bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4849: CAP-100: first-in-class anti-CCR7 antibody for CLL
Authors: Cuesta-Mateos Carlos , Muñoz-Calleja Cecilia , Loscertales Javier , Terron Fernando , Mol Wim
Published: August 2019

The homeostatic CC-chemokine receptor 7 (CCR7) is highly expressed in many hematological malignancies including chronic lymphocytic leukemia (CLL). CCR7 surface expression levels are much higher than those of CD20, and are correlated with poor prognosis. Upon engagement by its ligands (CCL19 and CCL21), CCR7 controls trafficking of CLL cells to locations where these chemokines are expressed, such as the lymph node (LN). In this protective microenvironment CCR7 ligands contribute to CLL cell survival and proliferation. Indeed, high CCR7-mediated in vitro migration of patient CLL cells correlates with LN involvement and adverse prognostic factors. Thus, strategies targeting CCR7 could provide an additional therapeutic approach for CLL.

Therefore, we have generated CAP-100, the first humanized immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand-mediated signaling from both CCL19 and CCL21. The antibody effectively inhibited CLL cells migration and survival. Furthermore, in in vivo pre-clinical studies, CAP-100 was shown to inhibit entry of CCR7-expressing cells to LNs. The antibody showed potent cell killing activity against CLL cells while preserving normal blood cells. This Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) clearly outperformed standard-of-care in CLL, such as rituximab. ADCC and migration inhibition were both independent of prognostic markers for high risk CLL, such as TP53 mutation including 17p deletion and relapse/refractoriness to various standard therapies, including BTK-inhibitors. Finally, when given as monotherapy in disseminated NHL xenograft tumors in SCID mice, CAP-100 exhibited tumor growth inhibition in established tumors and provided significant survival.

Collectively, our results demonstrate that CAP-100, the first-in-class anti-CCR7 mAb, is a potent antagonist with biological activity in CLL primary cells, including those from relapsed/refractory patients. Moreover, these results highlight the relevance of the CCR7-CCL19/CCL21 pathway as a therapeutic target in CLL. CAP-100’s unique propensity to block migration of CLL cells to the LN, in combination with its potent cell killing activity provides the biological rationale for use of CAP-100 in CLL, either as monotherapy or in combination with novel agents such as BTK-inhibitors. Clinical trials in CLL and other CCR7-expressing NHL will be initiated soon.

Citation Format: Carlos Cuesta-Mateos, Cecilia Muñoz-Calleja, Javier Loscertales, Fernando Terron, Wim Mol. CAP-100: first-in-class anti-CCR7 antibody for CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4849.
Abstract 4928: Relationship between protein biomarkers of potential chemotherapy response and microsatellite status, tumor mutational burden, and PD-L1 expression in patients with cancer
Authors: Nikanjam Mina , Arguello David , Gatalica Zoran , Barkauskas Donald A. , Kurzrock Razelle
Published: August 2019

Introduction: Chemotherapy and checkpoint inhibitor immunotherapies are increasingly used in combinations. We determined associations between the presence of anti-PD-1/PD-L1 therapeutic biomarkers and protein markers of potential chemotherapy response.

Methods: Data was extracted from a clinical-grade testing database (Caris Life Sciences; Feb 2015 - Nov2017): immunotherapy response markers (microsatellite instability high (MSI-H) and tumor mutation burden-high (TMB-H) (by next generation sequencing (NGS)) and PD-L1 protein expression (immunohistochemistry); and protein chemotherapy response markers (ERCC1, TOPO1, TOP2A, TS, TUBB3, RRM1, and MGMT; immunohistochemistry). Relationships were determined by the Mantel-Haenszel chi-squared test or Fischer’s exact tests.

Results: Overall, 28,034 patients (40 tumor types) were assessed. MSI-H was found in 3.3% of patients, TMB-H, 8.4%, and PD-L1 expression in 11.0% of patients. Based on concurrent biomarker expression, combinations of immunotherapy with platinum (ERCC1 negativity) or with doxorubicin, epirubicin, or etoposide (TOP2A positivity), have a higher probability of response while combinations with irinotecan or topotecan (TOPO1 positivity), with gemcitabine (RRM1 negativity), and fluorouracil, pemetrexed, or capecitabine (TS negativity) may be of less benefit. The potential for immunotherapy and taxane (TUBB3 negativity) combinations is present for MSI-H but not TMB-H or PD-L1-expressing tumors; for temozolomide and dacarbazine (MGMT negative), PD-L1 is frequently co-expressed, but MSI-H and TMB-H are not associated.

Conclusion: Protein markers of potential chemotherapy response along with NGS for immunotherapy response markers can help support rational combinations as part of a precision oncology approach.

