AI News, Adrián González Sánchez, MBA MSc artificial intelligence

34th Annual Meeting Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 2

Cancer cells require outside supply of some non-essential amino acids (NEAAs) to survive.

In this study, we tested the effects of NEAA-deprived diets and checkpoint inhibitor anti-PD-1 and anti-PD-L1 in colon cancer using syngeneic mouse model (Balb/c) bearing tumors of mouse colorectal cancer cell line CT-26.

Three diets were tested, including a natural rodent diet Teklad ENVIGO Global 16% Protein Rodent Diet (control 1), a formulated NEAA-complete diet COMPLETE (control 2, using amino acid mix in place of protein), and a formulated NEAA-deprived diet FTN203 (treatment, using amino acid mix in place of protein).

We found 1) On day 24 post tumor implantation, NEAA-deprived diet FTN203 significantly reduced tumor growth when used alone, compared to the group fed with Teklad ENVIGO (by 81%, P=0.0054, unpaired t-test after Welch correction) and COMPLETE (by 81%, P=0.013), respectively;

Our data supports the use of dietary NEAA deprivation to improve the efficacy of anti-PD-1 or anti-PD-L1 immunotherapy for colorectal cancer without noticeable side effects.

The study CA-XLI-6 was approved by the CRO's Ethics Board under IACUC approval number 19-015.9.

In the Phase 3 CORRECT study, which led to the approval of the multi-kinase inhibitor, Regorafenib, in 3rd/4th line metastatic colorectal cancer, the OS was 6.4 months and the PFS was 1.9 months compared to an OS of 5.0 months and a PFS of 1.7 months for placebo.

These experiments determined whether combination therapy with RRx-001 and regorafenib not only enhanced anticancer activity in vitro with HCT-116 and HCT-15 colorectal cell lines and in vivo with HCT 116 and HCT 15 xenografts but also attenuated the toxicity of regorafenib in these two xenografts.

The results from these experiments demonstrate that 1) RRx-001 + regorafenib is more effective than either agent alone both in vitro and in vivo and that 2) the addition of RRx-001 to regorafenib attenuates the toxicity of regorafenib in vivo.

Treatment of metastatic solid tumors remains a challenge in oncology, especially in the context of locally given therapies.

We utilized a murine melanoma model (B16.OVA) where each mouse had bilateral tumors.

Improvement in the overall survival was seen in the group receiving both therapies (anti-PD-1 plus virus) compared to the monotherapy groups.

These two cancer immunotherapies seem to be a promising approach that could increase survival upon clinical translation.

We thank the Biomedicum FACS Core facility and the Animal the Finnish Centre for Laboratory Animal Pathology (FCLAP) at the University of Helsinki, for their expert assistance.

All experiments described here have been approved by The Gene Technology Board of Finland, by the Experimental Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland, under the license number ESAVI/7755/04.10.07/2016.

Pegzilarginase monotherapy increased MHC expression on antigen presenting cells (APCs) and the frequency of intratumoral CD8+ T cells relative to controls (p<0.01).

Combination pegzilarginase/immunotherapy induces robust anti-tumor immunity characterized by increased intratumoral CD8+ T cells and M1-polarized macrophages.

All mice were maintained under specific pathogen-free conditions in the Providence Portland Medical Center animal facility.

Clinically, cryoablation is used to treat various types of cancers including certain prostate, liver and kidney tumors.

Cryoablation of a single primary tumor resulted in a tumor-free survival rate of 40%.

IL-12 formulated with chitosan and delivered after cryoablation is superior in treating not only a primary tumor, but also in inducing an abscopal effect on a distant, untreated tumor.

This work is supported by BTG, the NC State University Provost’s Fellowship, and startup funds provided by the College of Engineering at NC State University.

The study was approved by the NC State University's Ethics Board, approval number 17-052.

The presence of tumor infiltrating lymphocytes in breast cancer (BC) is associated with improved survival.

We investigated the effect of nitric oxide synthase2 (NOS2) or cycloxygenase2 (COX2) inhibition using commercially available inhibitor on radiation and αPD-L1 induced tumor growth delay and lung metastases in murine model of TNBC using 4T1 cell line implanted in flank of Balbc mice.

We found that RT induced inflammation associated biomarkers NOS2 and COX2 in the TME, specifically in the tumor cells.

