bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1734: Modulation of immune responses to cancer by bi-specific IL-2/IL-13 superkines
Authors: Merchant Fahar , To Minh
Published: July 2021

Introduction: The IL-2 pathway plays a vital role in stimulating a pro-inflammatory (Th1) response against cancer through expansion and activation of effector CD8 T and NK cells. In contrast, the IL-4/IL-13 pathway stimulates myeloid derived suppressor cells (MDSCs) and M2 skewing of tumor associated macrophage (TAM) to foster an anti-inflammatory (Th2) response that is often exploited by cancers as a means to dampen the effects of the Th1 pathway. Therefore, suppression of MDSC and M2 TAM through inhibition of the IL-4/IL-13 pathway together with stimulation of effector immune cells through activation of the IL-2 pathway have the potential to invigorate a pro-inflammatory response in an otherwise immune suppressive tumor microenvironment (TME). To achieve this goal, we leveraged the versatility of our IL-2 and IL-13 superkine platforms to engineer long-acting bi-specific constructs to co-target surface receptors of these respective pathways.

Experimental Procedure: Studies included binding analyses by BLI/Octet and Biacore/SPR, signaling analyses using IL-2 and IL-4/IL-13 reporter assays, signaling analysis in human PBMC, and in vitro M1/M2 macrophage polarization assay.

Summary of Data: sIL2M-Fc-sIL13M is a bi-specific superkine composed of an IL-2 super-agonist (sIL2M) and IL-13 super-antagonist (sIL13M) linked together by human IgG1 Fc. sIL2M binds CD122 with superior affinity over IL-2 but does not engage CD25, which is expressed on immune-suppressive Tregs. sIL13M has higher affinity than IL-13 for IL13Rα1, which together with IL-4Rα forms a functional receptor complex. In pSTAT5 assay using primary human PBMCs, sIL2M-Fc-sIL13M showed enhanced potency over IL-2 in the activation of CD8 T-cells and NK cells while exhibiting limited activity on Treg. In an IL-4/IL-13 dependent pSTAT6 reporter assay, sIL2M-Fc-sIL13M demonstrated dose-dependent antagonism against stimulatory activity of both IL-4 and IL-13. This antagonistic effect was further validated with inhibition of IL-13 induced M2 polarization of macrophages in vitro.

Conclusion: sIL2M-Fc-sIL13M is a bi-specific superkine capable of concomitantly stimulating a Th1 response through activation of the IL-2 pathway and suppressing a Th2 response through inhibition of the IL-4/IL-13 pathway. Additional bi-specific superkine constructs are currently under testing, including those designed to enable accumulation in TME by engaging the decoy IL-13Rα2 that is overexpressed on a number of different tumor types.

Citation Format: Fahar Merchant, Minh To. Modulation of immune responses to cancer by bi-specific IL-2/IL-13 superkines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1734.
Abstract 174: In-depth transcriptomic comparisons provide novel insights into cancer metastasis
Authors: LIU KE , Glicksberg Benjamin S. , Paithankar Shreya , Chen Bin
Published: July 2021

Comparison of the transcriptome between primary and metastatic cancer is frequently performed to study cancer metastasis; however, distilling plausible biology from the comparison results remains a big challenge. We show that in the differential expression (DE) analysis performed on bulk RNAseq data a large proportion of DE genes are identified due to technical issues instead of biological differences between primary and metastatic cancer cells. Next, we develop a computational pipeline DEBoost to minimize such artifacts and apply it to five cancer types. We perform comprehensive analysis on the retained tumor-intrinsic DE genes to explore the biological mechanisms underlying the metastasis of different cancer types. Many of the DE genes are associated with the progression of primary cancer, suggesting the differential expression may have already been established before metastasis. Clustering analysis on DE genes identifies CNVs that account for the transcriptomic change and we further confirm 19p13.12 amplification drives the metastasis of primary Basal-like breast cancer via activating NOTCH signaling pathway. We also find that cell cycling is more active in metastatic prostate cancer than in primary cancer, suggesting the difference of responses to treatment like cell cycling inhibitors. Finally, we show that some metastatic cancer cells tend to express genes whose expression is normally observed at the metastasis site and we experimentally validate this phenomenon in colorectal cancer liver metastasis. Our pan-cancer liver metastasis study provides novel biological insights into cancer metastasis and meanwhile demonstrates the feasibility of characterizing tumor-intrinsic DE genes in cancer metastasis using our computational pipeline.