Citation Format: Mina Nikanjam, David Arguello, Zoran Gatalica, Donald A. Barkauskas, Razelle Kurzrock. Relationship between protein biomarkers of potential chemotherapy response and microsatellite status, tumor mutational burden, and PD-L1 expression in patients with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4928.
Abstract 4949: Immunological ignorance is an enabling feature of the oligo-clonal T cell response to melanoma neoantigen
Authors: Linette Gerald P. , Carreno Beatriz M. , Skidmore Zackary L. , Hundel Jasreet , Griffith Obi L. , Griffith Malachi , Magrini Vincent , Mardis Elaine R.
Published: August 2019

The impact of intratumor heterogeneity (ITH) and the neoantigen landscape on T cell immunity is poorly understood. ITH is a widely recognized feature of solid tumors and poses distinct challenges related to the development of effective therapeutic strategies, including cancer immunotherapy. Cutaneous melanoma is characterized by C->T transitions caused by UV exposure which leads to a large mutational burden encoding putative neoantigens. Here, we performed ultra-deep DNA sequencing of multiple metastases from melanoma patients and observed ubiquitious sharing of clonal and subclonal single nucleotide variants (SNV) encoding putative HLA class I restricted neoantigen epitopes among tumors. MEL66, a reference melanoma patient with a large mutational burden (53 SNV/Mb), provided six resected metastases for study. The spontaneous T-cell response features an oligo/monoclonal T cell-receptor (TCR) repertoire which targets a few clonal neoantigens; most neoantigen-specific T cell clones ignore the tumor. CD8+ T cells to additional HLA class I restricted neoantigens can be elicited by DC vaccination (NCT03092453) with tumor-encoded amino acid substituted (mutated) peptides resulting in a diverse, high frequency TCR repertoire. Isolation of T cell clones by limiting dilution from blood and TIL permits functional validation regarding neoantigen-specificity. Gene transfer of paired abTCR heterodimers specific for clonal neoantigens confirmed correct clonotype assignments based on high throughput CDR3 sequencing. Additional melanoma patients with 2-6 resected metastases were investigated and similar observations were found. Our observations implicate ineffective neoantigen cross-presentation as the basis for immunological ignorance. We postulate that therapeutic vaccination is required to increase the breadth and diversity of neoantigen-specific CD8+ T cells as an adjunct to checkpoint inhibitor treatment for cancer.

Citation Format: Gerald P. Linette, Beatriz M. Carreno, Zackary L. Skidmore, Jasreet Hundel, Obi L. Griffith, Malachi Griffith, Vincent Magrini, Elaine R. Mardis. Immunological ignorance is an enabling feature of the oligo-clonal T cell response to melanoma neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4949.
Abstract 495: All-in-one flow cytometry staining panel for immune-cell profiling in syngeneic tumor models
Authors: Weber Holger , Beshay Janette , Obodozie Cynthia
Published: August 2019

Checkpoint inhibitor treatment has become a common therapy of various cancer types; however, clinical data indicate that only few patients respond to this regimen due to attenuated anti-tumor immune response. Thus, it has been recognized as important to consider the immune response already during preclinical drug development to anticipate such clinical drawbacks and investigate the changes of immune cell populations after treatment. Flow cytometry is widely used for this kind of analysis. However, a comprehensive analysis of numerousness immune cell populations in one tumor is a major challenge when only limited material is available due to small tumor size of e.g. a tumor responding to the treatment or analysis at an early tumor developmental stage. A staining panel that allows the analysis of all major immune cell populations in a single staining would be a major advantage.

Here we present an all-in-one flow cytometry panel based on 15 colors which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), NK cells, macrophages (M1/M2), MDSCs (neutrophils, M-MDSCs) and dendritic cells. To demonstrate the value of the panel, the immune cell content of CT26wt tumors was compared after subcutaneous, orthotopic or intra-mammary fat pad implantation. During tumor growth profiling of the immune population is performed every 4 days, starting at day 7 after implantation when tumor volume is still very small (50 - 100 mm3). A comprehensive overview is given with valuable insight into the immune biology of the tumor over time and with respect to different tumor sizes.

The established all-in-one staining panel allows the direct comparison of immune cell populations with a single staining which improves the development of new immune-modulating drug candidates.

Citation Format: Holger Weber, Janette Beshay, Cynthia Obodozie. All-in-one flow cytometry staining panel for immune-cell profiling in syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 495.
Abstract 4961: Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12
Authors: Uribe-Herranz Mireia , Bittinger Kyle , Rafail Stavros , Pierini Stefano , Guedan Sonia , Bushman Frederic , June Carl , Facciabene Andrea
Published: August 2019

Adoptive T cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome or treatment with antibiotics, or by heterologous fecal transfer. Depletion of bacteria with vancomycin decreased the rate of tumor growth in mice from The Jackson Laboratory receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ DCs, which sustained systemic adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest potentially new avenues to improve response to ACT by altering the gut microbiota.

Citation Format: Mireia Uribe-Herranz, Kyle Bittinger, Stavros Rafail, Stefano Pierini, Sonia Guedan, Frederic Bushman, Carl June, Andrea Facciabene. Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells and IL-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4961.
Abstract 4962: Repurposing bazedoxifene to suppress gastrointestinal cancer growth
Authors: Thilakasiri Pathum , Nero Tracy , Parker Michael W. , Ernst Matthias , Chand Ashwini L.
Published: August 2019