We demonstrated that co-treatment of COX2 inhibitor with radiation and immunotherapy effectively activated the immune system by changing the TME to support tumor clearance.

We have shown in both murine tumor models and patients that focal tumor radiotherapy synergizes with CTLA-4 blockade to induce systemically effective anti-tumor immunity in otherwise resistant tumors [1,2,3].

The mouse mammary carcinoma TSA cells were irradiated in vitro with 8 or 20 Gy, followed by chromatin immunoprecipitation (ChIP) assay to determine if the transcription factor cFos was bound to Trex1 promoter region 4 hours after irradiation.

The transcription factor c-Fos was found to be phosphorylated in TSA cells treated with a radiation dose that induces TREX1 upregulation (20Gy), but not in 8Gy-treated cells.

Overall, these data identify cFos as the transcription factor that regulates Trex1 gene expression in a radiation dose-dependent way.

Radiotherapy has been routinely used to treat lung cancer alone or in combination with chemotherapy or surgery.

In this study, we combined radiotherapy with checkpoint inhibitor immunotherapy and a radio-enhancing nanoparticle NBTXR3 (R3) to extend the application of radiotherapy to treating metastasized lung cancer in mice.

In both the 344SQ-anti-PD-1 sensitive and 344SQ-anti-PD-1 resistant lung cancer models, we found that the combination of anti-PD1 and radiotherapy-induced robust immune responses, which resulted in significantly better control of both the local tumor (implanted on the right leg of the mice) and the metastasis (implanted on the left leg of the mice) than either immunotherapy or radiotherapy alone.

In conclusion, R3 considerably enhances the the ability of the combination of radiotherapy and immunotherapy to control both the local tumor and the metastasis in both the 344SQ-anti-PD-1 sensitive and 344SQ-anti-PD-1 resistant lung cancer models.

Therapeutic and immunogenic effects of radiation therapy (RT) are often constrained by tolerogenic activity of the tumor microenvironment.

The STAT3 activity in immune populations infiltrating HNSCC tumors was analyzed in patients' derived fixed or frozen fine-needle biopsies.

Using the multicolor immunofluorescent analysis, we found a significant increase in the percentage of CD163+ M2 macrophages with activated STAT3 and a concurrent reduction in a number of CD8+ T-cells in tumors post-treatment.

Our clinical observations and animal study results suggest that myeloid cell-targeted inhibition of STAT3 together with CpG-mediated immunostimulation dramatically augment the outcome of radiation therapy against HPV-positive and HPV-negative HNSCC.

The clinical protocol including the relevant informed consent form was approved by the institutional review board at City of Hope (IRB-14255) and the study was conducted in accordance with the amended Declaration of Helsinki and the International Conference on Harmonization Guidelines.

Despite the success of immune checkpoint blockade in treating many types of cancers, there remain many that are insensitive to these treatments.

We orthotopically implanted 4T1 mammary carcinoma cells into BALB/c mice and treated with anti-mCTLA4 antibody, focal radiation, or both.

The 4T1 tumor model is typically unresponsive to immune checkpoint inhibition.

Taken together, these data suggest that synergistic treatment with anti-mCTLA4 and focal radiation can induce the tumor immune microenvironment to change to a more inflammatory, and less suppressive, state than either treatment alone.

Radiation therapy (RT) to enhances the efficacy of immune checkpoint inhibition (ICI) in multiple immunologically “cold” preclinical tumor models[1,2].

Mice were engrafted with B78 melanoma tumors, 80-90 mm3, and were randomized to the following treatment groups: vehicle only (VO), anti-CTLA4 (C4), 90Y-NM600 TRT (2.5 Gy tumor dose), external beam RT (EBRT, 2.5 or 12 Gy), or combination TRT+C4.

Comparison of RT dose effects demonstrated increased ratios of CD8+ T cells to CD4+FoxP3+ regulatory T cells in the TRT group, compared to the EBRT groups at Day 1, as well as increased myeloid cell (CD11b+) and NK cell infiltrate with TRT.

Our results demonstrate a cooperative therapeutic interaction between TRT+ICI in a murine tumor model that is functionally immunologically cold and does not respond to ICI alone.

Research material support for this work was provided by Bristol Myers Squibb.