Citation Format: KE LIU, Benjamin S. Glicksberg, Shreya Paithankar, Bin Chen. In-depth transcriptomic comparisons provide novel insights into cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 174.
Abstract 176: Detecting neoepitopes from tumor RNA sequencing datasets
Authors: Thompson Drew , Vaske Olena M. , Rao Arjun , Beale Holly C.
Published: July 2021

Epitopes are peptides that present on the surface of the cell and can be recognized by immune cells to initiate the immune response. Identification of neoepitopes – tumor-specific, MHC-bound epitopes recognized specifically by T-cells – is valuable for predicting response to immunotherapies, including checkpoint blockade therapies. Tumors with more neoepitopes tend to be more responsive to immune checkpoint therapies compared to tumors with fewer neoepitopes. ProTECT is a previously published computational method that uses Illumina whole genome and transcriptome sequencing data from tumor and matched normal tissues to identify neoepitopes. Tumor and normal whole genome sequencing data are used to infer a patient's HLA haplotypes, as well as annotate variants as either somatic or germline. While whole genome sequencing is comprehensive, it is quite costly and not available for many samples. Here we adapt ProTECT to use only tumor RNA sequencing data and HLA haplotype information available to the clinician to identify neoepitopes in a tumor sample. Prior to running ProTECT, we use the computational tools Opossum and Platypus for variant calling instead of Radia (which is designed for variant calling using both RNA and DNA sequencing data as input). To determine which variants are somatic and therefore could represent tumor neoepitopes, variants found in RNA are compared to a panel of normals, for example the Genome Aggregation Database (gnomAD; containing variants from 125,748 exome sequences and 15,708 whole-genome sequences). With the resulting somatic variants and the HLA type, ProTECT proceeds as usual, with translation of variants into proteins, MHC:Peptide binding predictions and neoepitope ranking. We find that high quality neoepitopes are identifiable using an RNA-only approach, when genomic data is absent. Future work will validate the sensitivity of our method by benchmarking it against the original ProTECT predictions in the TCGA Prostate Adenocarcinoma cohort.

Citation Format: Drew Thompson, Olena M. Vaske, Arjun Rao, Holly C. Beale. Detecting neoepitopes from tumor RNA sequencing datasets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 176.
Abstract 1760: Role of Batf-dependant Th17 immune response in PTEN-null mouse model
Authors: Zhang Qiuyang , Liu Sen , Zhang Bing
Published: July 2021

Basic leucine zipper transcription factor ATF-like (BATF) belongs to the activator protein 1 (AP-1) family of transcription factors and has been shown to be predominantly expressed in cells of hematopoietic origin, especially in T cells. Mice deficient for BATF were first described to lack T helper cell type 17 (Th17). Th17 cells are a distinct lineage of CD4+ T helper cells that provide protective immunity to infections but are also involved in chronic inflammation and autoimmunity. However, the role of Th17 response in prostate cancer remains controversial. In this study, we took a genetic approach to explore the role of BATF-dependent Th17 cells in prostate cancers by interbreeding BATF-deficient (Batf-/-) mice with mice that are conditionally mutant for PTEN, an established preclinical model for prostate cancer. Batf+/+;Pten-/- and Batf+/-;Pten-/- mice presented similar prostate phenotypes thus were put into one group (named Batf+), which were compared to the Batf-/-;Pten-/- group (named Batf-). We found that Batf- prostates were clearly smaller than Batf+ prostates at 30 weeks. The genitourinary bloc weight was significantly heavier in Batf+ mice than in Batf- mice at 12 and 30 weeks. Batf+ mice developed significantly higher percent of invasive adenocarcinomas in different prostate lobes at 9, 12, and 30 weeks, respectively compared to Batf- mice. Tumors in Batf- mice exhibited lower rates of cellular proliferation and higher apoptosis, as well as reduced inflammatory cell infiltration and angiogenesis in the prostatic stroma. Moreover, Batf- mice exhibited significantly reduced mRNA and protein levels of Th17 cytokines, AP-1 family members and IL-23R, and reduced NF-kB pathway activity in both spleen and prostate tissues. CD4+ T cells from Batf- mice cultured under Th17 conditions for 4 days, expressed reduced AP-1 family-regulated genes compared to Batf+ mice. Prostate cancer cells (LNCaP) with overexpression of BATF in vitro exhibited increased IL-23R expression. Prostate tissues from Batf- mice cultured under IL-6 or Th17 conditions for 4 days had reduced IL-23R expression compared to that from Batf+ mice. Taken together, our results suggest that Batf promotes the initiation and progression of prostate adenocarcinoma via Th17-mediated inflammatory immune responses and that the Batf-IL-23-IL-17 axis is a critical pathway that is needed for prostate adenocarcinoma development and progression.

This work was supported by the NIH-National Institute of General Medical Sciences COBRE on Aging and Regenerative Medicine (P20GM103629, PD: S. Michal Jazwinski; PI: Q.Z.) and the Carol Lavin Bernick Faculty Grant from Tulane University (PI: Q.Z.).