The interleukin (IL)6 family of inflammatory cytokines, characterized by the shared use of the gp130 receptor, supports the progression of solid cancers. Specifically, gp130 signaling becomes rate limiting for the growth of gastrointestinal tumors arising from oncogenic driver mutations in Apc, but remains dispensable for the homeostatic renewal of the gastrointestinal mucosa. In the present study we investigate the effects of the selective estrogen receptor modulator bazedoxifene, clinically approved for the treatment of osteoporosis, as a putative small molecule inhibitor of gp130 receptor oligomerization and activation. We assessed the effects of bazedoxifene on IL6 and IL11-dependent STAT3 activation and cell proliferation in gastric and colon cancer cell lines. In patient-derived colon cancer organoid cultures, we assessed bazedoxifene effects on IL11-dependent growth. The gp130Y757F, Lgr5CreERT2- or Cdx2CreERT2-driven Apc flox mouse models of gastrointestinal cancer, were used to examine the effects of bazedoxifene on tumour growth. In silico modeling suggests that the inhibitory activity of bazedoxifene is due to its ability to mimic the interaction between the Site III interface of gp130 receptor and the tryptophan-157 in IL6, or tryptophan-168 in IL11 cytokine. We show that bazedoxifene interferes with IL6 and IL11-dependent proliferation of BAF/03 cells. Bazedoxifene suppresses gastric tumor growth in gp130Y757F mice irrespective of their gender, in which tumors arise through excessive IL11-dependent STAT3 signaling. Strikingly, in sporadic colon cancer models based on aberrant activation of the β-catenin/canonical WNT pathway, bazedoxifene treatment also reduces tumor burden in mice following conditional ablation of the Apc suppressor gene using the Lgr5CreERT2- or Cdx2CreERT2-driver alleles. Consistent with our observation that nuclear accumulation of β-catenin remains unaffected in colonic tumors of bazedoxifene-treated mice, bazedoxifenetreatment of human SW480 colon cancer cells harboring mutant APC did not affect canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof-of-concept for the repurposing of bazedoxifene for the treatment of gastrointestinal cancers that arise from bona fide oncogenic driver mutations.

Note: This abstract was not presented at the meeting.

Citation Format: Pathum Thilakasiri, Tracy Nero, Michael W. Parker, Matthias Ernst, Ashwini L. Chand. Repurposing bazedoxifene to suppress gastrointestinal cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4962.
Abstract 4970: Establishment of a patient-derived platform for preclinical immunooncology studies using autologous tumor and TIL coculture
Authors: Hinada Kaede , Garland Dennis , Nair Dileep , Hu Yong , Broudy Thomas
Published: August 2019

The use of tumor infiltrated lymphocytes (TILs) as an adoptive cell transfer therapy to treat cancer has been conducted in clinic trials for more than two decades. TIL therapy in combination with added immunotherapy treatments, such as IL-2 or anti-CTLA4, has further improved clinical response rates. Isolated from a patient’s own tumor specimen, TILs that recognizes tumor-specific known or unknown antigens can be expanded and infused back to tumor-bearing patients to attack autologous cancer cell populations. Despite the progress in clinical research, preclinical and translational models of autologous tumor/TIL coculture have been difficult to establish.

This study has established autologous tumor/TIL coculture methods directly from patient tumor specimens. Models were established from BioDuro’s viably cryopreserved tumor & TIL bank, containing more than 100,000 patient biospecimens. Each patient specimen contains a heterogeneous population of cells types, including tumor, TILs and TAFs (tumor associated fibroblasts). These cell populations were expanded under specific growth conditions to then serve as components for the patient-derived coculture systems established.

In this study, TILs and monocytes from a cohort of patient tumor specimens (including kidney, melanoma and lung) were analyzed. Leukocytes, T cells, monocytes and B cells were marked with CD45, CD3, CD14, CD20. Patients’ samples were also tested for PD-1 expression levels. The results show significant individual variation on cell subpopulations and PD-1 expression. Established methods were used for expansion of TILs, supporting either CD4 or CD8 phenotypes. Autologous cancer cells were expanded at the same time. TILs and tumor cells were co-cultured and high content fluorescent image cytometry was used to monitor tumor cell killing. This study found that autologous patient-matched TILs showed preferential tumor cell killing compared to patient-unmatched TILs. This response was observed in a dose dependent fashion.

In summary, the BioDuro biobank of viable tumor and TIL patient specimens served as a large and powerful immunooncology resource to establish patient-derived autologous tumor/TIL coculture assays. These assays, and the ability to readily study larger populations of cancer patients, provide a powerful in vitro platform to test immunooncology drug candidates.

Citation Format: Kaede Hinada, Dennis Garland, Dileep Nair, Yong Hu, Thomas Broudy. Establishment of a patient-derived platform for preclinical immunooncology studies using autologous tumor and TIL coculture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4970.
Abstract 4971: Dominant negative PD 1 armored CAR T cells induce remission in refractory diffuse large B-cell lymphoma (DLBCL) patients
Authors: Chen Tong
Published: August 2019

Anti-CD19 chimeric antigen receptor (CAR19) autologous T cell treatment has been demonstrated as effective therapy to relapse/refractory B cell leukemia and lymphoma. However, CAR T shows limited efficacy in treatment of solid tumor or challenging with immunosuppressive characters, such as PD-L1+/hi lymphomas.

