Emerging peptide therapeutics for the treatment of ovarian cancer

ABSTRACT

Introduction

The discovery of therapeutic proteomic targets has resulted in remarkable advances in oncology. Identification of functional and hallmark peptides in ovarian cancer can be leveraged for diagnostic and therapeutic targeting. These targets are expressed in different tumor cell locations, making them excellent candidates for theranostic imaging, precision therapeutics, and immunotherapy. The ideal target is homogeneously overexpressed in malignant cells with no expression in healthy cells, thereby avoiding off-tumor bystander toxicity. Several peptides are currently undergoing extensive evaluation for the development of vaccines, antibody-drug conjugates, monoclonal antibodies, radioimmunoconjugates, and cell therapy.

Areas covered

This review focuses on the significance of peptides as promising targets in ovarian cancer. English peer-reviewed articles and abstracts were searched in MEDLINE, PubMed, Embase, and major conference databases.

Expert opinion

Peptides and proteins expressed in tumor cells are an exciting area of research with great potential and may significantly influence precision therapeutics and immunotherapeutic strategies. Accurate utilization of peptide expression as a predictive biomarker has the potential to greatly enhance treatment precision. The ability to measure receptor expression paves the way for its use as a predictive biomarker for therapeutic targeting and requires critical validation of sensitivity and specificity for each indication to guide therapy.

KEYWORDS:

• Epithelial ovarian cancer
• drug development
• proteomics
• immunotherapy
• targeted therapy

1. Background

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic cancer despite important therapeutic advances in recent years [1]. In the past, EOC was categorized into types I and II. Type I tumors were considered indolent, genetically stable, and linked to specific precursor lesions, while type II was more aggressive with higher malignant potential. However, recent advances have revealed the clinical and molecular heterogeneities of various histological subtypes, rendering the dualistic classification to be an oversimplification [2]. The main histological subtypes possess unique genomic characteristics: high-grade and low-grade serous, endometrioid, clear cell, and mucinous carcinomas. Among these, high-grade serous ovarian cancer (HGSOC) is the most common histological subtype, accounting for up to 80% of all cases. HGSOC predominantly arises from the distal end of the fallopian tube from precursor lesions called serous tubal intraepithelial carcinoma (STIC) [3] and is most often diagnosed at an advanced stage when the disease has spread beyond the pelvis (FIGO stage III/IV) [4].

Considerable progress has been made in the genetic and molecular characterizations of EOC, allowing the development of biomarker-driven therapy. The Cancer Genome Atlas (TCGA) study reported that about 50% of HGSOC harbor some form of homologous recombination deficiency (HRD), including germline or somatic mutations in BRCA genes [5]. Germline BRCA1/2 mutations are present in 16% of HGSOC, representing the most significant known genetic risk factor for EOC [6]. Identifying patients who carry BRCA1/2 mutations is important for genetic counseling and therapeutic planning. These patients tend to respond better to platinum-based chemotherapy and poly-ADP ribose polymerase (PARP) inhibitors, leading to improved outcomes despite typically being diagnosed at later stages.

The mainstay of EOC treatment is debulking surgery, ideally achieving no macroscopic residual disease, followed by a combination of platinum and taxane-based chemotherapy. Neoadjuvant chemotherapy (NACT), followed by interval debulking surgery and adjuvant chemotherapy, is an alternative option for selected patients. NACT may be considered for a subgroup of women who are not candidates for upfront surgery due to comorbidities, poor performance status, advanced age, or extensive disease where complete cytoreduction is not feasible. An advantage of NACT is its ability to assess chemo-sensitivity, allowing identification of patients at higher risk of relapse [7]. Maintenance therapy with bevacizumab (antiangiogenic agent) and PARP inhibitors are also part of the armamentarium against EOC [8]. Patients with HRD-positive tumors are susceptible to PARP inhibitors exploiting the concept of synthetic lethality. PARP inhibitors are the most successful targeted therapy in EOC [9]. Despite important progress in this field, about 60% of the patients with stage III and 80% with stage IV will succumb to the disease within 5 years, emphasizing the urgent need to identify novel therapeutic strategies to improve outcomes [4].

In cells lacking functional homologous recombination repair (HRR), such as those with BRCA mutations, alternative, error-prone pathways, including non-homologous end-joining repair (NHEJ), repair DNA double-strand breaks. This can lead to the accumulation of genomic instability and eventual cancer cell death. Although NHEJ is faster than HRR and primarily occurs during the G1 phase, recent evidence suggests that it also operates throughout the cell cycle [10]. In addition to well-known NHEJ-associated proteins, such as Ku70/80, DNA-PKcs, DNA pol λ/μ, DNA ligase IV-XRCC4, and XLF, newer proteins are implicated in the process. These include PAXX, MRI/CYREN, TARDBP, IFFO1, ERCC6L2, and RNase H2. Among these proteins, MRI/CYREN serves a dual role by stimulating NHEJ during the G1 phase while inhibiting the pathway during the S and G2 phases [10].

Proteomics encapsulates a wide range of processes, including protein expression profiling, protein–protein interactions (PPI), protein function, and structure. Dysregulated protein or peptide expression, such as overexpression, loss of expression, and defective or mislocated protein, can lead to oncogenesis, proliferation, apoptosis, and drug resistance [11,12]. These proteins show variable expression within different compartments of the cells, with many of them being mis-localized in tumor cells compared to normal cells. The role of proteins and peptides in tumor proliferation differs depending on their location on the cell surface, cytoplasm, mitochondria, or nucleus [12].

The development of therapeutic peptides against very specific proteins in cancer cells has emerged as an attractive approach due to their on-target damage to the cancer cell and normal tissue sparing [13,14]. Chemical and biological methods have been applied to produce and modify peptides and proteins. Peptides and proteins can be engineered in different ways to damage cancer cells, for example, cell-targeting peptides can bind and modulate oncogenic proteins expressed on the EOC cell surface, facilitating the internalization of the therapeutic complex through peptide–protein interaction [15].

Peptides and proteins can help deliver a therapeutic payload to tumors when coupled with anticancer drugs, genes, or RNAs [16]. The nature of the payload can vary, ranging from conventional ‘chemotherapeutic agents’ to smart immune modulators. It is important to recognize that the impact of a precise local ‘bombing’ can trigger a local reaction that affects the cells around the target cell. Recent technologies have led to the development of peptides and protein-based therapeutics, such as vaccines [17], antibody-drug conjugates (ADCs) [18], bispecific T-cell engagers (BiTes) [19], and adoptive cellular therapies (see Figure 1) [20]. This review will specifically focus on peptides currently under active development for the treatment of ovarian cancer (see Figure 2).

Figure 1. Screening and identification of peptide candidates for cancer therapy. Different applications of peptides in cancer therapy include vaccines, antibody–drug conjugate, peptide–drug conjugate, cell therapy, and probes for tumor diagnosis and imaging.

Figure 2. Emerging peptides and proteins in ovarian cancer treatment. These targets exhibit diverse origins and functions and could be associated with the testis, placenta, normal adult tissue, tumor stroma, and random unique mutations.

