-2 with abolished IL-2Rα binding. These immunocytokines spatiotemporally reinvigorate PD-1+ CD8+ tumor-infiltrating lymphocytes (TILs) via cis-activation and concomitantly activate the innate immunity via IL-2/15Rβγ signaling. METHODS:Human peripheral blood mononuclear cell and cell lines were used to evaluate cis/trans activity of SOT201. Anti-PD-1 mAb responsive (MC38, CT26) and resistant (B16F10, CT26 STK11 KO) mouse tumor models were used to determine the anticancer efficacy, and the underlying immune cell activity was analyzed via single-cell RNA sequencing and flow cytometry. The expansion of tumor antigen-specific CD8+ T cells by mSOT201 or mPD1-IL2v and memory CD8+ T-cell generation in vivo was determined by flow cytometry. RESULTS:SOT201 delivers attenuated IL-15 to PD-1+ T cells via cis-presentation, reinvigorates exhausted human T cells and induces higher interferon-γ production than pembrolizumab in vitro. mSOT201 administered as a single dose exhibits strong antitumor efficacy with several complete responses in all tested mouse tumor models. While mPD1-IL2v activates CD8+ T cells with a 50-fold higher potency than mSOT201 in vitro, mSOT201 more effectively reactivates effector exhausted CD8+ T cells (Tex), which demonstrate higher cytotoxicity, lower exhaustion and lower immune checkpoint transcriptional signatures in comparison to mPD1-IL2v in MC38 tumors in vivo. This can be correlated with a higher rate of complete responses in the MC38 tumor model following mSOT201 treatment when compared with mPD1-IL2v. mSOT201 increased the relative number of tumor antigen-specific CD8+ T cells, and unlike mPD1-IL2v stimulated greater expansion of adoptively transferred ovalbumin-primed CD8+ T cells simultaneously limiting the peripheral CD8+ T-cell sink, leading to the development of memory CD8+ T cells in vivo. CONCLUSIONS:SOT201 represents a promising therapeutic candidate that preferentially targets PD-1+ TILs, delivering balanced cytokine activity for reviving CD8+ Tex cells in tumors. SOT201 is currently being evaluated in the Phase I clinical study VICTORIA-01 (NCT06163391) in patients with advanced metastatic cancer.

NACT positively increases the ICI-sensitive TCF1+PD1+CD8+ T-cell phenotype within metastatic HGSOC. A, Representative image of immunofluorescence of CD68, CD8, PD-L1, FoxP3, TCF1, CD57, PanCK, PD1, CD4, CD20, GZMB, and TIM-3 staining (immunofluorescence panel 2). Scale bars, 2 μm, 10 µm, and 100 µm. B, Violin plot showing the density of CD8+ and PD1+CD8+ within tumor core and tumor stroma of pTME and mTME of chemo-naïve and treated HGSOC. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. C and D, Representative image (C) and box plot showing the density of TCF1+PD1+CD8+ T cells and TIM-3+PD1+CD8+ and spatial distribution of TCF1+PD1+CD8+ T cells within tumor core and stroma in pTME and mTME of chemo-naïve and treated HGSOC (D). Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. E, Supervised hierarchical clustering of gene signatures associated with different stages of T-cell differentiation: T-cell stemness (orange), T-cell effector function (blue), T-cell proliferation (green), T-cell phenotype (red) as determined by RNAseq in mTME of chemo-naïve and treated HGSOC. For further details, see Supplementary Fig. S9. F and G, Representative image of digital pathology spatial distribution and violin plot showing the number of cell contacts between PanCK+ malignant cells and TCF1+PD1+CD8+ T cells and TIM-3+PD1+CD8+ within 0 to 30 µm in pTME and mTME of chemo-naïve and treated HGSOC.

Background Interleukin-2 (IL-2) immunotherapy can induce durable tumor remissions, but its clinical performance has been limited by significant drawbacks such as short serum half-life and high toxicity. Administration of IL-2 in complex with certain anti-IL-2 antibodies (IL-2cx) enhances circulation half-life while also selectivity directing the cytokine to particular immune cell subsets. In particular, IL-2cx has been developed that targets either cells expressing the CD25-containing high-affinity IL-2 receptor (ie, CD25-biased IL-2cx) or cells expressing the CD25-lacking intermediate-affinity IL-2 receptor (ie, CD25-blocking IL-2cx). Since regulatory T (Treg) cells primarily express the high-affinity IL-2 receptor whereas naïve effector T and natural killer cells mainly express the low-affinity IL-2 receptor, CD25-blocking IL-2cx have traditionally been considered as potential cancer therapeutics, particularly in combination with immune checkpoint inhibitors (ICIs).Methods Stimulation of antigen-primed T cells by IL-2cx in the absence or presence of ICIs was evaluated through adoptive transfer of primed ovalbumin-specific T cells and analysis of expansion. Effects of IL-2cx on Treg cell-mediated inhibition of CD8+ T cells were assessed by flow cytometry and thymidine incorporation. Tumor-bearing mice received combination treatments comprizing IL-2cx and ICIs, where complexes were delivered either before or after ICIs. Tumor growth and mouse survival were monitored, and immune cell phenotyping was performed. Toxicity was determined by tracking body weight, temperature, and lung edema. Substitution of IL-2cx with single-agent cytokine/antibody fusion proteins (immunocytokines, ICs) was also explored.Results We showed that CD25-biased IL-2cx and ICs synergize with ICIs to completely eradicate large, established tumors despite robust Treg cell expansion. Importantly, we found that timing is crucial, as administration of IL-2cx after (but not before) ICIs led to profound antitumor effects. Mechanistically, CD25-biased IL-2cx selectively stimulated expansion and effector functions of tumor-specific CD8+ T cells in a CD25-dependent manner, overcoming Treg cell-mediated suppression. Moreover, CD25-biased IL-2cx showed much lower toxicity than CD25-blocking IL-2cx, enabling a larger therapeutic window. Furthermore, we demonstrated that administration of a human IL-2-based IC significantly enhanced the antitumor activity of ICIs, establishing the translational relevance of our work.Conclusions Our findings support the temporally optimized use of CD25-biased IL-2-based therapeutics in combination with ICIs for cancer immunotherapy.