Citation Format: Qiuyang Zhang, Sen Liu, Bing Zhang. Role of Batf-dependant Th17 immune response in PTEN-null mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1760.
Abstract 1770: Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice
Authors: Yamaguchi Kosuke , Miyake Naomi , Teruya Yasuhiko , Yamane Kohei , Kinoshita Naoki , Sakamoto Tomohiro , Kodani Masahiro , Chikumi Hiroki , Yamasaki Akira
Published: July 2021

ULBP2 is one of the ligands for NKG2D (NKG2DLs). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Previously, we reported high concentrations of serum-soluble ULBP2 (sULBP2) were correlated with poor prognosis in NSCLC patients. As a possible mechanism, we demonstrated that sULBP2 directly suppressed the cytolytic activity of peripheral blood mononuclear cells (PBMCs). However, little is known about the in vivo function of human NKG2DLs on tumor cells. The present study investigates the function of human NKG2DLs in the syngeneic C57BL/6 mice. We generated ULBP2 stably expressing B16 melanoma cell lines (ULBP2-B16). As previously reported, 4-hour 51Cr releasing assay showed NK cell cytotoxicity against the tumor cells was enhanced by ectopic expression of ULBP2 on the tumor cells. IFN-gamma production in NK cells was decreased by non-contact co-culture with ULBP2-B16. To investigate the in vivo function of ULBP2, C57BL/6 mice were inoculated s.c. with mock-B16 cells or ULBP2-B16 cells. ULBP2-B16 tumors grew rapidly compared to mock-B16 tumors. NKG2D blockade by anti-NKG2D antibody injected i.p. suppressed tumor growth of ULBP2-B16 tumors. Spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. NKG2D was expressed on splenic NK cells, while it was rarely expressed on splenic CD8+ T cells. NKG2D was downregulated on NK cells in TILs (NK-TILs) from ULBP2-B16. NKG2D was highly expressed on CD8+ T cells in TILs (CD8+-TILs) from mock-B16 tumors, while its expression was suppressed on these from ULBP2-B16 tumors. Degranulation analysis revealed that ULBP2-B16 reduced activity of NK-TILs and CD8+-TILs compared to mock-B16. In conclusion, ectopic expression of ULBP2 on tumor cells promotes tumor progression via suppression of the activity of NK cells and CD8+ T cells. These findings suggest that neutralization of NKG2DLs may be a promising approach for the treatment of NKG2DLs-expressing tumors.

Citation Format: Kosuke Yamaguchi, Naomi Miyake, Yasuhiko Teruya, Kohei Yamane, Naoki Kinoshita, Tomohiro Sakamoto, Masahiro Kodani, Hiroki Chikumi, Akira Yamasaki. Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1770.
Abstract 1771: SIGLEC15 humanized knock-in mice are a powerful tool to evaluate new therapies for cancer and osteoporosis
Authors: Li Huilin , Zhao Lei , Tian Maopeng , Chromikova Veronika , Yu Zhaoxue (Luke)
Published: July 2021

SIGLEC15 (Sialic acid-binding immunoglobulin-like lectin 15) belongs to the Siglec family of sialic acid-binding, immunoglobulin-like proteins. SIGLEC15 is an effective immunosuppressive molecule which is highly expressed on M2 macrophages but can be upregulated in many human cancers. The SIGLEC15 signaling pathway is non-redundant to the PD-L1 pathway in cancer, and the surface expression of SIGLEC15 is generally non-overlapping with PD-L1 expression on tumors. SIGLEC15 engages a putative responder protein expressed on the surface of effector cells, such as CD8 T cells, inducing suppression of anti-tumor immune responses. Blocking of SIGLEC15 amplifies anti-tumor immunity in the tumor microenvironment and inhibits tumor growth. This opens a road for anti-SIGLEC15 antibody therapeutics which may provide a solution for patients with SIGLEC15-positive and PD-L1-negative tumors, a patient population less likely to respond to a PD-1/PD-L1 directed therapy. Biocytogen has generated a humanized SIGLEC15 mouse model to address the need for in vitro functional validation and in vivo efficacy evaluation of SIGLEC15-targeting strategies. In these mice, genomic DNA spanning exons 2~4 of mouse Siglec15 gene encoding the extracellular domain, was replaced by its human counterpart. Following the humanization procedure, human SIGLEC15 could be detected on bone marrow derived macrophages (BMDM). Basal leukocyte subpopulations, including T/B/NK cells, DC, granulocytes, and monocytes/macrophages, were similar between humanized and wild-type mice. In addition, Biocytogen has generated a humanized MC38 cell line, in which the mouse Siglec15 gene was replaced with the full-length human coding sequence. The successful humanization of M38 was confirmed by RT-PCR and flow cytometry. Using the hMC38 tumor model, we showed robust efficacy of the anti-SIGLEC15 antibodies in inhibiting tumor growth in vivo. Combined SIGLEC15 and PD-1/PD-L1 blockade showed greater tumor inhibition than monotherapies. In summary, SIGLEC15 humanized mice can be employed for in vivo efficacy evaluation of anti-SIGLEC15 antibodies in immune normalization approaches for cancer treatment. Besides the tumor models (B16F10 and other tumors underway), we are also actively exploring the feasibility of the SIGLEC15 humanized mice as a pre-clinical model for osteoporosis in a light of recent discovery of SIGLEC15's role in osteoclast differentiation.