PD-1/PD-L1 blockade have been shown as potentially effective anti-tumor therapies. We generated a lenti-vector incorporating an anti-CD19 secondary CAR molecule sequence with 4-1BB co-stimulatory domain and a dominant negative PD-1 sequence (Figure A). Compared with conventional CART cells, these ”armored” CART cells show enhanced capability of tumor killing after multiple-round challenging by PD-L1+CD19+ positive tumors, with a ”emory-like” phenotype(Figure B), suggesting dominant negative PD-1 molecule may protect CART cells from inhibition in tumor micro-environment.

Here we report 2 cases of refractory diffuse large B cell lymphomas (DLBCLs) successfully treated with this novel dominant negative PD-1 armored humanized anti-CD19 CART cells. These two patients all failed to achieve response after multiple rounds of chemotherapy and radiotherapy, and infused with autologous CAR T cells at 5.23×10^6/kg and 1.97×10^6/kg during disease progress. Both patients showed significantly tumor mass decrease and SUV max decline in PET/CT results with ongoing response. (From 34.48 to 3.89 at day 27, from 25.02 to 2.38 at day 31, respectively.) (Figure C).

Conclusion: These 2 cases revealed the significant anti-bulky lymphoma response of new type of CAR19 T cells with limited and tolerated cytokine release syndrome and central nervous system toxicity. This novel structure of dnPD1-hCAR19 may augment CART cells persistence in the patient after activation triggered by the lymphoma cells, suggesting that dnPD-1 molecule may enhance efficacy of CAR T cells in treatment of solid tumor. We are continuing to recruit more patients in clinical research.

Citation Format: Tong Chen. Dominant negative PD 1 armored CAR T cells induce remission in refractory diffuse large B-cell lymphoma (DLBCL) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4971.
Abstract 4976: Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM)
Authors: He Daniel Xianfei , Zheng Lei , Sun Ruilin , An Annie Xiaoyu , Fei Jian , Li Henry Qixiang , Ouyang Davy Xuesong
Published: August 2019

CD40 receptor is a member of TNF-receptor superfamily, broadly expressed on the surface of antigen-presenting cells (APC) including B cells, dendritic cells (DC), macrophages, monocytes, and non-APC like platelets, endothelial cells and even some tumor cells. CD40 is essential for APC activation and signalling via ligation of CD40 ligand located on helper T cells and CD40 on APC to mediate licensing (activating) of APC. Ligation of CD40 on DCs, for example, can induce increased surface expression of costimulatory and MHC molecules, production of pro-inflammatory cytokines, and enhanced T cell activation. CD40 ligation on resting B cells can increase its antigen-presenting function and proliferation. In contrast to its critical role in the induction of effective innate and adaptive immune responses, CD40 signalling on certain malignant cells, particularly B cell lymphomas, triggers tumor apoptosis. The dual function of CD40 signalling provide unique opportunities for the use of agonistic CD40 antibodies in cancer immunotherapy. Indeed, several CD40 monoclonal antibodies are now in different stages of clinical development, some shown promising preliminary results in combination with other cancer immunotherapies. However, we are lacking preclinical models to evaluate efficacy of therapeutic CD40 agonistic antibodies, which usually don’t cross-bind to mouse targets. To fill this gap, we developed a human CD40 knock-in model (CD40 HuGEMM), of which mouse exon 2-5 are replaced by human counterparts. These KI mice express a chimeric CD40 with human extracellular domain & mouse trans-membrane and intracellular sequences. We have characterized the human CD40 expression of CD40 HuGEMM on a broad spectrum of mouse cells, such as lymphocytes, macrophages, DCs, and endothelial cells. In addition, we confirmed that human CD40 antibodies bind to the chimeric m/hCD40 recombinant protein, and lead to increased expression of MHCII on the B cells. Finally, the in vivo efficacy study indicates that human CD40 antibody treatment lead to robust anti-tumor response and contribute to better survival of the mice. Taken together, our CD40 HuGEMM provides a powerful preclinical model to assess the efficacy of the human-specific CD40 agonistic antibodies. It may also serve as a model for proof of concept studies of CD40 agonistic antibodies with other therapies.

Citation Format: Daniel Xianfei He, Lei Zheng, Ruilin Sun, Annie Xiaoyu An, Jian Fei, Henry Qixiang Li, Davy Xuesong Ouyang. Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4976.
Abstract 4977: Dynamic transcriptional programs controlling single NK cell killing dynamics
Authors: Hall Matthew S. , Decker Joseph T. , Grody Emanuelle I. , Blaisdell Rachel B. , Jeruss Jacqueline S. , Shea Lonnie D.
Published: August 2019