2. Medical need

Unfortunately, despite optimal front-line treatment, most women who present with advanced disease experience recurrence. Platinum-free interval (PFI) has been a key factor for selecting subsequent therapy and inferring prognosis. However, PFI should not be used in isolation, but rather in conjunction with other important clinical and molecular features such as histology, HRD status, previous therapies, outcomes of prior surgery, and patient-reported symptoms [21]. The Gynecologic Cancer InterGroup (GCIG) sixth Ovarian Cancer Conference on Clinical Research recommended replacing the platinum-free interval with a therapy-free interval (TFI) specific to particular therapies such as platinum, PARP inhibitors, and other specific and molecular factors [22]. Predictive biomarkers, exposure or response to previous therapies are important stratification factors for choosing the most effective treatment path.

Primary or acquired treatment resistance has been a major clinical challenge, and subsequent treatment options are limited, leading to broad cross-resistance. For example, in platinum-resistant EOC, response rates to single-agent chemotherapy are as low as 10–15%, and median overall survival (OS) is estimated at only 12 months [8]. Hence, there is an urgent need to identify novel therapeutic approaches, which may be effective in this setting for EOC. Proteomic technologies, such as mass spectrometry and protein array analysis, have significantly advanced our ability to understand molecular signaling events and proteomic profiles [23]. Proteomic analysis of ovarian cancer and its adaptive responses to therapy can reveal new therapeutic options that have the potential to mitigate drug resistance and improve patient outcomes.

3. Existing treatment

The foundation of current treatment for newly diagnosed patients with EOC consists of debulking surgery to achieve no macroscopic residual disease, followed by platinum-based chemotherapy. Systemic therapy has been evolving with the emergence of targeted therapies. The first peptide receptor targeted in EOC was the vascular endothelial growth factor (VEGF), which regulates the angiogenesis pathway. The monoclonal antibody anti-VEGF, bevacizumab, has changed therapy in first-line and recurrent settings. There is good evidence supporting the addition of bevacizumab to chemotherapy and its use as a maintenance therapy based on progression-free survival (PFS) benefit as observed in the GOG-0218 (NCT00262847) and ICON7 (ISRCTN91273375) trials [24,25]. Patients with high-risk factors such as stage IV, inoperable stage III, and macroscopic residual disease after primary debulking surgery experienced the most significant benefit with bevacizumab.

PARP inhibitors block the DNA repair pathway through multiple mechanisms, leading to DNA damage accumulation and cell death, particularly in cells with impaired DNA repair by virtue of BRCA mutations or Homologous Repair Deficiency (HRD) [26]. They have transformed the management of the EOC, establishing a new standard of care by being synthetically lethal in HGSOC with BRCA1/2 mutation and other forms of HRD, which is a characteristic present in about 50% of these tumors [5]. However, PARP inhibitors have also shown activity in HGSOC without BRCA mutations or HRD, although the BRCA-mutated tumors derive the greatest benefit [27]. Due to overlapping toxicity, the sequential use of PARP inhibitor after response to platinum-based chemotherapy has been adopted and is well tolerated. Initially, PARP inhibitors were assessed for the treatment and maintenance of recurrent ovarian cancer in platinum-sensitive disease [26]. However, recurrent EOC is not curable for the majority of patients. Thus, PARP inhibitors have been moved to first-line maintenance treatment, to prolong progression-free survival and ideally prevent or delay recurrence to improve survival [28].

Three different PARP inhibitors are approved as first-line maintenance treatments: niraparib, olaparib, and rucaparib. Niraparib was approved for the first-line maintenance regardless of the presence of BRCA mutation or HRD, based on the PRIMA trial (NCT02655016) which assessed niraparib or placebo for 3 years [29]. The most recent analysis with 3.5 years of follow-up maintains a median PFS benefit in all-comers of 13.8 months in niraparib versus 8.2 months in placebo arm (HR 0.66; 95% CI, 0.56–0.79; P < .001). The benefit was greater in patients with BRCA mutated or HRD tumors treated with niraparib, with a median PFS of 24.5 months versus 11.2 months for those treated with placebo (HR 0.52; 95% CI, 0.40–0.68; p < 0.001) [30].

The SOLO-1 trial (NCT01844986) evaluated maintenance therapy with 2 years of olaparib or placebo in patients with BRCA-mutated EOC. There was a clinically meaningful improvement in OS favoring olaparib after 7 years of follow-up. At an OS data maturity of 38.1%, median OS was not reached with olaparib compared to 75.2 months with placebo (HR 0.55; CI 0.40–0.76) [31]. Kaplan–Meier curves indicated that 67% of olaparib arm and 46.5% of placebo arm patients were alive at 7 years. This benefit was seen despite 44.3% of the patients in the placebo arm receiving subsequent PARP inhibitor. Time to first subsequent therapy (TFST) was delayed with maintenance olaparib with 64 months versus 15.1 months in the placebo arm (HR 0.37; CI 0.28–0.48) [31].

The PAOLA-1 trial (NCT02477644) evaluated olaparib plus bevacizumab versus bevacizumab alone, regardless of HRD status. In the overall population, the median OS was 56.5 months with olaparib plus bevacizumab and 51.6 months with bevacizumab alone, which was not statistically significant [32]. In an exploratory analysis, median OS improved with olaparib plus bevacizumab in HRD-positive patients, regardless of BRCA mutation status (HR 0.71; 95% CI 0.45–1.13). No benefit in OS was seen with the addition of olaparib in patients who were negative for HRD and received bevacizumab. Subsequent PARP inhibitor was received by 45.7% in the control arm [32].

Unfortunately, most women with advanced ovarian cancer eventually relapse and progress to develop resistant disease. Outcomes in patients with platinum-resistant EOC remain poor with single agent standard-of-care chemotherapies, with low response rates and short PFS of 3–4 months [33], and median OS of approximately 12–18 months [34]. In the open-label phase III AURELIA trial, the addition of bevacizumab to single-agent chemotherapy (pegylated liposomal doxorubicin, weekly paclitaxel, or topotecan) demonstrated significant improvement in PFS and ORR in patients with platinum-resistant EOC [33]. Median PFS was 3.4 months in the single-agent chemotherapy group and 6.7 months in the bevacizumab plus chemotherapy group (HR 0.48, 95% CI 0.38–0.60; non-stratified log-rank p < 0.001). There was no difference in median OS, 13.3 months versus 16.6 months in single-agent chemotherapy and bevacizumab plus chemotherapy group, respectively (HR 0.85, 95% CI, 0.66–1.08; p < 0.174). OS was likely affected by the 40% crossover of the single-agent chemotherapy group receiving bevacizumab at the time of analysis [33].

The treatment strategy for non-high-grade serous or endometrioid ovarian cancer is continually evolving to incorporate more precise therapies tailored to the specific histological subtype. The sixth GCIG ovarian cancer consensus conference (OCCC6) favors primary cytoreductive surgery in histological subtypes with low chemo-sensitivity, like ovarian clear cell carcinoma (OCCC) and low-grade serous ovarian cancer (LGSOC) [22]. Precision in molecular characterization is changing the treatment landscape of LGSOC, especially with the new evidence of tyrosine-kinase inhibitors (TKIs). MEK/BRAF and FAK inhibitors are being investigated in the recurrent setting with the demonstration that about 60% of LGSOC harbor activating mutations in the MAPK pathway [35]. The phase III MILO-ENGOT-ov11 trial (NCT01849874) failed to demonstrate PFS improvement with binimetinib (MEK inhibitor) compared to investigator-choice chemotherapy after platinum-based treatment [36]. However, the phase III GOG 281/LOGS trial evaluating trametinib showed significant improvement in PFS compared with SOC, regardless of mutational status [37]. Another strategy being evaluated is the combination of VS-6766 (RAF/MEK dual inhibitor) and defactinib (FAK inhibitor) (NCT04625270).