Citation Format: Huilin Li, Lei Zhao, Maopeng Tian, Veronika Chromikova, Zhaoxue (Luke) Yu. SIGLEC15 humanized knock-in mice are a powerful tool to evaluate new therapies for cancer and osteoporosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1771.
Abstract 178: Desmosome mutations in melanoma promote cellular proliferation and disease progression
Authors: Baron Maayan , Ideker Trey
Published: July 2021

Desmosomes are transmembrane protein complexes that contribute to cell-cell adhesion in the epithelia and other tissues under mechanical stress. Aberrant desmosome expression is often associated with developmental diseases leading to impaired tissue integrity. Recently, similar findings have been reported in cancer; Mutations in desmosomes genes have been observed in various cancer types including skin cancer, head and neck and lung cancer, however mostly epigenetic alterations have been used to associate desmosomes as suppressors of tumor metastasis. Here, we report that desmosomes are frequently mutated in seven cancer types. In melanoma, we find that over 70% of tumors have non-synonymous mutations in desmosomes, and that the desmosome mutational burden is associated with a strong decrease in mRNA expression levels in primary tumor samples (R = -0.23). Differential gene expression analysis and functional characterizations between mutant and wild-type tumors implicates the mutated cells in promoting cell proliferation at early stages of tumorigenesis. These results emerge uniquely from a systems-level analysis integrating multiple proteins in complexes and multiple cell types in heterogeneous tumors.

Citation Format: Maayan Baron, Trey Ideker. Desmosome mutations in melanoma promote cellular proliferation and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 178.
Abstract 1789: Triggering anti-tumor immunity by overexpressing of MAVS in combination with PD-L1 ICB in immune insensitive colorectal cancers
Authors: Hwang Bin-Jin
Published: July 2021

While the use of immune checkpoint blockade (ICB) has gained significant traction as a viable therapy for cancers, many more cancers remain refractory to these immunotherapies due to their highly immunosuppressive tumor microenvironment (TME). From both the earlier published report and our analysis of clinical cohorts of colorectal cancer, we found that the expression of MAVS (Mitochondrial Antiviral Signaling Protein) was significantly reduced. Because MAVS are known to play a key role in the initiation of anti-viral innate immunity, we hypothesized that MAVS may be critical in anti-tumor immune response and tumor cells are under evolutionary pressure to reduce MAVS expression. As such, the stimulation of the MAVS pathway in tumor cells may be a necessary precursor to trigger anti-tumor immune response and reverse local immunosuppressive TME. To circumvent issues associated with intracellular double stranded RNA ligand stimulation, we exploited the ability of MAVS to constitutively polymerize and elicit broad downstream interferon signaling through an overexpression strategy. This approach provided a novel genetic mean to elicit ligand free innate immune signaling in the TME, which we found to elicit robust stimulation of multiple interferon genes. Overexpression of MAVS in colorectal cell lines stimulated significant anti-tumors responses in vivo, which we found to be primarily mediated through the induction of tumor antigen-specific adaptive immunity. Utilizing viral vectors overexpressing MAVS, we demonstrate significant induction of interferon responses among different types of tumor cells and different types of cells in the tumor microenvironment. Intra-lesional injection of tumors using these vectors elicited profound anti-tumor responses against primary tumors, as well as, an abscopal effect against syngeneic implanted tumors. Overexpression of MAVS also stimulated the expression of PD-L1 as an immunosuppressive feedback mechanism due to stimulation of the MAVS pathway. As such, we tested the combination of MAVS overexpression and PD-L1 ICB in colorectal cancers and found that this combination allowed for significantly improved anti-tumor immunity. In conclusion, our findings suggest that a combination of MAVS cancer gene therapy and ICB may offer an alternative approach to activate immunity in "immunologically cold" tumors.

Citation Format: Bin-Jin Hwang. Triggering anti-tumor immunity by overexpressing of MAVS in combination with PD-L1 ICB in immune insensitive colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1789.
Abstract 1820: Uncovering mechanisms of immune evasion in a novel immunogenic model of KRAS-mutant lung cancer
Authors: Boumelha Jesse , de Carné Sophie , Mugarza Edurne , Romero-Clavijo Pablo , Molina-Arcas Miriam , Downward Julian
Published: July 2021

Oncogenic KRAS mutations drive tumourigenesis in 30% of non-small cell lung cancer (NSCLC). Despite much effort, targeted therapies that aim to directly inhibit signalling pathways downstream of KRAS have limited clinical benefits for NSCLC patients, but the recent approval of PD-1/PD-L1 antibodies has led to striking durable responses. However, only a fraction of patients respond and therefore a deeper understanding of the mechanisms that drive immune evasion are required in order to broaden the clinical efficacy of immunotherapy. Increasing evidence suggests that oncogenic signalling pathways greatly influence the tumour immune landscape to impair anti-tumour immune responses. We therefore aim to understand the mechanisms by which KRAS signalling mediates immune evasion in lung cancer. Current mouse models of KRAS-mutant lung cancer are poorly immunogenic, limiting investigations into tumour-immune interactions. To overcome this, we generated a novel transplantable KRAS-mutant lung cancer model, KPAR1.3, which triggers spontaneous anti-tumour immune responses and is sensitive to immune checkpoint blockade. To identify mechanisms of immune evasion we carried out an in vivo pooled CRISPR-Cas9 screen targeting 240 KRAS-regulated genes using this novel immunogenic model. This identified a number of genes that increased sensitivity or caused resistance to anti-tumour immune responses. As an alternative approach we utilised the recently developed class of mutant-specific KRAS-G12C inhibitors to assess the impact of inhibiting KRAS signalling on anti-tumour immune responses in KPAR1.3 tumours. KRAS-inhibition stimulated adaptive immunity in vivo, which contributed to the response of KPAR1.3 tumours in immune-competent mice. Together these data suggest that targeting KRAS, or KRAS-driven mechanisms of immune evasion, could broaden the clinical efficacy of immunotherapy in KRAS-mutant NSCLC.