NK cells have a vast heterogeneity in function and phenotype in vivo and their healthy function is critical in the prevention of nascent cancers. NK cell-based immunotherapies, such as adoptive NK cell therapy, are emerging and greater insights into the mechanism of NK cell activation could increase their efficacy. In vitro cytotoxicity assays have revealed that a small subset of NK cells kill the majority of cancer target cells, yet little is known about the dynamic state or triggers of these highly cytotoxic cells. Here, we present methodology for discovery of the molecular precursors and drivers of single NK cell killing events. We study the NK-92 cell line both as a model of primary NK cells and to gain direct insights for improving NK-92 based adoptive cell therapies. In our method, we first performed single cell RNA sequencing on NK-92 cells seeded sparsely on a monolayer of HeLa cancer cells along with a control NK-92 cell population. Clustering cells by principal component analysis revealed groups of NK cells that upregulated genes associated with NK cell cytotoxicity in the HeLa cell activated sample that are absent in the control group. Landmark genes associated with the cytotoxic NK cell clusters included PRF1, GZMK, and ITGB2. Utilizing live cell imaging, we identified categories of dynamic functional phenotypes in NK-92 cells exposed to a HeLa cell monolayer including non-motile and non-killing, highly motile but non-killing, single killing, serial killing, and exhausted. In order to link the systems-level single cell gene expression data to functional NK cell behaviors, we next performed high content live cell imaging of NK-92 cells bearing cas9-based endogenous genetic reporters as they surveilled and killed HeLa cells. Utilizing a custom analysis algorithm, we tracked the motility and killing activity of individual NK-92 cells, and correlated this activity to the dynamic gene expression. Next, we aim to identify the causality and sequence of transcriptional changes associated with NK cell killing and extend our results to the analysis of the critical transcription factor networks governing dynamic NK cell functional phenotypes. We note that the single cell functional discovery strategy that we have shown can be applied more broadly to other model systems of the cancer microenvironment.

Citation Format: Matthew S. Hall, Joseph T. Decker, Emanuelle I. Grody, Rachel B. Blaisdell, Jacqueline S. Jeruss, Lonnie D. Shea. Dynamic transcriptional programs controlling single NK cell killing dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4977.
Abstract 4985: TLR agonists for anticancer immunotherapy
Authors: Zhang Wenqiu , Kim Hyunjoon , Khanna Vidhi , Ferguson David M. , Griffith Thomas , Panyam Jayanth
Published: August 2019

Introduction: Toll-like receptors (TLRs) are important pattern recognition receptors through which innate immune cells recognize invasive microorganisms. The immunostimulatory property of TLR agonists allows for activation of dendritic cells (DCs) and enables their use as anticancer vaccine adjuvants. Because TLR 7 and 8 can be activated by synthetic small molecules, they are of particular interest as a target in vaccine adjuvant discovery. However, a key drawback of these synthetic small molecules is that they also induce immunosuppressive cytokines, resulting in immunosuppression.

Methods: We have previously developed a suite of highly substituted imidazoquinolines, which potently activate TLR 7 and/or 8. In this study, we tested some selected TLR7, TLR8 and TLR7/8 dual agonists for their ability to activate DCs without inducing immune suppressive cytokines. Murine bone marrow-derived dendritic cells (BMDCs) were generated from C57BL/6 mice. BMDCs were treated with one of seven TLR agonists for 72 hrs. Cells were collected and stained for flow cytometry analysis, and the culture supernatants were examined for secretion of pro-inflammatory cytokines, IL-12p70 and IFN-γ, and an immunosuppressive cytokine, IL-10, by ELISA. We also investigated activation of T cells by these agonists. Human peripheral blood mononuclear cells (hPBMCs) from healthy donors were incubated with one of seven TLR agonists overnight and cells were analyzed by flow cytometry for the expression of T cell activation marker CD69 and production of IFN-γ.

Results: All the TLR agonists investigated activated BMDCs, as evidenced by the upregulation of costimulatory markers CD40, CD80 and CD86 on DCs. In addition, all the agonists examined induced the secretion of IL-12p70 and IFN-γ. TLR 8 agonists showed the least induction of IL-10 secretion. All the TLR agonists activated both CD4 and CD8 T cells. TLR 8 agonists induced higher production of IFN-γ in both type of T cells than other candidates.

Conclusion: Both TLR 8 and TLR 7/8 agonists activated BMDCs and stimulated strong pro-inflammatory cytokine production. However, TLR8 activation was associated with less immunosuppression. These results suggest that these new imidazoquinoline esters are promising candidates for use as anti-cancer vaccine adjuvants.

Citation Format: Wenqiu Zhang, Hyunjoon Kim, Vidhi Khanna, David M. Ferguson, Thomas Griffith, Jayanth Panyam. TLR agonists for anticancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4985.
Abstract 4987: Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and reduce response to immuno-oncology treatments
Authors: Sitnikova Suzanne I. , Morrow Michelle , Valge-Archer Viia , Wilkinson Robert W. , Dovedi Simon J. , Robinson Matthew J.
Published: August 2019

Immuno-oncology research relies heavily on murine syngeneic tumor models. However, whilst the median age for a cancer diagnosis is 65 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that aging has been shown to have a significant impact on the immune response. Using aged mice bearing CT26 tumors, we analysed how aging impacts the immune composition of the tumor, spleen and tumor-draining lymph nodes by flow cytometry. We found many age-related changes between aged (60-72 weeks old) and young (6-8 weeks old) mice, such as a reduction in the naïve T cell population and a decreased CD8/Treg ratio in aged animals. Profiling of co-inhibitory and co-stimulatory receptor expression levels on immune cells in aged versus young mice also identified altered expression profiles in both the periphery and tumor. We hypothesised that these differences may contribute to impaired anti-cancer immune responses in aged mice. To investigate this, we compared the anti-tumor efficacy of immune checkpoint blockade (PD-L1 and CTLA-4) and T-cell costimulation (OX-40) in aged versus young mice. Our data demonstrate that aged mice retained their capacity to generate effective anti-tumor immune responses, albeit often attenuated when compared to the responses observed in young mice. These differences highlight the potential importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