OCCC is chemoresistant, with response rates lower than 10% in relapsed disease [38]. Compared to HGSOC, OCCC has lower p53 and BRCA1/2 mutations but a higher frequency of mutations in ARID1A, PIK3CA, and PTEN. This unique genetic profiling suggests a potential benefit of targeted therapies and immunotherapy in patients with OCCC [39]. Likewise, mucinous ovarian carcinoma (MOC) responds poorly to platinum-based chemotherapy [40]. Biomarker-driven therapies with targeted agents are emerging in histologies with relative chemoresistance. Targeting proteins and pathways have shown modest efficacy with the limitation that TKIs are not selective and generate significant bystander effects and toxicity [41].

4. Scientific rationale

Peptides are composed of short sequences of amino acids and are held together by peptide bonds. They are structural segments of proteins and are subdivided into oligopeptides and polypeptides [42]. Therapeutic peptides and proteins bind to cell receptors with high affinity and trigger intracellular effects. They are vital for cellular activity, such as cell growth, energy metabolism, material transport, signal transmission, and immune regulation [43–45]. Most of these peptides and proteins are expressed on the tumor cell surface and are classified as tumor-associated antigens (TAA). Other emerging targets are the cancer-testis antigens (CTA), which are expressed in a wide range of cancer types. In contrast, their expression in normal tissues is restricted to immune privileged sites such as testis and placenta [46]. Some of these peptides and proteins are ideal targets for cancer-specific immunotherapy.

The National Cancer Institute (NCI) reported a list of cancer antigens [47]. This list comprises 75 proteins ranked according to the ideal cancer antigen criteria. The characteristics analyzed were 1) therapeutic function; 2) Immunogenicity; 3) Oncogenicity; 4) Specificity; 5) expression level and percentage of positive cells; 6) Stem cell expression; 7) Number of patients with antigen-positive tumors; 8) Number of epitopes; 9) Cellular location of expression. For instance, a high expression level on the cell surface with no circulating antigen and oncogenic ‘self’ protein are favorable characteristics for developing immunotherapeutic strategies [47].

The ideal antigen is also composed of multiple epitopes with the potential to bind most major histocompatibility complex (MHC) proteins. The MHC class I and class II proteins bind to the tumor antigen epitopes and present the peptides on the cell surface, activating anti-tumor immunity. Furthermore, this mechanism is important for immune recognition of the intracellular proteome on the cell surface (immunopeptidome) via peptide/MHC interactions [48]. All these basic requirements are essential to select peptides for the development of immunotherapies (see Figure 3).

Figure 3. Key requirements for cancer targeting peptides. These criteria were described as important characteristics of peptides/proteins for developing effective immunotherapy.

Peptides and proteins can be considered in cancer therapy in four main ways: (1) radioisotopes, dyes, and other molecular labeled peptides as probes for diagnosis and imaging [49]; (2) peptide vaccines to elicit a tumor-specific immune response [50]; (3) platform for transporting other peptides and proteins, DNAs, RNAs, and cytotoxic therapy into tumor cells [51]; (4) engineering peptide antigens to specific targets by cell therapy [52].

TAAs and CTAs have enabled the development of multiple antibody-based therapeutic strategies. Optimization in peptide vaccine development brought this approach to the therapeutic front again. Improvements are evident in peptide selection, vaccine delivery, adjuvant formulation, and the use of combination therapies [53]. Antigenic peptides from specific target proteins are engineered as vaccines to bind to the MHC on antigen-presenting cells (APCs). At the injection site, APCs take up target peptides and present antigen to immune cells to trigger the anti-tumor effects of helper or cytotoxic T cells. T cells traffic to distant tumor sites and elicit effective tumor cell death. Peptides are suitable molecules because of their low molecular weights, good cellular uptake, and low cost [54].

An emerging category of targeted therapy is precision use of antibody-drug conjugates. Antibody drug conjugates are comprised of three elements: a monoclonal antibody (mAb), a linker, and a payload. Innovative linkers and payloads have enhanced drug delivery to tumor cells with heterogeneous antigen expression [55]. The bystander killing effect is the ability to provide cytotoxic activity against off-target cancer cells because of diffusion of the free cytotoxic payload, spreading from the antigen-positive cells. This effect addresses the antigen heterogeneity often observed in solid tumors. Although a payload can belong to any class of drugs, ADC development has mainly explored potent cytotoxic products [56].

Cell-based therapy, particularly adoptive cell therapy (ACT), is a personalized form of immunotherapy in which autologous antigen-specific T cells are transferred after ex vivo modification and expansion. There are three different types of ACT: expanded natural tumor-infiltrating lymphocytes (TILs), T-cell receptor (TCR) therapy, and chimeric antigen receptors T-cell (CAR-T) therapy [57]. CAR-T cell therapy redirects T cells to recognize cell surface antigens in an HLA-independent manner. For this, CAR-T cells utilize antibody fragments that bind to specific antigens on the cancer cell surface. Conversely, TCR-T cell therapy can recognize membrane and intracellular antigens as long as these are presented by MHC molecules. Thus, TCR-T is MHC restricted to recognize targets and activate T cell function [58]. ACTs in the EOC are currently in early-phase clinical trials targeting TAAs and CTAs [59].

5. Competitive environment: a review of drugs in phase II & III development (Table 1)

5.1. Mucins

The mucin family is a group of high molecular weight glycoproteins synthesized by specialized epithelial cells. They are characterized by a repeated amino acid structure with variations of tandem repeats between different mucins. They are classified into secreted or membrane-bound mucins. The main physiological function of this family is lubrication of epithelial surfaces and their protection. However, some mucins are aberrantly expressed in many tumor tissues and have their role in signal transduction pathways [60]. When overexpressed on the membrane, mucins confer antiadhesive and anti-recognition properties leading to immune surveillance evasion. For example, adenocarcinomas release high mucin levels to shield their cells from the toxic microenvironment [61]. The extracellular domain of membrane-bound mucins expressed in specific tumors is a potential target for antibody-mediated therapies. Particularly in EOC, the glycoproteins MUC1 and MUC16 play an important role in tumor development and progression [62 p.1].

Table 1. Competitive environment of emerging peptides for ovarian cancer treatment in phases I, II, and III of development.