The study was approved by the University of Wisconsin institutional animal care and use committee protocol 005670.

Combination of radiation with immuno-therapy had shown promising results in preclinical experiments as well as in clinical trials.

The meta-analysis was performed using the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guideline.

In total, information on OS and LC was reported in, respectively, 26 and 6 studies, with 50 and 13 arms, featuring 2528 and 525 patients.

Higher 1-year was observed in ICI-RT treatments vs mono-RT, with corresponding incidence rates were 59% [95% CI: 54-64%] vs 32% [95% CI: 25-40%] (P Concurrent treatment was associated with higher 1-year OS comparing to sequential regimen with incidence rates 69% [95% CI: 60-78%] vs 52% [95% CI: 45-58%] (P=0.039);

RT and ICI combinations were associated with improved 1-year OS vs mono-RT.

Hormone receptor+ (HR+) breast cancer (BC) is the most frequent cause of BC death [1], and immunotherapy with immune checkpoint blockers (ICBs) in HR+ BC patients has been disappointing [2].

The synergy between RT and palbociclib (a highly selective CDK4/CDK6 inhibitor approved for the treatment of BC in humans) was tested: (1) in vitro, in 2 human BC cell lines with differential sensitivity to palbociclib and RT (MCF7 and MBA-MD-231 cells), to evaluate the effects on cell cycle distribution, cell death, cell senescence and IFN secretion;

We identified the lowest doses of RT and palbociclib that mediate robust short-term cytostatic/cytotoxic effects in MDA-MB-231 and MCF7 cells. Optimized dose combinations and sequencing experiments pointed to RT followed by palbociclib as the approach with superior therapeutic potential.

Our preliminary results suggest that RT should precede CDK4/6 inhibition to achieve superior disease control.

Prostate cancer responds poorly to checkpoint blockade due to its immunologically “cold” microenvironment with low CD8+ T cell infiltration along with relatively high numbers of myeloid-derived suppressor cells (MDSCs) and regulatory CD4+ T cells (Tregs) [1–3].

6-week old male FVB mice (n=3 per group) were implanted with subcutaneous (s.c.) MyC-CaP murine prostate tumors then given a single intravenous injection of either 50 (3.1 Gy) or 250 (15.5 Gy) μCi of 90Y-NM600.

CD8+ T cell tumor infiltration increased by 13% at Day 14 compared to Day 0 (p=.039) following MTRT administration in both dose groups while GR-1+ cells (including MDSCs) transiently decreased by 31% at Day 4 (p=.007).

Our data indicates that MTRT disrupts the suppressive tumor microenvironment of prostate tumors by depleting suppressive populations and increasing CD8+ T cell infiltration.

Focal radiotherapy (RT) in combination with CTLA-4 blockade activates T-cells that reject tumors in both mice and patients unresponsive to CTLA-4 blockade alone [1, 2].

For single cell sequencing (SC-seq) and bulk TCR sequencing (TCR-seq) experiments, mice were inoculated in one or both flanks with 50,000 4T1 cells, respectively.

In tumors treated with RT alone or in combination with anti-CTLA-4, both the TCRα and TCRβ repertoires increased in clonality, whereas a smaller increase in TCRβ clonality was found after anti-CTLA-4 monotherapy.

Overall, these results suggest that RT promotes the differentiation of the intratumoral TCR repertoire but in the absence of CTLA-4 blockade it does not drive the expansion and functional differentiation of T cell phenotypes necessary for immune-mediated tumor rejection.

Radiation therapy (RT) used at immunogenic doses (8GyX3) leads to cytosolic DNA accumulation which activates interferon type I (IFN-I) production via cGAS/STING pathway in cancer cells [1-3].

TEX were isolated 48 hours after last dose of radiation or mock treatment using ultracentrifugation.

Genomic and mitochondrial DNA were identified in both TEX and TSA cytosolic fraction regardless of RT.

Our data suggest that the immunostimulatory DNA cargo of RT-TEX has unique features that reflect molecular changes occurring in radiation-treated cells.

TLR7 agonists are potent immune activators that are highly attractive for anti-cancer immunotherapy with potential to act synergistically with other cancer treatments such as vaccines, radiotherapy, and chemotherapy.