Citation Format: Jesse Boumelha, Sophie de Carné, Edurne Mugarza, Pablo Romero-Clavijo, Miriam Molina-Arcas, Julian Downward. Uncovering mechanisms of immune evasion in a novel immunogenic model of KRAS-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1820.
Abstract 1839: Nanobodies against EGF enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer cell models
Authors: Guardiola Salvador , Sánchez-Navarro Macarena , Giralt Ernest , Rosell Rafael , Codony-Servat Jordi
Published: July 2021

Background: The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGFR mutations. However, acquired resistance appears invariably due to several mechanisms. The strategy of using EGF-targeted nanobodies (Nbs) to block the initial step of the EGFR pathway constitutes a new research area. Nbs also offer several advantages compared to traditional mAbs, such as their reduced size, higher stability and tissue penetration, low production costs and lack of Fc effector function, which provide key advantages for targeting soluble tumoral growth factors. In this study we investigated the efficacy of anti-EGF Nbs to reduce osimertinib-resistance.

Experimental procedures: Cell viability and colony formation assays were performed in PC9 and PC9-osimertinib-resistant NSCLC cell lines tested with anti-EGF Nbs alone or in combination with osimertinib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study protein expression and activation.

Summary of data: Two anti-EGF Nbs, generated in our laboratory, inhibited cell viability and colony formation in PC9 and PC9-derived osimertinib-resistant cell lines. The combination of these Nbs with osimertinib improved the antitumoral efficacy of this EGFR-TKI in cell viability and colony formation experiments. The combination of anti-EGF Nbs with osimertinib clearly decreases the activation of proteins involved in the EGFR pathway, such as, EGFR, Akt and Erk 1/2. In addition, it increases cellular apoptosis, and decreases the expression of Hes1, a marker of cancer stem cells involved in metastasis and osimertinib resistance mechanisms.

Conclusions: We conclude that the combination of anti-EGF nanobodies and osimertinib exhibits a good efficacy in inhibiting cell viability and colony formation, while, inhibiting expression and activation of proteins involved in osimertinib resistance.

Citation Format: Salvador Guardiola, Macarena Sánchez-Navarro, Ernest Giralt, Rafael Rosell, Jordi Codony-Servat. Nanobodies against EGF enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1839.
Abstract 1877: IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models
Authors: Deken Marcel A. , Carruthers Alan M. , Niewola-Staszkowska Karolina , Gernon Gráinne , Sandig Hilary , Shah Pritom , Lahn Michael M. , Johnson Zoë
Published: July 2021

CD73, an ecto-5'-nucleotidase, is frequently overexpressed in the tumor microenvironment and can be found on tumor, stromal cells and immune cells. Its function is to catalyze the conversion of adenosine monophosphate (AMP) to adenosine and phosphate, both at the cell surface and as a soluble factor. CD73 has gained interest as a therapeutic target in cancer due to the immunosuppressive role of adenosine in the tumor microenvironment. IOA-237 is a fully human, phage derived monoclonal antibody fully cross-reactive to mouse and human and with high potency against soluble and cell surface CD73. This novel antibody demonstrates in vitro and in vivo characteristics that suggest a best-in-class potential. We demonstrate that IOA-237 is able to significantly reduce tumor outgrowth in multiple mouse models of solid tumors. In the low CD73 expressing CT26 mouse colorectal cancer model, monotherapeutic application of IOA-237 showed tumor outgrowth reduction compared with isotype control. Best-in-class potential was confirmed with head-to-head comparison against a clinical stage anti-CD73 mAb, MEDI9447. Furthermore, inhibition of CD73 by IOA-237 was combined with inhibitors of novel immuno-oncology targets to study complementary modes of action in mouse models of solid tumors. These data indicate the potential of IOA-237 in monotherapeutic and combination therapeutic approaches and warrants further development. Additional in vitro and in vivo efficacy studies are ongoing and non-clinical safety studies are being planned.