Peptide	Agent/study	Therapeutic Class	Stage of development	Indication	NCT no.
Mucin-16	Oregovomab (FLORA-5 trial)	Vaccine	Phase III	EOC – First line with chemotherapy	NCT04498117
	Oregovomab	Vaccine	Phase II	PSOC	NCT05335993
Survivin	Maveropepimut-S (PESCO trial)	Vaccine	Phase II	PROC, PSOC	NCT03029403
Folate receptor α	Mirvetuximab soravtansine (MIRASOL trial)	ADC	Phase III	PROC, high FRα expression	NCT04209855
	Mirvetuximab soravtansine (PICCOLO trial)	ADC	Phase II	PSOC, high FRα expression	NCT05041257
NaPi2b	Upifitamab rilsodotin (UPLIFT trial)	ADC	Phase I/II	PROC	NCT03319628
	Upifitamab rilsodotin(UPNEXT trial)	ADC	Phase III	Maintenance in PSOC, high NaPi2b expression	NCT05329545
	Upifitamab rilsodotin (UPGRADE trial)	ADC	Phase I	PSOC	NCT04907968
Mesothelin	Gavocabtagene autoleucel (TC-210)	Cell therapy	Phase I/II	EOC with high MSLN expression	NCT03907852
NY-ESO-1	TBI-1301	Cell therapy	Phase Ib	EOC, HLA matched, high NY-ESO-1 expression	NCT02869217

Note: Abbreviation: EOC: Epithelial ovarian cancer; PSOC: platinum-sensitive ovarian cancer; PROC: platinum-resistant ovarian cancer; ADC: Antibody–drug conjugate; FRα: Folate receptor alpha; MSLN: mesothelin; HLA: Human Leukocyte Antigen.

MUC-1, also known as EMA (epithelial membrane antigen), has two subunits, MUC1-N and MUC1-C. MUC1-N is released into the extracellular compartment to form the mucous gel. MUC1-C is a transmembrane protein. The cytoplasmic portion of MUC1-C interacts with several signaling pathways through GSK3 (glycogen synthase kinase 3 beta), -catenin, GRB2 (growth factor receptor-bound protein 2), SRC, and ESR1 (estrogen receptor 1). MUC1-C also contains a p53 binding region that activates p53-dependent growth and suppresses the apoptotic genes, thereby promoting the survival of tumor cells upon exposure to DNA-damaging agents [63]. To date, therapeutic strategies targeting MUC1 did not show clinical benefit, although there is a biological rationale. Sehouli and colleagues recently published the results of the phase 2 trial, a double-blind, randomized, placebo-controlled assessing Gatipotuzumab as a switch maintenance therapy in recurrent HGSOC (NCT01899599). Gatipotuzumab is a monoclonal antibody that binds to tumor-associated MUC1 (TA-MUC1) with high affinity. Gatipotuzumab did not improve outcomes over placebo in TA-MUC1 positive OC patients [64].

MUC16 is the largest transmembrane glycoprotein with limited expression in the epithelium of the upper respiratory tract, ocular surface, mesothelial cells, and reproductive organs. MUC16 overexpression on the EOC cell surface and its presence in blood (CA125 antigen) make it an attractive therapeutic target and a well-established serum biomarker for EOC [65]. Oregovomab is a monoclonal antibody drug that binds to the glycosylated N-terminal region of membrane MUC16 resulting in immune cell-mediated killing of MUC16-expressing tumor cells. Initially, oregovomab showed favorable outcome in combination with carboplatin and paclitaxel compared to carboplatin and paclitaxel in a phase II trial (NCT01616303) [66]. However, oregovomab monotherapy failed to show clinical benefit in phase II and III trials. A phase 3 clinical trial (NCT04498117) of oregovomab is ongoing for newly diagnosed EOC patients in conjunction with carboplatin and paclitaxel chemotherapy. Other trials are evaluating the combination of oregovomab with bevacizumab (NCT04938583) and niraparib (NCT05335993) in platinum-sensitive recurrence.

DMU4C064A is an anti-MUC16 thiomab-ADC, utilizing a cysteine-engineered humanized IgG1 conjugated to an anti-mitotic agent (monomethyl auristatin E – MMAE) payload. DMU4C064A has shown interesting results in a phase I trial of platinum-resistant EOC (NCT02146313) [67]. The confirmed ORR in a small phase 2 trial was 45%, including 1 CR and 8 PRs, median PFS was 5.8 months. All eight responders with evaluable archival tumor tissue were MUC16 IHC 2+/3+. The most common related AEs for all dose levels were fatigue (34%), nausea (32%), and diarrhea (23%). Four dose-limiting toxicities occurred [67].

Most molecules developed bind to epitopes present on the terminal fraction of the glycoprotein and are later released. The challenge is to bind to the retained extracellular fraction on the membrane. A hybridoma that generates a mAb specific to the extracellular retained fraction of the MUC16 antigen (MUC16ecto) has been utilized to create a CAR specific to MUC16ecto, which can be utilized to engineer autologous T cells targeted to the retained, surface-exposed antigen. A phase I clinical trial proposes the use of adoptive T cell therapy using IL-12 secreting MUC16ecto directed CARs for recurrent EOC [68].

5.2. Survivin

One important mechanism of immune evasion of tumor cells is resistance to apoptosis. Thus, molecules involved in the regulation of apoptosis are potential targets for therapy. Survivin is the smallest member of the inhibitors of apoptosis (IAPs) family of proteins involved in tumor cell differentiation, programmed cell death, regulation of cell division, invasion, and metastasis [69]. Survivin is primarily found in embryonic tissues and is relatively undetectable in normal adult tissues; however, it is overexpressed in many tumor types [70]. The gene BIRC5 encodes wild-type survivin other five splice variants. It is unclear whether splicing variants are an adaptation of tumor cells to support their proliferation and escape from immune surveillance. All isoforms differ from each other in expression patterns and cellular localization [71]. Initial research was focused on the role of survivin as an intracellular protein. More recently, a proteomic study identified survivin on the outer membrane of tumor cells, making it a specific immunotherapeutic target [72]. The percentage of survivin expression in EOC varies from 74% to 90% [73] and is correlated with poor prognostic factors such as high grade, histologic type, and p53 mutation [74,75].

Preclinical and clinical studies have evaluated vaccine therapy against survivin in EOC due to its high expression. Vaccine therapy leads to the presentation of survivin epitopes by MHC I complexes on the cell surface, triggering the anti-tumor effects of helper or cytotoxic T cells [70]. Maveropepimut-S (formerly DPX-Survivac) is a unique peptide cancer vaccine composed of 5 HLA class I peptides derived from survivin, a T helper peptide, and an adjuvant poly dIdC [76]. When the vaccine is administered subcutaneously, a unique oil-based delivery system forms a depot at the injection site. This formulation compels the antigen-presenting cells (APCs) to internalize the lipid vesicles containing survivin peptides at the injection site and transport them to the lymph nodes, where they can be presented as naive immune cells. Survivin-specific immune cells enter the bloodstream, infiltrating the tumor tissues. The combination of Maveropepimut-S with cyclophosphamide in EOC was assessed in phase II, open-label, single-arm, DeCidE1 trial (NCT02785250). Twenty-two pre-treated patients with recurrent EOC were included regardless of platinum sensitivity status. Among 19 evaluable patients, the ORR was 21.1%. Translational analyses provide evidence of tumor infiltration by diverse survivin-specific T cells [77]. More recent results from phase I and the platinum-resistant cohort of the phase II PESCO trial (NCT03029403) were presented [17]. In this study, Maveropepimut-S was combined with low-dose cyclophosphamide and the anti-PD1, Pembrolizumab. The patients were heavily pretreated with a median of 4 prior lines. Twenty-three patients were evaluable, and the ORR was 22%, including one complete response in a patient with microsatellite instability-high (MSI-high). The clinical benefit rate in HGSOC was 68%. Correlative studies detected immune responses specific against survivin, whereas the complete responder had the highest and long-lasting response. Treatment with Maveropepimut-S is usually well tolerated, with the most common adverse events being grade 1 injection site reaction (ISR) and fatigue [17]. The high-grade serous or endometrioid platinum-sensitive and the exploratory cohort of the PESCO trial are ongoing (NCT03029403). The phase 2 AVALON trial is ongoing, testing the combination of Maveropepimut-S and low-dose cyclophosphamide in platinum-resistant patients (NCT05243524).