Citation Format: Suzanne I. Sitnikova, Michelle Morrow, Viia Valge-Archer, Robert W. Wilkinson, Simon J. Dovedi, Matthew J. Robinson. Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and reduce response to immuno-oncology treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4987.
Abstract 4992: The occurrence of immune-related adverse events indicates better efficacy of immune checkpoint blockade in solid tumors: A systematic review and meta-analysis
Authors: Yang Xu , Wang Dongxu , Lin Jianzhen , Xiong Jianping , Long Junyu , Bai Yi , Bian Jin , Huang Hanchun , Yang Xiaobo , Mao Yilei , Sang Xinting , Zhao Haitao
Published: August 2019

Introduction: Immune-related adverse events (irAEs) were mostly induced by immune checkpoint blockade (ICB) and were associated with the immune response. Whether irAEs are associated with clinical benefits during ICB treatment in cancer patients remains unclear. This systematic review and meta-analysis aimed to investigate the relationship between irAEs and clinical efficacy.

Methods: We systemically searched and selected irAEs and clinical outcome data from cancer patients treated with ICB in the PubMed, Embase, and Cochrane Central Trial databases up to July 26, 2018. Keywords used for the search included PD-1, PD-L1, CTLA-4, immune checkpoint and immune-related adverse events. The relationships between the occurrence of irAEs and the objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were assessed using relative risk (RR) or hazard ratio (HR) values and 95% confidence intervals (CIs) in random-effect models.

Results: Nineteen eligible observational articles were included in this systematic review and fourteen studies including 3022 participants (1264 with irAEs and 1758 with non-irAE) were included in our meta-analysis. The occurrence of irAEs was significantly associated with higher ORR (RR = 2.36, 95% CI: 1.85-3.02; P < 0.001), better PFS (HR

= 0.48, 95% CI: 0.26-0.70; P < 0.001), and longer OS (HR = 0.42; 95% CI = 0.18-0.67; P = 0.001) than those in the non-irAE group. Subgroup analysis showed that the effect size of ORR in anti-PD-1 therapy (RR = 2.68, 95% CI, 2.21-3.26) was inferior to that of the anti-CTLA-4 therapy (RR = 1.91, 95% CI, 0.44-8.32). The meta-regression models also found that ICB type (anti-CTLA-4 or anti-PD-1) were associated with significant heterogeneity (P = 0.005).

Conclusion: In conclusion, irAEs indicate greater ORR, PFS and OS than non-irAEs in ICB treatment, and the relationship in anti-PD-1 therapy was stronger than in anti-CTLA-4 therapy. With appropriate management of irAEs, the clinical predictor, continued treatment may be possible to maximize its potential ICB benefits.

Note: This abstract was not presented at the meeting.

Citation Format: Xu Yang, Dongxu Wang, Jianzhen Lin, Jianping Xiong, Junyu Long, Yi Bai, Jin Bian, Hanchun Huang, Xiaobo Yang, Yilei Mao, Xinting Sang, Haitao Zhao. The occurrence of immune-related adverse events indicates better efficacy of immune checkpoint blockade in solid tumors: A systematic review and meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4992.
Abstract 5: Pre-clinical development of CDK9 inhibitor - LS007 for the treatment of acute myeloid leukemia
Authors: Hui Wang , Shudong Wang
Published: August 2019

Acute myeloid leukemia (AML) is the most common form of acute leukaemia with an increasing incidence and dismal long-term prognosis with age. The most aggressive subtype, MLL-AML, is characterized by translocations of the mixed-lineage leukaemia gene (MLL) in chromosome band 11q23 and resistance to conventional chemotherapy with a short overall survival of only 9 months. Cyclin-dependent 9 (CDK9) activity is required for MLL-driven oncogenic transcription. Inhibition of CDK9 directly targets the transcriptional machinery required for MLL-fusion proteins (e.g. MLL-AF4, MLL-AF9 and MLL-ENL), thereby leading to apoptosis.

We have developed a highly effective drug candidate - LS007 that inhibited CDK9 with a Ki = 4 nM. It suppressed proliferation and induced apoptosis in a range of AML cell lines and patient samples. LS007 was highly efficacious against several AML cell line xenografts and patient-derived xenografts, and well tolerated in mice after oral administration. In a Molm-13 xenograft model, after 21 days of orally daily dosing at 25 mg/kg and 50 mg/kg LS007

inhibited tumor growth (p < 0.001) with T/C = 44 % and 18 %, respectively. Similarly, in a HL-60 xenograft model, LS007 exhibited a significant tumor growth inhibition at 45 mg/kg and 60 mg/kg daily by oral administration with T/C = 40 % and 20 %, respectively.