The clinical relevance of combined chemotherapy and immunotherapy in mouse models of TMBLo and TMBHi ovarian cancer. A and B, Bar plots showing single-nucleotide variants positions (SNVs) (A) and the somatic mutations prevalence (mutations per megabase) (B) in ID8 (n = 3) and Brca1−/−Trp53−/−/Myc/Hras SO1 (n = 3) C57BL/6 syngeneic mouse ovarian cancer cell lines. Mean and SEM are shown. Statistical significance was calculated by multiple t test. P values are indicated. C, Experimental design for the analysis of TLS aggregates development and efficacy of combined chemotherapy and aPD1 and/or aTIM-3 therapy in TMBLo ID8 and TMBHi SO1 experimental syngeneic mouse models. D and E, Representative immunostaining for CD4, CD8, CD20, and CD21 (D) and a box plot showing density of TLS aggregates within chemo-naïve (n = 5) and treated TMBHi SO1 (n = 9) ovarian tumors (E). Scale bars, 100 µm and 2.5 mm. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F–H, Representative dot plot (F) and flow cytometry analyses for percentages of CD62L+CD44+ central memory (CM) and CD62L−CD44− terminally differentiated CD8+ T cells (TEMRA) (G) and TCF1+PD1+CD8+ and TIM-3+PD1+CD8+ T cells (H) in tumor samples of the TMBHi SO1 experimental model in the presence or absence of carboplatin and taxane chemotherapy (NACT). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. I and J, Overall survival (OS) of TMBHi SO1 experimental model (I) and flow cytometry analyses for percentage of TCF1+PD1+CD8+ T cells after NACT, aPD1, aTIM-3 and combined therapy (J). Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. K and L, Representative immunostaining for CD4, CD8, CD20, and CD21 (K) and a box plot showing density of TLS aggregates within chemo-naïve (n = 8) and treated TMBLo ID8 (n = 8) (L) ovarian tumors. Scale bars, 100 µm and 2.5 mm. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. M, Flow cytometry analyses for TCF1+PD1+CD8+ and TIM-3+PD1+CD8+ T cells in tumor samples of the TMBLo ID8 experimental model in the presence or absence of carboplatin and taxane chemotherapy (NACT). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. N, Overall survival (OS) of TMBLo ID8 experimental model after NACT, anti-PD1, and combined therapy. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. P values are indicated. (Panel C created with BioRender.com.)

Multidrug resistance (MDR) represents one of the major concerns in cancer therapy as it may cause reduced efficacy of chemotherapeutic drugs due to the overexpression of ABC transporters, particularly P-glycoprotein (P-gp). This study explores the potential of novel amphiphilic diblock (DB) copolymers composed of poly[N-(2-hydroxypropyl)methacrylamide]-based copolymers (PHPMA) and poly(propylene oxide) (PPO) to overcome MDR mechanisms. The DB copolymers and their doxorubicin (Dox) conjugates significantly increased Dox accumulation in P-gp positive cells, markedly sensitizing them to Dox cytotoxic activity. The underlying mechanisms included depletion of intracellular ATP with subsequent inhibition of P-gp mediated drug efflux, an altered mitochondrial membrane potential, and increased ROS production. Moreover, the DB-Dox conjugates inhibited tumor growth in vivo more effectively compared to the corresponding PHPMA-based drug delivery system. Copolymers with additionally loaded PPO in the micelle core demonstrated superior efficacy in terms of P-gp inhibition, ATP depletion, and chemosensitizing effect in vitro, as well as antitumor activity in vivo. DB copolymers effectively depleted ATP levels both in vitro and in vivo using patient-derived xenograft (PDX) models, underscoring their capacity to enhance the effectiveness of standard chemotherapy and translational potential.