5.3. Folatereceptor alpha (FRα)

FRα is a folate-binding protein located on the cell surface membrane promoting the folate transport into cells required for DNA and RNA synthesis, cell growth, and proliferation. Minimal expression of FRα in non-malignant tissues can be found in the choroid plexus, thyroid, salivary glands, alveoli of the lungs, kidneys, breasts, colon, and bladder [78]. The FRα expression pattern can lead to potential target-dependent toxicities due to the distribution of the agent, such as ocular or pulmonary toxicity [55]. However, no clear target-dependent toxicity is exclusively related to FRα [78].

Aberrant or overexpression of FRα is seen in ovarian, endometrial, breast, and non-small cell lung cancers [79]. It is upregulated in tumor cells in response to increased folate demand. About 80–96% of EOC overexpress FRα, making it an interesting tumor antigen with selective drug delivery [80]. Thus, molecularly targeted approaches exploiting FRα in EOC are under development. Farletuzumab (MORAb-003), a fully humanized anti-FRα monoclonal antibody, demonstrated safety as a single agent in heavily pretreated EOC patients in a phase I trial [81]. Subsequently, a phase II trial revealed a favorable ORR of 75% with the combination of farletuzumab, carboplatin, and a taxane followed by farletuzumab maintenance therapy in relapsed platinum-sensitive EOC [82]. This study led to the phase III trial that randomized women with relapsed platinum-sensitive EOC to receive carboplatin plus a taxane with either farletuzumab or placebo. The study did not meet its primary PFS endpoint [83]. However, subgroup analysis demonstrated PFS (HR, 0.49) and OS (HR, 0.44) superiority over placebo in patients who had low CA-125 (3× upper limit of normal). The hypothesis is that CA-125 directly binds to farletuzumab, thereby interfering with the antibody-mediated tumor cellular cytotoxicity [83]. Thus, a phase II trial (NCT02289950) assessed the addition of farletuzumab to carboplatin plus paclitaxel or carboplatin plus pegylated liposomal doxorubicin (PLD) in patients with platinum-sensitive recurrent ovarian cancer low CA-125 levels [84]. The combination of farletuzumab plus chemotherapy did not demonstrate superiority over placebo plus chemotherapy. Taken together, these results demonstrated that farletuzumab as a monoclonal antibody alone has some activity, but it would require improvements to achieve better outcomes.

An ADC called farletuzumab ecteribulin (MORAb-202) was developed and comprised of farletuzumab conjugated to the potent cytotoxic microtubule inhibitor eribulin as the payload by a cathepsin B-cleavable linker. Data from the dose expansion cohorts of the phase Ib Study 101 trial (NCT03386942) with farletuzumab ecteribulin in platinum-resistant EOC in the Japanese population was recently presented [85]. Tumor FRα expression (>5%) was required. Two levels of farletuzumab ecteribulin were evaluated (cohort 1 at 0.9 mg/kg and cohort 2 at 1.2 mg/kg). In cohort 1, the ORR was 25.1%, while in cohort 2 was 52.4%. Although farletuzumab ecteribulin demonstrated antitumor activity regardless of FRα expression levels, interstitial lung disease (ILD)/pneumonitis was the most common treatment-related adverse event experienced by 37.5% and 66.7% of the patients in cohort 1 and cohort 2, respectively [85].

Concurrently, another FRα-targeting ADC, mirvetuximab soravtansine (IMGN853) showed promising outcomes. Mirvetuximab soravtansine is composed of a humanized FRα-binding monoclonal antibody (M9346A) conjugated to the cytotoxic maytansinoid effector molecule DM4 by a cleavable disulfide linker. Preclinical in vitro and in vivo studies demonstrated the antitumor activity of this agent in models of ovarian cancer with an interesting bystander effect in tumor cells lacking FRα expression [86]. In the first-in-human phase I trial (NCT01609556), mirvetuximab soravtansine was administered to patients with FRα-positive solid tumors, including women with EOC [87]. The most frequent treatment-related adverse events were fatigue, diarrhea, and blurred vision, mostly in grades 1 or 2. The expansion cohort as part of this phase I trial demonstrated an ORR of 26% in highly pretreated patients with platinum-resistant and FRα-overexpressing EOC [88]. These results led to the phase III FORWARD I trial (NCT02631876) in patients with platinum-resistant EOC with high or medium levels of FRα expression, defined as 75% and 50–74% of the cells with detectable membrane staining, respectively, at 10× microscope magnification [18]. Patients were randomized to receive chemotherapy (paclitaxel, pegylated liposomal doxorubicin, or topotecan) or mirvetuximab soravtansine. FORWARD I did not meet the primary endpoint of superior PFS in the mirvetuximab soravtansine arm [18].

In contrast to the phase I trial, FORWARD I used a simplified immunohistochemical test (x10 scoring) with a cutoff of 50% observable membranous staining, regardless of staining intensity. An exploratory rescoring analysis using the percentage of cells that stained positive for FRα and cellular staining intensity found that 10× scoring selected patients with lower levels of FRα expression [89]. Even with this finding, in the subset of patients with high FRα expression per protocol, secondary endpoints favored mirvetuximab soravtansine arm, including ORR (24% versus 10%), CA-125 responses (53% versus 25%), and patient-reported outcomes (27% versus 13%) [89]. These results were not significant due to the statistical analysis plan, and hence the study is being redone with the appropriate biomarker [90]. MIRASOL is an ongoing phase 3 confirmatory trial (NCT04209855) of mirvetuximab soravtansine monotherapy versus investigator’s choice of chemotherapy in patients with FRα-high platinum-resistant EOC [91]. Preliminary results of MIRASOL demonstrated the superiority of mirvetuximab soravtansine compared to chemotherapy. There is a statistically significant improvement in ORR (42.3% versus 15.9%), median PFS (5.62 versus 3.98 months, HR 0.65, p = 0.0046), and median OS (16.4 versus 12.7 months, HR0.67, p = 0.0001). The most common adverse events were ocular and gastrointestinal [92].