Citation Format: Marcel A. Deken, Alan M. Carruthers, Karolina Niewola-Staszkowska, Gráinne Gernon, Hilary Sandig, Pritom Shah, Michael M. Lahn, Zoë Johnson. IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1877.
Abstract 1886: Anti -GPRC5D/CD3 T cell-redirecting bispecific antibody with potent in vitro and in vivo antitumor efficacy against multiple myeloma
Authors: Kodama Tatsushi , Kochi Yu , Nakai Waka , Mizuno Hideaki , Baba Takeshi , Habu Kiyoshi , Sawada Noriaki , Tsunoda Hiroyuki , Shima Takahiro , Miyawaki Kohta , Kikushige Yoshikane , Mori Yasuo , Miyamoto Toshihiro , Maeda Takahiro , Akashi Koichi
Published: July 2021

Background: Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), was previously identified as expressed by mRNA in patients with multiple myeloma with only low expression detected in normal tissues, but confirmation of protein expression remained elusive. In this study, we determined the cell surface expression of GPRC5D on malignant and normal hematological cells. In addition, we evaluated the antitumor activity and mechanism of GPRC5D TRABs in in vitro and in vivo mouse models.

Method & Results: We established specific monoclonal antibodies against human GPRC5D and identified its expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, did not find it at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. To investigate whether GPRC5D could be therapeutic target, we generated IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRABs). GPRC5D TRABs induced T cell activation and the killing of a wide variety of GPRC5D expressing tumor cells in vitro. In mouse models with reconstituted human immune cells, GPRC5D TRABs showed strong antitumor efficacy against GPRC5D-positive tumors through the activation of T cells.

Conclusion: These findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

Citation Format: Tatsushi Kodama, Yu Kochi, Waka Nakai, Hideaki Mizuno, Takeshi Baba, Kiyoshi Habu, Noriaki Sawada, Hiroyuki Tsunoda, Takahiro Shima, Kohta Miyawaki, Yoshikane Kikushige, Yasuo Mori, Toshihiro Miyamoto, Takahiro Maeda, Koichi Akashi. Anti -GPRC5D/CD3 T cell-redirecting bispecific antibody with potent in vitro and in vivo antitumor efficacy against multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1886.
Abstract 1896: The genomic features of Chinese cancer patients harboring HLA LOH
Authors: Zhao Jian , Hu Jie , Zhang Xiuqin , Gao Xuan , Tong Fan , Fu Xiaorui , Yi Yuting , Guan Yanfang , Xia Xuefeng , Huang Jian'an
Published: July 2021

HLA LOH is an immune escape mechanism subject to microenvironmental selection pressures in tumor evolution. In previous study, the prevalence of HLA LOH in Chinese cancer patients has been characterized. However, the genomic features of patients harboring HLA LOH remains unknown.

Methods

The sequencing data of 1021 cancer genes from 1504 advanced patients across 11 diverse cancer types was analyzed to investigate the internal relationship between HLA LOH and genomic alteration. Exon 2, exon 3, and bilateral introns of HLA-A/B/C genes were well covered in the 1021-gene panel. HLA LOH was analyzed using the LOHHLA algorithm.

Results

Consistent with previous study, we found that the occurrence of HLA LOH is related to genomic instability. In colorectal cancer, the HLA LOH ratio of MSS samples was significantly higher than that of MSI-H samples. Among the remaining 10 cancer types, the trend was consistent (Table 1). These results revealed that HLA LOH is a potential immune escape mechanism for MSS patients. In NSCLC and BRCA, HLA LOH is associated with an elevated TMB (p<0.001, p<0,05). The alteration frequency of TP53 and LRP1B in HLA LOH group was significantly higher than that in HLA stable group (p<0.001, p=0.005, respectively). The occurrence of HLA LOH is also related to alterations in several important signaling pathways. The alteration frequencies of RTK/RAS pathway, p53 pathway, Notch pathway, Hippo pathway and Nrf2 pathway in the HLA LOH group were significantly higher than that in HLA stable group. These results are indicating that HLA LOH may mediate tumorigenesis by interfering with normal signaling transduction.

Conclusions and Discussion

Statistical results revealed that the occurrence of HLA LOH was related to genomic instability, including high TMB level and alterations in oncogenic signaling pathways, which may facilitate tumor formation. Overall, these findings may provide useful information for clinical practice and immunotherapy.

The correlation between MSI stage and HLA LOHCancer TypesMSIHLA LOHHLA stablePercent with HLA LOHP valueColorectal cancerMSS617146.2%0.025MSI-H31416.7%Remaining 10 cancer typesMSS48549949.3%0.031MSI-H51525.0%

Citation Format: Jian Zhao, Jie Hu, Xiuqin Zhang, Xuan Gao, Fan Tong, Xiaorui Fu, Yuting Yi, Yanfang Guan, Xuefeng Xia, Jian'an Huang. The genomic features of Chinese cancer patients harboring HLA LOH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1896.
Abstract 1909: Pan-TGF-β inhibition with PD-1 blockade as a combination treatment strategy to augment anti-tumor immune response in hepatocellular carcinoma
Authors: Malkova Natalia , Theilhaber Joachim , Levit Mikhail , Tolstykh Tatiana , Hebert Andrew , Atchison Kevin , Maisonet Adalis , Sinicropi-Yao Sara Sara , Protopopov Alexei , Pollard Jack , Sidhu Sukhvinder , Lin Tun Tun , Pao Lily , Wiederschain Dmitri , Sharma Sharad K.
Published: July 2021

