Science Translational Medicine

Engineering CAR T cells to secrete VEGF-neutralizing scFvs enhances antitumor activity against solid tumors

Chimeric antigen receptor (CAR) T cell therapy has shown limited efficacy against solid tumors, which often reside in highly immunosuppressive tumor microenvironments (TMEs). TMEs can be highly abundant in vascular endothelial growth factor A (VEGF), which contributes to immunosuppression and abnormal tumor vasculature. Here, we found that CAR T cells engineered to secrete an anti-VEGF single-chain variable fragment (CAR-αVEGF T cells) achieved superior antitumor efficacy against multiple in vivo models of ovarian cancer and glioma, outperforming conventional CAR T cells with and without combination anti-VEGF antibody therapy. Microscopy, flow cytometry, and transcriptomic analyses revealed that armoring the CAR T cells with anti-VEGF single-chain variable fragments enhanced their activation and mitochondrial fitness and enriched immune-stimulatory signatures among endogenous immune cells in the tumor-bearing brain. Moreover, CAR-αVEGF T cells circumvented multiple detrimental effects associated with on-target CAR T cell therapy, including infiltration of suppressive myeloid cells, exaggerated vasculature abnormalities, and hypoxia. Together, our results provide rationale for the clinical translation of CAR-αVEGF T cells as a safe and potent therapy for solid tumors characterized by elevated VEGF.

Heterogeneous cellular responses to hyperthermia support combined intraperitoneal hyperthermic immunotherapy for ovarian cancer mouse models

The benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) in ovarian cancer remains controversial, hindering the development of rational combination therapies based on hyperthermia (HT). This study reports the preliminary results of the neoadjuvant HIPEC (NHIPEC) trial (ChiCTR2000038173), demonstrating enhanced tumor response in high-grade serous ovarian cancer with NHIPEC. Through single-cell RNA sequencing analysis, we identified both homogeneous and heterogeneous cellular responses to HT within the tumor and microenvironment. Epithelial-mesenchymal transition–activated tumor cells and matrix metallopeptidase 11 (MMP-11) + cancer-associated fibroblasts (CAFs) exhibited greater reductions and higher sensitivity to HT. CUT&Tag and RNA sequencing integration unveiled the differential binding programs and transcriptional regulatory mechanisms of HSF1 under normothermia (NT) and HT in tumor cells and CAFs. Furthermore, HT ameliorated the immunosuppressive tumor microenvironment, and in vivo mouse models confirmed the combined antitumor effects of HT and programmed cell death ligand 1 blockade. These findings provide an innovative strategy for rational combination therapy with HT in ovarian cancer.

Cationic nanogel–based nasal therapeutic HPV vaccine prevents the development of cervical cancer

Therapeutic vaccines against cervical cancer caused by human papillomavirus (HPV) are still an unmet medical need, despite a prophylactic HPV vaccine being available, and the now-licensed systemic vaccines may have a limited effect on the reproductive tract. To specifically inhibit cervical cancer development, the concept of mucosal immunity based on the reproductive-respiratory axis was adopted to develop a nasal HPV therapeutic vaccine. We used a cationic nanogel for the nasal vaccine delivery system and targeted HPV16 E7, an oncoprotein in HPV-driven cervical cancer to demonstrate the feasibility of a nasal therapeutic vaccine. The vaccine was combined with cyclic di–adenosine monophosphate as a cell-mediated immunity-inducing adjuvant. Intranasal immunization with the nanogel vaccine induced E7-specific CD4 + and CD8 + T cells in mouse cervicovaginal tissue. An antitumor effect due to the infiltration of vaccine-induced E7-specific T cells was also observed in an orthotopic tumor model in mice. Furthermore, intranasal immunization of nonhuman primates with the nanogel vaccine using a spray device that is also applicable to humans induced E7-specific T cells in the reproductive tissues. Our findings demonstrated that this nasal therapeutic vaccine effectively controlled cervical cancer and will contribute to preclinical evidence for clinical testing in the near future.

Mitigating T cell DNA damage during PARP inhibitor treatment enhances antitumor efficacy

Poly(ADP-ribose) polymerase inhibitors (PARPis) are a class of agents targeting DNA damage repair that have become standard therapy for epithelial ovarian cancer (EOC) and multiple other solid tumors. In addition to targeting DNA damage repair, PARPis actively modulate antitumor immune responses, with efficacy being partially dependent on T cell activity. Here, we found that patient T cells sustain DNA damage during PARPi treatment, which reduces treatment efficacy. Leveraging paired pre- and posttreatment tumor samples from a clinical trial of patients with EOC treated with neoadjuvant niraparib as monotherapy, we showed that the PARPi caused DNA damage, slowed proliferation, and increased apoptosis in T cells, which we validated both in vitro and in mouse models. A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) knockout screen in primary human T cells identified PARP1 as the principal mediator of PARPi-induced T cell death. T cell–specific deletion of PARP1 or mutating Parp1 at its binding sites in transgenic mice led to reduced T cell DNA damage during PARPi treatment, resulting in improved efficacy of PARPis, alone or in combination with immune checkpoint inhibition. We then engineered PARPi-tolerant CAR T cells using cytosine base editing, which decreased PARPi-induced PARP1 trapping and led to reduced PARPi-induced DNA damage, resulting in superior antitumor efficacy in xenograft models compared with parental CAR T cells. This study highlights the relevance of PARPi-induced DNA damage to T cells and suggests opportunities to improve the efficacy of PARPis as monotherapy or in combination with immunotherapy.