Additional studies with mirvetuximab soravtansine in platinum-resistant EOC were designed based on FORWARD I outcomes. The single-arm phase III SORAYA trial (NCT04209855) included platinum-resistant HGSOC with high FRα expression [93]. Prior bevacizumab was required, and prior PARP inhibitors were allowed. High FRα expression was defined as 75% of the cells with 2+ staining intensity by immunohistochemistry score. The ORR was 32.4%, and mPFS was 4.3 months (95% CI, 3.7–5.2). The most common treatment-related adverse events were the low-grade reversible ocular and gastrointestinal events [93]. Multiple clinical trials assessing mirvetuximab soravtansine in combination with other targeted agents or chemotherapies are ongoing. For example, combination with bevacizumab in platinum-resistant patients was assessed in the phase Ib FORWARD II trial (NCT02606305) and showed favorable tolerability and efficacy [94]. Mirvetuximab soravtansine is also being evaluated in the platinum-sensitive setting. For instance, the phase 2 PICCOLO trial (NCT05041257) is a single-arm study of mirvetuximab soravtansine in FRα-high recurrent platinum-sensitive EOC.

Cellular therapy targeting FRα had been investigated for the first time in phase I trial of CAR-T in EOC. Although this study demonstrated safety, there were no tumor responses. This study was done with the first generation of CAR design that lacked a costimulatory domain that can improve the persistence of engineered T cells [95]. A phase I trial (NCT03585764) with the third-generation CAR-T design is ongoing in patients with FRα-high expression (≥70% of tumor cells with ≥2+ FRα staining). Preclinical strategies to further improve the efficacy of anti- FRα CAR T cells have been investigated, including oncolytic adenovirus armed with EGFR-targeting bi-specific T cell engagers (OAd- BiTEs) to redirect anti-FRα CAR T to FRα-negative cancer cells [96].

An emerging field of intraoperative molecular imaging (IMI) exploits FRα on the EOC surface to detect cancer lesions during surgery that would not be detected by standard approach. Pafolacianine is a fluorescent drug that targets FRα and is the first optical imaging agent approved by the FDA in EOC patients. The approval was based on the single-arm phase III study that demonstrated a sensitivity of 83% to detect ovarian cancer lesions and a false-positive rate of 24.8% [49]. The use of IMI is expected to improve the rate of complete surgical resection in EOC, affecting survival.

5.4. NaPi2b

NaPi2b is a multi-transmembrane type II sodium-dependent phosphate transporter. It is responsible for the transcellular transport of inorganic phosphate, maintaining phosphate metabolism homeostasis. High phosphate level in the tumor microenvironment compared to normal tissues is a marker for tumor progression [97]. NaPi2b is overexpressed on the cell surface of 90% EOCs and other human malignancies, including thyroid, lung, breast, and other cancers [98]. Lifastuzumab vedotin (LIFA, or DNIB0600A), is an anti-NaPi2B ADC composed of a humanized IgG1 conjugated to monomethyl auristatin E, a potent microtubule inhibitor, via a cleavable valine-citrulline linker. Results from the phase I trial (NCT01363947) that enrolled 24 patients with platinum-resistant EOC showed radiologic and CA125 responses. All responses were observed in patients with NaPi2b-high expression by immunohistochemistry [99]. Subsequently, patients with platinum-resistant EOC were randomized to receive LIFA or PLD in phase II study (NCT01991210). Although the response rate was higher in the LIFA arm (34%) versus the PLD arm (15%), the duration of response was short, and there was no significant difference in PFS. The sponsor discontinued the development of LIFA due to lack of clinically significant improvement [100].

A new ADC targeting NaPi2b, upifitamab rilsodotin (UpRi, or XMT-1536), received increased attention. Upifitamab rilsodotin uses a new ADC dolaflexin platform technology that presents a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect [101]. The first-in-human phase 1/2 UPLIFT study (NCT03319628) is currently enrolling patients with platinum-resistant EOC or metastatic non – small cell lung cancer to evaluate the safety and efficacy of UpRi in tumors that express NaPi2b. It is expected that about 60% of the patients with HGSOC have NaPi2b-high expression based on IHC tumor proportion score of at least 75% [102]. Preliminary data showed antitumor activity in the platinum-resistant EOC Phase Ib expansion cohort (NCT03319628), including patients treated with prior bevacizumab and PARP inhibitors. The most common adverse effects are fatigue, nausea, vomiting, pyrexia, and transient aspartate transaminase elevations [103].

UpRi is also under investigation in recurrent platinum-sensitive HGSOC. The phase I/II UPGRADE umbrella trial (NCT04907968) evaluates UpRi in combination with other EOC therapies, including carboplatin, and the phase 3 UP-NEXT trial (NCT05329545) evaluates UpRi as maintenance monotherapy after platinum-based chemotherapy in recurrent platinum-sensitive HGSOC.

5.5. Mesothelin

Mesothelin (MSLN) is a membrane-bound glycoprotein with limited expression in normal mesothelial cells of the pericardium, peritoneum, pleura, cornea, and conjunctiva [104–106]. It is overexpressed in 55–100% of EOC. The physiological and biological functions of MSLN in normal tissue is uncertain. Differently, MSLN is involved in several mechanisms of oncogenesis and is considered as a TAA [107]. In EOC, the interaction between MSLN and MUC16 (CA125) plays a role in cell adhesion, facilitating intra-peritoneal metastasis [108].

Anetumab ravtansine is an anti-MSLN ADC composed of a fully human IgG1 conjugated to maytansinoid DM4 payload linked by a cleavable sulfo-PDB. Phase 1 trial (NCT01439152) in patients with solid tumors, including EOC, showed safety and preliminary clinical activity in patients with high levels of tumor MSLN expression [109]. In a phase Ib trial (NCT02751918), anetumab ravtansine was combined with PLD in patients with platinum-resistant EOC. Adverse events included reversible corneal disorders, neutropenia, thrombocytopenia, and gastrointestinal events. A total of 11 pts (52%) had confirmed partial responses (PR) and 7 (33%) had SD [110]. The randomized phase II (NCT03587311) study of bevacizumab with weekly anetumab ravtansine (ARB) or weekly paclitaxel (PB) in platinum-refractory or resistant mesothelin-positive EOC was terminated due to better outcomes in the control arm at the time of futility analysis. The ORR was 18% in the ARB arm versus 55% in the PB arm. The estimated median PFS favored the control arm, with 5.3 months in the ARB arm versus 9.6 months in the PB arm. This study included heavily pre-treated patients, and 42% had received prior bevacizumab [111].

Another emerging mesothelin-targeting ADC is the BMS-986148, which contains a fully human IgG1 anti-MSLN mAb conjugated to tubulysin (a cytotoxic anti-microtubule) via a valine-citrulline linker [112]. The phase I/IIa CA008–002 trial (NCT02341625) assessed the safety, tolerability, and preliminary efficacy of BMS-986148 with or without nivolumab in patients with mesothelin-expressing tumors, including 27 patients with recurrent EOC. The combination of BMS-986148 plus nivolumab demonstrated an ORR of 20%. The most common treatment-related adverse events were increased aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase [113].

Anti-MSLN monoclonal antibody has been tested in EOC. Amatuximab (MORAb-009) is a mouse-human chimeric monoclonal antibody with a high affinity for MSLN. Preclinical evaluation demonstrated inhibition of the adhesion between MSLN and MUC16 and cell-mediated cytotoxicity [114]. A phase I clinical trial evaluating the mechanisms of amatuximab in patients with mesothelin-expressing cancers reported an increase in serum CA125 levels in patients with mesothelioma and ovarian cancer, thereby preventing the intraperitoneal/intrapleural spread of disease [115]. Despite the rationale and early evidence of efficacy, amatuximab has still not been evaluated in phase II trials [116].