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for more than 700,000 deaths per year. Although transforming growth factor beta (TGF-β) can function as a tumor-suppressor in premalignant cells, patient data suggest that it plays a role in the progression of HCC. Recently, immunotherapy targeting PD-1 has been approved for HCC, however, success is limited to only ~17% objective response rate in the treated HCC patients. Analysis of genomic data from two HCC patient cohorts revealed that TGF-β pathway is highly activated in patients with advanced HCC. Collectively, these findings indicate that targeting TGF-β in combination with PD-1 blockade may be a compelling therapeutic strategy. Therefore, using two syngeneic mouse models of HCC (Hepa1-6 and H22), we examined the effect of a pan-TGF-β neutralizing antibody (1D11) in combination with PD-1 blockade. Results from our study show that co-inhibiting TGF-β with PD-1 resulted in significant tumor growth inhibition in both HCC models. Flow cytometry analysis of the tumor revealed the combination treatment group exhibited a reduction in potentially suppressive tumor-associated-macrophages relative to either single agent groups. In addition, we show that combination therapy was associated with increase in the intra-tumoral levels of proinflammatory cytokines - IFNγ, IL-1β, TNF-α as well as enhanced expression of anti-tumor immune related genes. Based on these data, clinical testing of SAR439459 (TGF-β antibody) and Cemiplimab (anti-PD1) combination has recently been initiated in HCC patients (NCT03192345).

Citation Format: Natalia Malkova, Joachim Theilhaber, Mikhail Levit, Tatiana Tolstykh, Andrew Hebert, Kevin Atchison, Adalis Maisonet, Sara Sara Sinicropi-Yao, Alexei Protopopov, Jack Pollard, Sukhvinder Sidhu, Tun Tun Lin, Lily Pao, Dmitri Wiederschain, Sharad K. Sharma. Pan-TGF-β inhibition with PD-1 blockade as a combination treatment strategy to augment anti-tumor immune response in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1909.
Abstract 1915: Novel synthetic DNA vaccines in prostate cancer immunotherapy
Authors: Bordoloi Devivasha , Xiao Peng , Choi Hyeree , Ho Michelle , Perales-Puchalt Alfredo , Khoshnejad Makan , Kim J.Joseph , Humeau Laurent , Srinivasan Alagarsamy , Weiner David B. , Muthumani Kar
Published: July 2021

Prostate cancer (PCa) represents the most predominant cancer type among males with an estimated 191,930 cases diagnosed in 2020 with 33,330 deaths globally. Notably, different treatment modalities such as surgery, chemo, radiation and hormone therapy exhibited improved outcomes in early stage PCa patients; however, there exist high recurrence rate in the treated patients and leads to castration resistant prostate cancer, the treatment choices for which are highly restricted. This scenario has pressed us to consider synthetic DNA vaccine-based immunotherapy approach, with ability to induce antigen-specific tumor-killing immune cells. Considering the high heterogeneity of prostate cancer cells, we hypothesized that synthetic DNA vaccines targeting multiple prostate specific antigens would elicit improved immune responses in those who are at high risk as well as advanced stages of prostate cancer. Therefore, we generated synthetic enhanced DNA immunogens as DNA constructs (SEV) targeting STEAP1, PAP, PARM1, PSCA, PCTA and PSP94 through utilization of bioinformatics approaches. The SEVs for prostate cancer antigens elicited potent cellular immunity as well as polyfunctionality of antigen-specific T cells in mice. Further, they were capable of generating robust humoral immune responses to each antigen. The anti-tumor activities of the SEVs were studied in prostate tumor which showed that the induced immune responses delayed tumor progression and enhanced infiltration of anti-tumor CD8+ T cells in the tumor microenvironment in a mouse model challenge system. These data highlight further study of these novel immunogens as synthetic DNA immunogens for immunotherapy of PCa, and perhaps other human cancers.

Citation Format: Devivasha Bordoloi, Peng Xiao, Hyeree Choi, Michelle Ho, Alfredo Perales-Puchalt, Makan Khoshnejad, J.Joseph Kim, Laurent Humeau, Alagarsamy Srinivasan, David B. Weiner, Kar Muthumani. Novel synthetic DNA vaccines in prostate cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1915.
Abstract 1922: Protective tumor neo-epitopes are processed from conformationally stable neo-antigen segments
Authors: Landry Samuel J.
Published: July 2021