Characterization of ascites- and tumor-infiltrating γδ T cells reveals distinct repertoires and a beneficial role in ovarian cancer

γδ T cells are clonotypically distinct in ascites and tumors of patients with ovarian cancer and their functionality correlates with clinical outcome.

Metabolic adaptation of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD + ), which reduced T cell proliferation and function. Because NAD + metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD + on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD + -mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Dual FAK and EPHA2 targeting by brigatinib tackles PARP inhibitor adaptive survival response in high-grade serous ovarian cancer

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are an important therapy for high-grade serous ovarian cancer (HGSOC). However, PARPi resistance frequently emerges, necessitating previously unrecognized approaches to improve HGSOC responses. Here, we showed that the anaplastic lymphoma kinase (ALK) inhibitor brigatinib enhances PARPi activity in HGSOC cells by disrupting an adaptive survival mechanism orchestrated by Fos-related antigen 1 (FRA1) in response to PARPi. This effect of brigatinib occurred through an ALK-independent pathway, wherein brigatinib induced a dual blockade of focal adhesion kinase (FAK) and EPH receptor A2 (EPHA2) tyrosine kinases, leading to the suppression of protein kinase B (Akt) and extracellular-regulated kinase (ERK) signaling accompanied by disruption of a phosphorylation event crucial for FRA1 protein stability. Moreover, in HGSOC patient-derived xenograft (PDX) models, brigatinib and PARPi combination therapy induced tumor regression and improved overall survival compared with PARPi alone, particularly in models with high FAK and EPHA2. These findings support dual targeting of FAK and EPHA2 as a strategy to achieve effective and durable PARPi responses and identify a promising biomarker-based combinatorial approach using brigatinib and PARPi for HGSOC, particularly the subset characterized by high FAK and EPHA2.

Sequential treatment with PARPi and WEE1i enhances antitumor immune responses in preclinical models of ovarian cancer

The antitumor activity demonstrated by DNA damage response inhibitors (DDRis) can be partially attributed to their capacity to enhance immune responses. However, the toxicity of DDRis to lymphocytes, particularly when a DDRi is combined with other treatments targeting cell cycle checkpoint kinases, indicates a need for the development of different DDRi treatment schedules. Here, we systematically assessed changes to the tumor immune microenvironment (TIME) in response to DDRis across various treatment timelines in ovarian cancer. Using single-cell analysis, we found that the sequential treatment with an inhibitor of poly(ADP-ribose) polymerase (PARPi), followed by an inhibitor of the cell cycle checkpoint kinase WEE1 (WEE1i), resulted in more effective cancer eradication and stronger antitumor immune responses in vivo, compared with mono- and concurrent therapy. Both sequential and concurrent treatment schedules could induce lethal DNA damage and activate the cGAS-STING pathway in cancer cells, but T cell viability was greater under sequential treatment. Proteomic analysis showed that T cells more quickly recovered from DNA damage after DDRi treatment compared with cancer cells. Both immune checkpoint therapy and CAR T cells were more effective when combined with sequential treatment compared with monotherapy treatment in a syngeneic high-grade serous ovarian cancer mouse model and in a treatment-resistant ovarian cancer patient-derived xenograft model. Our study demonstrated that sequential treatment with PARPi and WEE1i spared T cells from severe DNA damage and activated the cGAS-STING pathway in cancer cells, suggesting that antitumor immunity and control of tumor growth can be optimized through changes in treatment schedules.

Genome-wide repeat landscapes in cancer and cell-free DNA

Genetic changes in repetitive sequences are a hallmark of cancer and other diseases, but characterizing these has been challenging using standard sequencing approaches. We developed a de novo kmer finding approach, called ARTEMIS (Analysis of RepeaT EleMents in dISease), to identify repeat elements from whole-genome sequencing. Using this method, we analyzed 1.2 billion kmers in 2837 tissue and plasma samples from 1975 patients, including those with lung, breast, colorectal, ovarian, liver, gastric, head and neck, bladder, cervical, thyroid, or prostate cancer. We identified tumor-specific changes in these patients in 1280 repeat element types from the LINE, SINE, LTR, transposable element, and human satellite families. These included changes to known repeats and 820 elements that were not previously known to be altered in human cancer. Repeat elements were enriched in regions of driver genes, and their representation was altered by structural changes and epigenetic states. Machine learning analyses of genome-wide repeat landscapes and fragmentation profiles in cfDNA detected patients with early-stage lung or liver cancer in cross-validated and externally validated cohorts. In addition, these repeat landscapes could be used to noninvasively identify the tissue of origin of tumors. These analyses reveal widespread changes in repeat landscapes of human cancers and provide an approach for their detection and characterization that could benefit early detection and disease monitoring of patients with cancer.