Background/Objectives: Here, we report the design, synthesis, and in vitro biological evaluation of a novel stimuli-sensitive nanotherapeutics based on cisplatin analog, cis-[PtCl2(NH3)(2-(3-oxobutyl)pyridine)] (Pt-OBP), covalently linked to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via a pH-sensitive hydrazone bond. Methods: Two polymer–drug conjugates, P-Pt-A and P-Pt-B, were synthesized, differing in spacer length between the polymer chain and hydrazone bond, which in turn modulates their drug release kinetics. Results: The spacer based on hydrazone bond demonstrated satisfactory stability under blood-mimicking conditions while enabling selective release of the active drug intracellularly or even in the mildly acidic tumor microenvironment. Pt-OBP exhibits comparable or even superior cytostatic and cytotoxic activity to carboplatin across a panel of murine and human cancer cell lines, with the highest potency observed in FaDu cells representing human head and neck squamous cell carcinoma. Mechanistically, Pt-OBP induced significant phosphorylation of γ-H2AX and activation of caspase-3, indicating its ability to cause DNA damage with subsequent apoptosis induction. P-Pt-A retained moderate biological activity, whereas the slower-releasing P-Pt-B exhibited reduced potency in vitro, consistent with its drug release profile. Conclusions: Notably, free Pt-OBP induced rapid apoptotic cell death, surpassing carboplatin at early time points, and the polymeric conjugates achieved comparable pro-apoptotic activity after extended incubation, suggesting effective intracellular release of the active drug.

NACT-mediated progenitor TCF1+PD1+CD8+ T-cell phenotype associates with effector cytotoxic functions within metastatic HGSOC. A–C, Representative dot plot (A) and box plot (B) showing percentage of TIM-3+PD1+CD8+ T cells and GZMB+CD8+ T cells within native pTME and mTME of chemo-naïve and treated HGSOC as determined by flow cytometry. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by the Mann–Whitney test. P values are indicated. C, Marker heatmap dot plots obtained after t-SNE and showing the relative expression of the indicated marker in the different phenotypic clusters within mTME of chemo-naïve and treated HGSOS as determined by flow cytometry. D, Design of experimental and sequencing workflow in 11 patients with HGSOC before and after NACT. scRNAseq was performed on dissociated solid tumor specimens using 10× Genomics Chromium platform. E and F, Uniform manifold approximation and projection (UMAP) plot of all cells (n = 51,476) passing the quality control, colored by type of therapy (E) and cell type (F). G and H, TILs projections (G) and predicted subtype frequencies (H) in biopsies from patients with chemo-naïve and treated HGSOC. CM, central memory; EM, effector memory; MAIT, mucosal-associated invariant T cells; PTEX, progenitor exhausted T cells; TEMRA, terminally exhausted T cells; TEX, exhausted T cells. I and J, Radar plot showing percentage of CD8+ T cells expressing respective T-cell marker (KLRB1, TCF7, CCR7, IL7R, LMNA, FGFBP2, XCL1, CD200, CRTAM, TOX, PDCD1, HAVCR2, and GNLY; I) and UMAP showing expression of PDCD1, HAVCR2 and TCF7 in CD4+ and CD8+ T-cell clusters in chemo-naïve and treated HGSOC samples (study cohort 5; J), as determined by scRNA-seq. (Panel D created with BioRender.com.)

Abstract Epithelial ovarian carcinoma (EOC) is one of the top five causes of cancer-related death in women. Most patients with EOC achieve initial remission after primary or interval cytoreductive surgery combined with platinum-taxane chemotherapy. However, mutations in BRCA1 or BRCA2 genes, which lead to homologous recombination (HR) defects, play a crucial role in platinum sensitivity and justify the use of poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy, commonly linked to extended progression-free survival (PFS). Besides their direct cytotoxic and cytostatic effects, PARP inhibitors (PARPi) have shown significant immunostimulatory properties by disrupting DNA repair in cancer cells, and opening possibilities for synergy with immune checkpoint inhibitors (ICIs). In this study, we investigate the immunomodulatory effects of PARPi using multiparametric flow cytometry, multiplexed immunolabeling, single-cell transcriptomics, and functional assays in an experimental BR5Brca1-/- syngeneic mouse model and human EOC tumor samples. We examine the molecular and cellular mechanisms that can be exploited to develop more effective combination therapies. PARPi may increase the mutational burden in EOCs due to unresolved DNA damage and the release of damage-associated molecular patterns (DAMPs), thereby increasing T-cell infiltration. In addition, PARPi appear to promote potent type I interferon (IFN) secretion through the activation of cGAMP synthase and the stimulator of the interferon genes (STING) pathway. In combination with ICIs, PARPi showed a beneficial effect on the balance between adaptive anti-tumor immunity and innate myeloid components, leading to an improved cytotoxic T-cell response, as observed in mouse models and HGSOC tumor samples. These observations emphasize the role of strategically designed combinations of PARPi and immunotherapeutic agents, which could be the key to overcoming immunosuppression in the EOC microenvironment, thereby improving clinical outcomes. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Michal Hensler, Romana Mikyskova, Lenka Kasikova, Josef Pasuvka, Jana Drozenova, Katerina Mojzisova, Marek Kovar, Iain McNiesh, Michael Halaska, Lukas Rob, Sandra Orsulic, Milan Reinis, Lorenzo Galluzzi, Radek Spisek, Jitka Palich Fucikova. PARP inhibitors as immune modulators in metastatic ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C046.