Immunogenic tumor neo-epitopes are the basis of successful checkpoint immunotherapy for cancer, and they potentially offer a target for personalized cancer vaccines. However, the frequency of immunologically protective neo-epitopes appears to be very low in comparison to the frequency of tumor mutations. Vaccine designers typically select neo-epitopes by demonstrable gene expression and elevated MHC class I binding, anticipating that corresponding CD8+ T cells will be protective; but CD4+ T-cells are more often correlated with therapeutic effects. CD4+ T cells are most well known for helping B cells make antibodies, and B cells also have recently been implicated in protection from tumors. Antigen-presenting cells, including dendritic cells, B cells, and macrophages present epitope peptides in complexes with class II MHC molecules (MHCII) to CD4+ T cells, and CD4+ T cells signal responding cells to eliminate tumor cells. Studies on immunodominant CD4+ T-cell epitopes from foreign antigens suggest that immunogenicity depends on antigen conformational stability. Likewise, the immunogenic CD4+ neo-epitopes of tumors could be associated with mutations affecting residues in conformationally stable protein segments. In order to test this hypothesis, we surveyed existing tumor neo-epitope data to analyze the conformational context of neo-epitopes that are associated with protection from tumors. First, protective neo-epitopes are three-fold more likely to occur in a crystal structure (67%) than other potential neo-epitopes (22%). Second, the protective neo-epitopes are enriched within or adjacent to conformationally stable segments of the neo-antigens, as reported by the Antigen Processing Likelihood algorithm. Third, the distribution of mutations between functionally similar hotspots encoding R248 and R273 in the gene for the p53 tumor suppressor is consistent with increased immunologic selection in tumors subjected to increased immune response. These results suggest that neo-epitope evaluation for cancer prognosis and vaccines should include the analysis of conformational stability in the neo-antigen.

Citation Format: Samuel J. Landry. Protective tumor neo-epitopes are processed from conformationally stable neo-antigen segments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1922.
Abstract 1928: Erdafitinib in combination with BCLXL/BCL2 inhibitor induce apoptosis in urothelial carcinoma cells without FGFR mutation
Authors: ohtsu Akira , Arai Seiji , Sawada Tatsuhiro , Suzuki Kazuhiro
Published: July 2021

Although Erdafitinib has recently emerged as a treatment for metastatic urothelial carcinoma, it has been shown to be effective against FGFR mutations, but the target patient population is small, and a new treatment for urothelial carcinoma without FGFR mutations is needed. We found that Erdafitinib degrades MCL1 in urothelial carcinoma cells. Furthermore, we found that in combination with Navitoclax, a BCLXL/BCL2 inhibitor, it induced potent apoptosis in urothelial carcinoma cell lines without FGFR mutation. This combination therapy may represent a new treatment for urothelial carcinoma without FGFR mutations.

Citation Format: Akira ohtsu, Seiji Arai, Tatsuhiro Sawada, Kazuhiro Suzuki. Erdafitinib in combination with BCLXL/BCL2 inhibitor induce apoptosis in urothelial carcinoma cells without FGFR mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1928.
Abstract 1931: Evaluation of cholecystokinin receptor expression and function in non-small cell lung cancer
Authors: Miller Alexander C. , Subry Shelby J. , Assar Manijeh M. , Pearman Krista , Norris Jeffrey William
Published: July 2021

Introduction: Lung cancer is the leading cause of cancer-related death in the United States, with nearly 85% of cases histologically classified as non-small cell lung cancer (NSCLC). A recent epidemiological study provides evidence of an increased risk ratio for NSCLC in association with adherence to a Western dietary pattern, which has a higher dietary fat content, compared to vegetarian or Tex-Mex dietary patterns. Our laboratory and others have measured increased tumor burden in murine models of NSCLC when feeding diets are high in fat compared to a standard rodent cereal diet. In our laboratory, increased tumor burden is associated with increased tumor multiplicity, but no change in proliferative markers (Ki-67 and pS6) were observed. Dietary fat increases both tumorigenesis in mouse models of pancreatic cancer and circulating levels of the gastrointestinal hormone cholecystokinin (CCK). Inhibition of CCK receptors antagonizes progression of precursor lesions in murine models of pancreatic cancer. Although CCK receptor expression has not been detected in normal human pulmonary tissue, the CCK-B receptor has been identified immunohistologically in human lung tumors. Further, the CCK-A receptor is expressed in normal murine pulmonary epithelium.

Hypothesis: At concentrations approximating circulating levels in mice fed a high fat diet, CCK reduces apoptosis of NSCLC cells in vitro.

Materials and Methods: The murine NSCLC cell line IO33 was grown in serum-replete RPMI 1640 or serum-deprived RPM 1640 with 0 to 1000 pM exogenous CCK-8 peptide added. Expression of CCK-A and CCK-B receptors were measured in IO33 and human NSCLC cell lines by qPCR. Apoptosis was assessed using a Caspase-3/7 peptide cleavage assay.

Results: Expression of CCK-A and CCK-B receptors are reported in IO33 cells and 11 human lung cancer cell lines. Apoptosis of IO33 cells was significantly decreased by greater than 30% at CCK concentrations greater than 100pM (P < 0.03), which is the circulating concentration of CCK in mice fed a high fat diet.

Conclusions: CCK receptors are expressed in human and murine NSCLC cells and circulating CCK may reduce NSCLC cell apoptosis.

Citation Format: Alexander C. Miller, Shelby J. Subry, Manijeh M. Assar, Krista Pearman, Jeffrey William Norris. Evaluation of cholecystokinin receptor expression and function in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1931.