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Table S1. Differential expression analysis for CD8 T cells.

Supplementary Data from Antibody-Peptide Epitope Conjugates for Personalized Cancer Therapy

Table S5. List of Genes Used to Generate NK Cell Activation Scores.

Table S2. Differential expression analysis for Treg CD4 T cells.

Table S3. Differential expression analysis for non-Treg CD4 T cells.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC.Significance:This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Table S4. Differential expression analysis for NK cells.

Supplementary Materials and Methods, Figures, and Figure and Table legends

Antibody–peptide epitope conjugates (APEC) are a new class of modified antibody–drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs.Significance:This study develops a high-throughput preclinical platform to identify patient-specific antibody–peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

Objective: Uterine serous cancer (USC) represents about 10% of all uterine cancers, yet it accounts for up to 40% of all uterine cancer deaths. This lethality is due in part to the highly aggressive clinical course of this disease. Recently, galectin 3 (Gal3), a glycoprotein, was shown to contribute to malignant features in other tumors including ovarian cancer. In this study, we test the hypothesis that Gal3 facilitates malignant features in USC and might provide an opportunity for therapeutic intervention. Methods: The TCGA database was used to define the relationship between LGALS3 mRNA expression and prognosis. Gal3 knockout cells were established via CRISPR/Cas9-mediated deletion from USC cell lines, ARK1 and ARK2 (Gal3-KO). Gal3’s role in cell proliferation, cell cycle, and cell death were assessed. Gal3’s influence on apoptotic priming was done by BH3 profiling. Gal3 small molecule inhibitors (SMIs) were used to as an orthogonal approach to genetic knockdown. The effect of Gal3 loss on migration, invasion, and angiogenesis was assessed with transwell, Matrigel-invasion and HUVEC (human umbilical vein endothelial cells) tube-forming assays. The impact of Gal3 loss on cancer stem cells (CSC), phenotypically characterized by CD44, CD117, and ALDH activity, was measured by flow cytometry, and functionally by colony-forming and sphere-forming assays. An in vivo limiting dilution tumorigenic assay was performed with ARK1 Gal3-CTRL and KO cells in immunocompromised mice. Statistical analysis was done and a significance level of p < 0.05 was used. Results: The TCGA database revealed a worse prognosis for patients with USC and high LGALS3 mRNA. Cell Counts revealed Gal3-KO cells proliferated at a slower rate compared to Gal3-CTRL cells, and the difference wasn’t attributed to an increase in cell death. Cell cycle analysis showed an extended G2/M arrest in Gal3-KO cells suggesting Gal3 may influence cell cycle checkpoints. Unlike SKOV3 ovarian cancer cells, loss of Gal3 did not affect apoptotic sensitivity of USC cells as determined by BH3 profiling. Gal3 knockout reduced the number of migrating and invading cells in vitro. Gal3 SMIs (GB1107 and TD139) often differed in their response when compared to the knockout strategy. Conditioned media (CM) from ARK2 Gal3-CTRL promoted tube formation and invasion of HUVECs. This effect was reduced with CM from ARK2 Gal3-KO cells, suggesting Gal3 influences the tumor vascular microenvironment. Gal3 loss markedly reduced CD117+ cells and cells displaying high ALDH activity. Functionally, these findings were supported by a reduction in the number and size of colonies and fewer spheres formed by Gal3-KO cells compared to Gal3-CTRL cells. ARK1 Gal3-KO cells formed fewer and smaller tumors compared to Gal3-CTRL in mice. Conclusion: Gal3 has the potential to promote the more aggressive features of USC and patients would likely benefit from an anti-Gal3 based strategy. Citation Format: Yusuke Matoba, Dominique T. Zarrella, Venkatesh Pooladanda, Eugene Kim, Shaan Kumar, Mengyao Xu, Xingping Qin, Raj Kumar, Artem Kononenko, Irva Veillard, Kristopher Sarosiek, Oladapo Yeku, David R. Spriggs, Bo R. Rueda. Loss of galectin 3 reveals its potential roles in the aggressive pathology of uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2438.

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. Significance: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Antibody–peptide epitope conjugates (APEC) are a new class of modified antibody–drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs. Significance: This study develops a high-throughput preclinical platform to identify patient-specific antibody–peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

In this direct replication of Mueller and Oppenheimer’s (2014) Study 1, participants watched a lecture while taking notes with a laptop ( n = 74) or longhand ( n = 68). After a brief distraction and without the opportunity to study, they took a quiz. As in the original study, laptop participants took notes containing more words spoken verbatim by the lecturer and more words overall than did longhand participants. However, laptop participants did not perform better than longhand participants on the quiz. Exploratory meta-analyses of eight similar studies echoed this pattern. In addition, in both the original study and our replication, higher word count was associated with better quiz performance, and higher verbatim overlap was associated with worse quiz performance, but the latter finding was not robust in our replication. Overall, results do not support the idea that longhand note taking improves immediate learning via better encoding of information.