Biophysical properties of the nanoparticles were characterized and anti-tumor efficacy was investigated in three murine syngeneic subcutaneous cancer models (CT26, MC38, and B16-F10).

Nanoparticles containing 1V270 displayed a synergistic effect with local radiotherapy and all (8/8) mice carrying CT26 tumors displayed complete tumor rejection.

The novel immunotherapeutic nanoparticle formulation induced a potent anti-tumor immune response;

Immune checkpoint inhibition targeting PD-1/PD-L1 has shown promise in breast cancer but is largely limited to triple negative breast cancer (TNBC).[1] This limitation is primarily due to inherently low levels of tumour infiltrating lymphocytes (TILs), particularly in HR+ disease.[2] Focal radiation therapy (RT) has been shown to generate anti-tumour T cells and increase TILs using mouse models of breast cancer (BC).[3,4] Mechanistically, radiation-induced increase in cytosolic DNA leads to activation of cGAS/STING pathway and production of IFN-훃, which is essential for priming of anti-tumour CD8+ T cells.[5] This process is under the control of TREX1, and dependent on RT dose and fractionation.[5] Inter-tumor variability in optimal RT dose for activating the IFN-I pathway and heterogeneity in immunological response amongst BC subtypes highlight the importance of precision use of RT.[6] We are testing the hypothesis that improved in vitro and in vivo assays allow testing of the pro-immunogenic response to radiation in individual BC patients.

Spheroids were established from human adenocarcinoma cell lines MCF-7 (ER+,PR+,HER2-) and MDA-MB-231 (TNBC) by seeding 1,000 cells in a mixture of 2% Matrigel in DMEM.

Spheroids and organoids were successfully established using human cell lines and PDTX, respectively (Figure 1).

Developing a platform that utilizes pre-treatment tumor tissue from a patient to determine the functional response to different RT doses and aid in the selection of the best combination treatment with anti-PD-1/PD-L1 is a critical step towards precision use of RT to enhance responses to immunotherapy.

The authors which to acknowledge financial support from the Breast Cancer Research Foundation (award BCRF-18-053).

Polarizing the tumor microenvironment (TME) towards an inflamed and immunosupportive type is highly attractive for anti-cancer therapies.

The Immunogel was formulated to provide sustained release of a TLR7/8 agonist (NT03-gel) and combined sustained release of a TLR7/8 agonist and TGFβ-inhibitor (NT04C-gel).

The NT03-gel demonstrated a sustained intratumoral drug release of 31% after 24 hours and 81% after 7 days.

By enabling intratumoral sustained drug release, the Immunogel has the ability to polarize the TME towards an inflamed immunosupportive state, which is key for improving anti-cancer immunotherapies.

All experimental procedures involving animals were approved by The Danish Animal Experiments Inspectorate.

To date, pembrolizumab plus axitinib (P+A) and nivolumab plus ipilimumab (N+I) are the only immunotherapy combination therapies demonstrating superior overall survival (OS) compared to sunitinib in Phase III clinical trials in subjects diagnosed with advanced/metastatic renal cell carcinoma (mRCC).

An anchored matching-adjusted indirect comparison (MAIC) was conducted using individual patient-level data (IPD) from KEYNOTE-426 and pseudo-IPD (digitized KM curves) in the published data from CHECKMATE-214.

After matching, P+A resulted in a lower hazard of disease progression or death than N+I (HR=0.81;

Following the adjustment of cross-trial differences, P+A demonstrated lower hazards in the PFS and OS compared to N+I in both the ITT, and intermediate and poor risk populations.

Talimogene laherparepvec (T-VEC) is an oncolytic HSV-1 approved for the treatment of melanoma.

Immunocompetent C57/BL6 mice bearing D4M3A melanoma tumors were treated with an adapted T-VEC encoding murine GM-CSF (mT-VEC;106 PFU) given by intra-tumoral administration biweekly with or without MEK inhibitor trametinib (0.1 mg/kg) by oral gavage for 2 weeks.

In an immunocompetent murine HSV-1-sensitive D4M3A melanoma model we observed a significant decrease in tumor growth and increased survival following the addition of trametinib to T-VEC therapy.

Oncolytic virus immunotherapy is improved by MEK inhibition and appears to sensitize tumors to immune checkpoint blockade.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO2) are key immunoregulatory enzymes that catabolize the essential amino acid tryptophan (Trp) to kynurenine (Kyn), an immunosuppressive metabolite.