The pharmacokinetic profiles of LS007 was further assessed in the cynomolgus monkeys. After oral doses at the dose of 2, 4 and 8 mg/kg, LS007 was detected in the plasma with the maximum concentration (Cmax) from 55.9 to 279.5 ng/mL and the area under the plasma drug concentration-time graph (AUC) up to 1862.7 h·ng/mL. The mean terminal half-life (T 1/2) was around 3 h. LS007 possessed the favorable pharmacokinetic profiles with oral bioavailability F = 56% in the cynomolgus monkey (at dose 4 mg/kg), suggesting its therapeutic potential as an orally bioavailable anti-AML agent. The GLP- toxicological studies of LS007 in the cynomolgus monkey were completed, and the maximum tolerated doses were identified, which were used to estimate the safe starting dose for the Phase I clinical trials.

With its excellent anti-cancer efficacy along with appreciable absorption and safety profiles, LS007 offers a very exciting prospect as a clinical development candidate. The application of Investigational New Drug (IND) has been approved allowing the commencement of clinical trials of LS007 in patients with AML.

Citation Format: Wang Hui, Wang Shudong. Pre-clinical development of CDK9 inhibitor - LS007 for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5.
Abstract 500: Predicting patient response to immuno-oncology agents in vitro using 3D cultures
Authors: Appleton Kathryn M. , Guo Qi , Elrod Ashley , Lotstein Alina , Holmes Lillia , DesRochers Teresa M.
Published: August 2019

Immuno-oncology (I/O) based therapeutics, including cellular therapies and checkpoint inhibitors have surged in the last 2 years, but the technology to accurately test them in a pre-clinical setting is significantly lacking. While animal models have tried to provide accurate testing platforms, the ultimate goal of a matched patient tumor and immune system is not achievable in mice. To overcome this issue, we have developed two 3D tissue systems for in vitro testing that combine a patient’s tumor cells and autologous immune cells for accurate testing and prediction. We hypothesize that our 3D cell culture systems can recapitulate the patient’s tumor microenvironment to detect I/O response. Our spheroid-based system allows us to monitor how primary T-cells are affected by paired tumor cells and/or the PD-1 inhibitor pembrolizumab using flow cytometry. We have successfully detected pembrolizumab binding to T-cells in a dose dependent manner, clonal expansion of lymphocyte populations, as well as increased expression of activation markers on CD3+ cells following combination with tumor cells and exposure to pembrolizumab. This model also accurately detects CD3+CD8+ T-cell mediated tumor cell death and can be used to track changes in secreted cytokines and chemokines such as Granzyme B and IFN gamma. Our second model, a 3D microtumor platform, allows us to detect immune cell migration and infiltration and therapy related cell death. Our results show pembrolizumab can increase lymphocyte infiltration while simultaneously decreasing microtumor growth in matched patient samples whose tumor cells express PD-L1 and whose lymphocytes are CD8+. Cytokine secretion detected by multiplex technology from our microtumor model supports our observed enhanced T-cell activation in the presence of pembrolizumab. The data generated from our two complex 3D in vitro models can recapitulate in vivo biology in order to derive correlations to I/O drug response. These models can be utilized for preclinical testing of new I/O agents as well as for patient response predictions to I/O therapies.

Citation Format: Kathryn M. Appleton, Qi Guo, Ashley Elrod, Alina Lotstein, Lillia Holmes, Teresa M. DesRochers. Predicting patient response to immuno-oncology agents in vitro using 3D cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 500.
Abstract 502: Aldo-keto reductase 1B8 (AKR1B8) deficiency causes intestinal immune deficiency
Authors: Wang Xin , Cao Deliang
Published: August 2019

Background: Intestinal epithelial cells (IECs) are crucial mediators of intestinal immunity. IECs defects may disrupt development and maturation of intestinal immunity. Aldo-keto reductase 1B10 (AKR1B10) is a regulator of de novo fatty acids synthesis and lipid synthesis by mediating acetyl-CoA carboxylase-α (ACCA) stability. Aldo-keto reductase 1B8 (AKR1B8) is an ortholog in mice of human AKR1B10, which has similar regulatory function in fatty acid/lipid synthesis. AKR1B8 deficiency causes IECs defects in proliferation and self-renewal, which may lead to dysregulation of intestinal immunity.

Methods: AKR1B8 -/- mice mouse stain was produced by homozygous recombination. Immune cells were isolated from colonic lamina propria (cLP) and mesenteric lymph nodes (MLN). Flow cytometry and immunofluorescence staining were applied for immune cell sorting and measurements. Microbiota composition were quantified by next generation sequencing.

Results: AKR1B8 deficiency causes infiltration of neutrophils, mast cells and basophils in the colon. Also, AKR1B8 deficiency causes impaired γδT cells and T helper cells (Th cells) immunity, such as IFNγ, IL17, IL4 or IL22 production, in colon and mesenteric lymph nodes, while enhances distinct arm of CD8 activity and Treg cells. These alterations may be due to the changes of MHCII+ DCs and CD103+ DCs in AKR1B8 deficient mice. Meanwhile, AKR1B8 inhibits the phosphorylation of p-AKT (T308 and S473) and p-ERK (1/2) in IECs, and suppresses the regulation IKKα/NF-κB signaling cascades. Furthermore, AKR1B8 deficiency induces gut microbiota shifts with higher ratio of Firmicutes/Bacteroidetes.

Conclusions: AKR1B8 is a critical mediators for IECs signaling and immune homeostasis. Loss of AKR1B8 in IECs induces Th cells immune deficiency. This study identified a new compensatory mechanism under IECs defects for maintaining intestinal immune homeostasis.