Genomic instability analysis in DNA from Papanicolaou test provides proof-of-principle early diagnosis of high-grade serous ovarian cancer

Late diagnosis and the lack of screening methods for early detection define high-grade serous ovarian cancer (HGSOC) as the gynecological malignancy with the highest mortality rate. In the work presented here, we investigated a retrospective and multicentric cohort of 250 archival Papanicolaou (Pap) test smears collected during routine gynecological screening. Samples were taken at different time points (from 1 month to 13.5 years before diagnosis) from 113 presymptomatic women who were subsequently diagnosed with HGSOC (pre-HGSOC) and from 77 healthy women. Genome instability was detected through low-pass whole-genome sequencing of DNA derived from Pap test samples in terms of copy number profile abnormality (CPA). CPA values of DNA extracted from Pap test samples from pre-HGSOC women were substantially higher than those in samples from healthy women. Consistently with the longitudinal analysis of clonal pathogenic TP53 mutations, this assay could detect HGSOC presence up to 9 years before diagnosis. This finding confirms the continual shedding of tumor cells from fimbriae toward the endocervical canal, suggesting a new path for the early diagnosis of HGSOC. We integrated the CPA score into the EVA (early ovarian cancer) test, the sensitivity of which was 75% (95% CI, 64.97 to 85.79), the specificity 96% (95% CI, 88.35 to 100.00), and the accuracy 81%. This proof-of-principle study indicates that the early diagnosis of HGSOC is feasible through the analysis of genomic alterations in DNA from endocervical smears.

BLM overexpression as a predictive biomarker for CHK1 inhibitor response in PARP inhibitor–resistant BRCA -mutant ovarian cancer

Poly(ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in breast cancer gene ( BRCA )–mutant high-grade serous ovarian carcinoma (HGSC). However, most patients eventually develop resistance to PARPis, highlighting an unmet need for improved therapeutic strategies. Using high-throughput drug screens, we identified ataxia telangiectasia and rad3-related protein/checkpoint kinase 1 (CHK1) pathway inhibitors as cytotoxic and further validated the activity of the CHK1 inhibitor (CHK1i) prexasertib in PARPi-sensitive and -resistant BRCA -mutant HGSC cells and xenograft mouse models. CHK1i monotherapy induced DNA damage, apoptosis, and tumor size reduction. We then conducted a phase 2 study (NCT02203513) of prexasertib in patients with BRCA -mutant HGSC. The treatment was well tolerated but yielded an objective response rate of 6% (1 of 17; one partial response) in patients with previous PARPi treatment. Exploratory biomarker analyses revealed that replication stress and fork stabilization were associated with clinical benefit to CHK1i. In particular, overexpression of Bloom syndrome RecQ helicase ( BLM ) and cyclin E1 ( CCNE1 ) overexpression or copy number gain/amplification were seen in patients who derived durable benefit from CHK1i. BRCA reversion mutation in previously PARPi-treated BRCA -mutant patients was not associated with resistance to CHK1i. Our findings suggest that replication fork–related genes should be further evaluated as biomarkers for CHK1i sensitivity in patients with BRCA -mutant HGSC.

An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

High-risk human papillomavirus (HPV) DNA testing is widely acknowledged as the most sensitive cervical cancer screening method but has limited availability in resource-limited settings, where the burden of cervical cancer is highest. Recently, HPV DNA tests have been developed for use in resource-limited settings, but they remain too costly for widespread use and require instruments that are often limited to centralized laboratories. To help meet the global need for low-cost cervical cancer screening, we developed a prototype, sample-to-answer, point-of-care test for HPV16 and HPV18 DNA. Our test relies on isothermal DNA amplification and lateral flow detection, two technologies that reduce the need for complex instrumentation. We integrated all test components into a low-cost, manufacturable platform, and performance of the integrated test was evaluated with synthetic samples, provider-collected clinical samples in a high-resource setting in the United States, and self-collected clinical samples in a low-resource setting in Mozambique. We demonstrated a clinically relevant limit of detection of 1000 HPV16 or HPV18 DNA copies per test. The test requires six user steps, yields results in 45 min, and can be performed using a benchtop instrument and minicentrifuge by minimally trained personnel. The projected per-test cost is <$5, and the projected instrumentation cost is <$1000. These results show the feasibility of a sample-to-answer, point-of-care HPV DNA test. With the inclusion of other HPV types, this test has the potential to fill a critical gap for decentralized and globally accessible cervical cancer screening.