Anti-MSLN cancer vaccines have received increasing attention in EOC, mesothelioma, and pancreatic cancer. CRS-207 vaccine uses attenuated Listeria monocytogenes that are engineered to express human MSLN, which is processed by MHC. Two clinical trials assessing CRS-207 safety and efficacy in EOC (NCT00585845 and NCT02575807) were terminated due to low accrual or lack of clinical activity. Mesothelin continues to be a promising target for vaccine therapy, and preclinical efforts are being made to design and develop other types of vaccines, such as MSLN-specific DNA vaccines combined with immunomodulators [117].

In general, clinical trials investigating MSLN-targeting CAR-T cells in solid tumors showed safety and tolerability. Despite preclinical evidence of anti-tumor activity, phase I studies have demonstrated a limited effect [118]. A preclinical study evaluated the efficacy of two second-generation MSLN-directed CAR-T cells containing MSLN CD28 and 4-1BB in orthotopic mouse models of ovarian cancer. CAR-T cells could control SKOV3 ovarian cancer with prolonged survival and remission in vivo [20]. Recently, a study with three patients with MSLN-positive EOC showed the safety and efficacy of the second-generation anti-MSLN CAR-T cells. In this study, one patient achieved CR, and two patients achieved SD. There were no grades 2–4 adverse events. The phase I clinical trial is being designed [119]. An ongoing clinical trial, including patients with EOC, assesses the safety of a novel cell therapy consisting of autologous genetically engineered T cells expressing an antibody that recognizes the mesothelin called Gavo-cell (NCT03907852).

5.6. PD-1/PD-L1

Programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are transmembrane proteins that regulate immune homeostasis. PD-L1 is overexpressed on tumor cells and many other cells in the tumor microenvironment (TME), such as macrophages, dendritic cells, T cells, and cancer-associated fibroblasts. PD-L1 binds to the PD-1 on tumor-infiltrating lymphocytes (TILs), inhibiting T cells function and leading to an immunosuppressive TME [120]. Blocking the PD-1/PD-L1 interaction with immune checkpoint inhibitors (ICI) is the main strategy to normalize the TME [121]. However, studies assessing these agents have so far been disappointing. A meta-analysis evaluating the efficacy of PD-1/PD-L1 inhibitors in ovarian cancer included 15 trials and demonstrated limited efficacy from single anti-PD-1/PD-L1 therapy with an ORR of 19%. When combined with chemotherapy, the anti-PD-1/PD-L1 therapy showed a better ORR of 36% [122].

The low efficacy of ICI may be explained by the different tumor immunophenotypes based on patterns of intraepithelial T cells infiltrations: 1) ‘hot’ tumor with T cell infiltration in the tumor and stroma; 2) excluded tumor with T cell infiltration restricted to the stroma; 3) non-inflamed or ‘cold’ tumor with no T cells infiltration [123]. Therefore, strategies to modulate the TME may improve the immune response. For example, alterations in tumor endothelial cells also promote an immunosuppressive TME where T cells cannot infiltrate the tumor [124]. In tumors with BRCA deficiency, a STING-dependent innate immune response can be triggered and induce a type I interferon and pro-inflammatory cytokine production. In addition, in clinical models, PARP inhibition can upregulate PD-L1 in a dose-dependent manner, leading to suppressed T cell activation and increased cancer apoptosis [125]. Exploring these mechanisms, several trials combining antiangiogenics, PARP inhibitors, and ICI are ongoing, and preliminary results showed improved efficacy [126].

5.7. Other emerging peptides

Trophoblast antigen 2 (Trop2) is a cell surface transmembrane glycoprotein encoded by the TACSTD2 gene. Trop2 binds to different ligands and promotes signal transduction for tumor cell proliferation, invasion, and metastasis [127]. Trop2 is overexpressed in 82–92% of ovarian cancer with minimal expression in normal tissues [107]. Sacituzumab govitecan (IMMU-132) is an ADC composed of humanized IgG antibody targeting Trop2 conjugated to SN38, the active metabolite of irinotecan, by a cleavable CLA2 linker. Preclinical data demonstrated significant antitumor activity of sacituzumab govitecan in chemotherapy-resistant EOC xenograft models overexpressing Trop2 [128]. This result, combined with the phase I/II IMMU-132-01 basket study (NCT01631552) demonstrating clinical activity in epithelial tumors resistant to chemotherapy [129], supports further studies in Trop2 positive EOC. The arm B of the phase Ib SEASTAR study (NCT03992131) enrolled platinum-resistant EOC to receive the combination of rucaparib plus sacituzumab govitecan. Recent results from a case series of this study described two patients with homologous recombination proficient EOC that had a partial response and stable disease with the combination [130]. Several Trop2-directed therapeutics are emerging as promising agents in Trop2-positive tumors treatment [131].

NY-ESO-1, New York esophageal squamous cell carcinoma 1, is a strong immunogenic CTA overexpressed in more than 40% of EOC patients and is correlated with poor prognosis [132]. Clinical activity of NY-ESO-1-based vaccines has been demonstrated in early clinical trials [133–135]. However, limited objective responses have been obtained with this approach. There are mechanisms where tumors escape the immune surveillance, including antigen expression downregulation and epitope spreading due to prolonged immune pressure by natural tumor antigen immune responses [136]. A phase I study evaluating the NY-ESO-1 peptide vaccine in combination with anti-PD-1 nivolumab is ongoing (NCT02737787). Also, adoptive T-cell therapy directed against NY-ESO-1 epitopes has been engineered and is investigated in multiple clinical trials (NCT02869217, NCT03691376, and NCT02457650).

XPO1 (chromosome region maintenance protein 1) is an intracellular protein responsible for transporting proteins from the nucleus to the cytoplasm to maintain cellular homeostasis. Many of these proteins are tumor suppressors, such as p53, RB1, and p27. Any dysregulation in this transport can lead to oncogenesis [137]. XPO1 is overexpressed in ovarian cancer and is associated with platinum resistance and poor outcomes [138]. Early XPO1 inhibitors achieved limited benefit owing to toxicities, although more selective inhibitors of XPO1 have been developed, with promising results.

Selinexor is a second-generation selective inhibitor of nuclear export (SINE) and is known to cause nuclear retention of tumor suppressor proteins, notably p53. In advanced endometrial cancer, selinexor improved PFS as maintenance therapy, particularly in patients with p53 wild-type [139]. A preclinical study assessed the XPO1 inhibition with Selinexor or KPT-185 in EOC cell lines and mouse xenograft models. In this study, inhibition of XPO1 resulted in apoptosis and suggested overcoming platinum resistance. XPO1 inhibitor – mediated apoptosis occurred through both p53-dependent and p53-independent signaling pathways [138]. Single-agent selinexor was assessed in a phase II trial (NCT02025985) in heavily pretreated recurrent gynecological malignancies [140]. This study enrolled 66 patients with ovarian cancer and showed a disease control rate of 30%, with 8% of patients with PR. The most common treatment-related AEs were thrombocytopenia, fatigue, anemia, and nausea [140]. Preclinical evidence suggested increased responses with a combination of therapy strategies [141]. The synergism between selinexor and RG-7388, an MDM2 inhibitor, reactivates p53, reduces cell viability, and induces cell death in ovarian cancer cell lines through up-regulation of p53 and p21 [142].