Citation Format: Xin Wang, Deliang Cao. Aldo-keto reductase 1B8 (AKR1B8) deficiency causes intestinal immune deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 502.
Abstract 5050: Testosterone supplementation in relation to prostate cancer in a US commercial insurance claims database
Authors: Cook Michael B. , Beachler Daniel C. , Parlett Lauren E. , Cochetti Philip T. , Finkle William D. , Lanes Stephan , Hoover Robert N.
Published: August 2019

Background: Testosterone supplementation (TS) has dramatically increased in the United States (US). We conducted a study of TS and prostate cancer risk using a large US commercial insurance research database.

Methods: From the HealthCore Integrated Research Database (HIRDSM), we selected men aged 30 years or greater who were new users of TS during 2007-2015. We selected two male comparison groups: 1) unexposed (matched 10:1); 2) new users of a phosphodiesterase type 5 inhibitor (PDE5i). Incident prostate cancer was defined as diagnosis of prostate cancer within four-weeks following prostate biopsy. Propensity scores and inverse probability of treatment weights were used in Poisson regression models to estimate adjusted incidence rates, incidence rate ratios (IRRs) and 95% confidence intervals (CI) using doubly robust estimation. Subgroup analyses included stratification by prostate cancer screening, hypogonadism, and follow-up time.

Results: The adjusted prostate cancer IRR was 0.77 (95%CI: 0.68, 0.86) when comparing TS with the unexposed group and 0.85 (95%CI: 0.79, 0.91) in comparison with the PDE5i group. Inverse associations between TS and prostate cancer were observed in a majority of subgroup analyses, although in both comparisons estimates generally attenuated with increasing time following initial exposure. Amongst TS users, duration of exposure was not associated with prostate cancer.

Conclusion: In this study, men who received TS did not have a higher rate of prostate cancer compared with the unexposed or PDE5i comparison groups. The inverse association between TS and prostate cancer could be the result of residual confounding, contraindication bias, or undefined biologic effect.

Citation Format: Michael B. Cook, Daniel C. Beachler, Lauren E. Parlett, Philip T. Cochetti, William D. Finkle, Stephan Lanes, Robert N. Hoover. Testosterone supplementation in relation to prostate cancer in a US commercial insurance claims database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5050.
Abstract 5057: Single cell RNA sequencing reveals altered natural killer-like, effector CD8+ T lymphocytes in smokers
Authors: Martos Suzanne N. , Campbell Michelle R. , Iannone Marie A. , Pittman Gary S. , Wan Ma , Bell Douglas A.
Published: August 2019

Tobacco smoke exposure is a risk factor for many human diseases and the global disease burden attributed to smoking remains substantial. In addition to DNA damage, smoking-induced epigenetic changes may contribute to etiology of complex smoking-associated diseases. Smoking alters the epigenome and transcriptome of human blood leukocytes. However, interpretation of bulk genomic approaches is limited because changes could indicate altered distribution of cell (sub)populations or changes in expression within (sub)populations. To characterize smoking-related gene expression changes in primary immune cells, we performed single cell RNA sequencing on human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Transcriptomes of 45,965 cells (78,183 reads per cell) revealed an altered population of Natural Killer (NK)-like T lymphocytes in smokers. Compared to NK cells, the NK-like T cell cluster had elevated expression of CD8A, CD8B, CD3D, CD3E, CD3G, CD6, and CD2, indicative of CD8+ T lymphocytes. Relatively rare in nonsmokers (2.2%), the transcriptionally unique subset of CD8+ T cells comprised 8.7% of PBMCs in smokers. Among CD8+ T cell subtypes, the increase in NK-like CD8+ T cells (Mann-Whitney p = 0.03) corresponded with a decrease in Naïve CD8+ T cells (p = 0.03). We did not observe changes in the frequencies of two additional CD8+ T cell clusters or in the overall frequency of CD8+ T cells. Mass cytometry of a 26-antibody leukocyte panel confirmed no differences in the frequencies of total CD8+ T (CD3+/CD8+/CD56-), total NKT (CD3+/CD56+) or CD8+ NKT (CD3+/CD8+/CD56+) cells between smokers and nonsmokers. This suggests smoking is associated with an increased number of CD8+ T cells that share characteristics with NK cells, but are not NKT cells. Consistent with an NK-like phenotype, altered effector CD8+ T cells had elevated expression of genes reported to be upregulated in T cells reprogrammed to NK-like cells. Compared to other effector CD8+ T cells, altered effector CD8+ T cells had reduced IL7R and increased FCGR3A (CD16), IFNG (interferon gamma), GZMB (granzyme B), and PRF1 (perforin) expression. In mice, granzyme B and perforin expressing CD8+ T cells contribute to the development of atherosclerotic plaques. Our data highlights a potential link between smoking-induced functional changes in human CD8+ T cells and atherosclerosis and /or immune surveillance in cancer.

Citation Format: Suzanne N. Martos, Michelle R. Campbell, Marie A. Iannone, Gary S. Pittman, Ma Wan, Douglas A. Bell. Single cell RNA sequencing reveals altered natural killer-like, effector CD8+ T lymphocytes in smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5057.