6. Potential development issues

Recent advances in proteomics have led to a comprehensive knowledge of potential tumor-targetable proteins and peptides. Their location in the tumor cell determines the accessibility and feasibility of therapeutic strategies [11]. Most of these proteins and peptides are expressed on the tumor cell surface and are classified as TAA, making them excellent targets for immunotherapy strategies. One of the barriers is finding the ideal target with low or minimal expression in normal cells. The presence of normal cells expressing these targets can result in detrimental off-target toxicity. The type of toxicity will depend on the distribution of each target, while the severity varies according to the intensity of expression in normal tissues. The identification and selection of the tumor target are crucial for the development of effective therapies. Heterogeneous intratumoral antigen expression may spare antigen-negative cells. This could be attenuated by ADCs therapies that may have a bystander-killing effect in the surrounding cells [143].

Determination of target positivity on tumor cells using reliable methods is a critical challenge. As demonstrated in the FORWARD I trial targeting the FRα with mirvetuximab soravtansine, the screening score systems may affect the eligibility of patients to receive the targeted therapy [89]. The use of a more robust scoring system incorporating the number of staining cells along with intensity should identify the patient who would benefit the most [144]. However, an abundance of TAA on the surface of tumor cells does not guarantee target activation. Target binding may be impaired by antigen down-regulation, truncation, or epitope mutation [145]. It is unclear which tumor tissue is most appropriate to measure target expression. Analysis of paired archival and fresh biopsies of tumor tissues showed a high concordance between target expression levels [146].

Immunotherapies, such as vaccines and ACT, targeting TAAs is an emerging field in EOC. However, certain characteristics of EOC may contribute to limited immunotherapeutic efficacy. High aneuploidy, high levels of copy number alterations, and immunosuppressive tumor microenvironments are associated with low immunogenicity [5]. Peptide-based vaccines can target either cell surface or intracellular protein, which is an advantage over antibody or CAR-T cell therapies that can only target surface antigens. Moreover, peptide vaccines are highly cost-effective and have more flexibility to target multiple epitopes [53]. However, the vaccine strategy relies on antigen presentation by the MHC to elicit a T cell-mediated immune response. One limitation of this approach is the MHC restriction of epitope and heterogeneity in MHC alleles in the population. Even though peptide-based vaccines are promising, they require optimization of peptide formulations, adjuvants, and administration routes to achieve better results [147].

Adoptive cell therapy in EOC is still in the initial research stage. There is evidence of mechanisms of immune escape by the tumor cells, such as loss of protein surface expression and overexpression of immune checkpoint ligands, which makes the combination of CAR-T cells with checkpoint blockade a rational treatment strategy. In solid tumor cell therapy, a bystander effect needs to be avoided. T cells have also been shown to be activated independently of T cell receptors or cytokines. This process of bystander activation leads to host injury mediated by a higher level of cytotoxicity [59]. Other obstacles to overcome in cell therapy for EOC are cell persistence, intratumoral trafficking, and immunosuppressive TME [58].

7. Conclusion

Despite the high initial responses, EOC is associated with high recurrence and a poor prognosis. Targeted immunotherapy strategies exploring peptides and proteins expressed by tumor cells have the potential to achieve antitumor activity. The differential expression between tumor cells (high levels) and normal tissues (low levels) is striking. Identifying promising candidate antigens with these features, in silico strategies using RNA-sequencing and protein-expression data can help predict the most suitable antigens for targeting. Several ongoing clinical trials in EOC incorporate precision peptide targeting to improve outcomes [148].

8. Expert opinion

Proteomics has allowed for the valuable characterization of different proteins and peptides, which have the potential to serve as novel biomarkers and therapeutic targets. Targeting tumor cell peptides and proteins has come a long way using diverse approaches from vaccination to adoptive T cell therapy. Effectively tailored therapy based on specific molecular aberrations and biomarkers may require EOCs to be sub-classified beyond the histology and HRD status.

Further optimization of biomarkers is warranted to improve their predictive and therapeutic values for clinical decision-making. Selecting populations based on the expression profile may also require reestablishing outcomes in these groups to distinguish between prognostic and predictive impact. Some studies have shown improved outcomes in patients with high-expressing antigen tumors. This underscores the importance of patient stratification based on receptor expression status and having a control population in the design of clinical trials. Existing tools are unable to accurately measure the complete range of tumor antigens. It is reasonable to speculate that a broader repertoire of tumor antigens can activate a more diverse and powerful antitumor immune response. In addition, antigen quality requirements are crucial to achieving a prolonged immune response.

EOC’s heterogeneous and immunosuppressive tumor microenvironments could prevent effective T cell infiltration and cause T cell exhaustion, even if the antigen is highly immunogenic. This is an important challenge for the successful application of adoptive cell therapies. Translational studies help understand the TME behavior to optimize therapies and design combinatorial strategies to overcome this ‘cold’ tumor immune phenotype. Induction of exhausted T cells in ‘hot’ tumors requires strategies to reconfigure these T cells to maintain tumor control. Advances in T cell profiling and longitudinal tracking may allow us to capture and comprehend the dynamic changes in antitumor immunity and translate them into therapeutic options.

Cancer vaccines have been modulated with different targeted tumor antigens, formulations, adjuvants, and delivery vehicles. Despite efforts to develop immunogenic cancer vaccines, the successful clinical translation into efficacious therapies has proven to be challenging. This difficulty arises mainly from highly heterogeneous tumor antigens and low immune responses. Among cancer vaccines, mRNA is a promising platform due to its high potency, rapid development potential, cost-effective manufacturing, and ability to present multiple epitopes simultaneously. These mRNA vaccines are also less HLA-restricted and more likely to stimulate a broader T cell response. It is important to investigate how cancer vaccines influence T cell repertoire by monitoring circulating T cells’ responsiveness toward the targeted antigens. This remains a very promising direction for further research in EOC.

The strategies discussed in this review are applicable to a wide range of solid tumors that express many of these proteins and peptides. Recent advances in technology have led to the development of emerging techniques capable of capturing the complex spatial, temporal, and intralesional heterogeneity present within tumors. Despite these achievements, there are still limitations that need to be addressed, particularly in terms of sensitivity and specificity. These factors continue to pose challenges that must be overcome to fully leverage the potential of these technologies in understanding and effectively targeting tumor heterogeneity. Integrated multi-omics approaches and comprehensive tumor profiling will help identify the ideal tumor antigens. In the future, a new wave of target-directed active treatments will expand our options for immunotherapy and translate into concrete clinical benefit.

Declaration of interest

A M Oza is a PI and participates in steering committees of trials sponsored by AstraZeneca, GSK, Clovis (uncompensated) and is the CEO of Ozmosis Research (uncompensated). He is also an advisor for Morphosys.

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

The authors were supported by the Princess Margaret Cancer Centre, University Health Network, and Princess Margaret Cancer Foundation.