One-way, mixed lymphocyte reaction (MLR) assays were performed on human peripheral blood mononuclear cells and mature dendritic cells treated with different concentrations of M4112 in combination with avelumab, a fully human anti–PD-L1 IgG1 antibody, to measure interferon-gamma (IFN-gamma) levels [1].

In combination with avelumab, M4112 treatment increased IFN-gamma production, indicating enhanced T-cell activation.

The dual-selective IDO1/TDO2 inhibitor, M4112, in combination with avelumab enhanced antitumor activity in mouse and human tumor models and normalized systemic Kyn/Trp levels.

Glioblastomas are among the most lethal of cancers [1], and antibodies against the programmed cell death protein 1 (anti-PD-1) represent a promising immunotherapeutic strategy.

We evaluated whether SGT-53 could augment anti-PD-1 therapy for murine glioblastomas (GL261) grown subcutaneously or intracranially in syngeneic mice.

Adding SGT-53 to anti-PD-1 immunotherapy resulted in increased intratumoral infiltration of immune effector cells and altered expression of a number of genes related to immune responses.

Tumor-targeted p53 gene therapy via SGT-53 was able to augment anti-PD-1 immune checkpoint blockade and convert mouse syngeneic glioblastoma tumors that were unresponsive (immunologically “cold” tumors) into tumors that were more responsive to anti-PD-1 immunotherapy.

All animal studies was performed under an IACUC-approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals in the AAALAC-accredited animal facility at Georgetown University, which adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council .

Around 10% of breast cancer cases are attributed to genetic disorders like mutations in BRCA-1/2 genes.

To study the effect of this common mutation on tumor biology in general and specifically the immunological landscape of the tumor microenvironment, we knocked out the BRCA1 gene in the murine EMT6 breast cancer cell line.

The percentage of TILs (=CD45+ cells) was similar in both lines (33.0% vs 29.6%, BRCA1-/- vs BRCA1wt ).

Further studies using microdialysis as read-out will give additional information on the influence of the BRCA1 gene knockout and the resulting modulation of the tumor microenvironment and will help to elucidate the tumor biology and clinical relevance behind these findings.

The study was approved by the local authorities (Regierungspräsidium Freiburg;

Cancer immunotherapy using immune checkpoint inhibitors has significantly improved the clinical outcomes of patients with multiple aggressive and advanced-stage cancers.

We tested the combination of anti-PD-1 with different dose of VEGFR2-targeting antibody in syngeneic breast cancer mouse models.

Antibody targeting VEGFR2 sensitizes breast tumors to PD-1 blockade in a dose-dependent manner.

Taken together, these results demonstrate that a dose-dependent synergism exists between anti-angiogenic therapy and immune checkpoint blockade, thus providing important insights into identifying the optimal strategies to combine immunotherapy with molecular targeted agents.

All patient-related procedures were performed with the approval of the Internal Review and the Ethics Boards of the Sun Yat-sen Memorial Hospital.

The treatment landscape has drastically changed in mRCC, moving from anti-VEGF therapies to an immunotherapeutic approach in the first line setting for IMDC intermediate or poor risk mRCC.

Eighty-six patients were treated with ipi/nivo for mRCC and of these patients, 34 patients also received cabozantinib, either alone or in combination with nivolumab.

Progression on immunotherapy alone did not appear to confer resistance to cabo/nivo treatment for five of the six patients who had a disease response assessment.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, accounting for 338,000 new cases worldwide with a 5-year survival rate of less than 10%.

Using a combination of humanized anti-MUC1.IgE, anti-PD-L1, and PolyICLC, we investigated the efficacy of this unique triple combination in a pre-clinical model of pancreatic cancer using transgenic mice expressing human MUC1 and FcεRI (hMUC1/hFcεRI).

We noted that combined immunotherapy with a humanized IgE directed towards a tumor antigen expressed on pancreatic adenocarcinoma (MUC1), PolyICLC, and anti-PD-L1 induces antigen-specific cellular immune responses that mediate rejection of tumor cells in a pre-clinical model of pancreatic cancer.

Taken together, this is the first study to show that specific stimulation of the IgE/FcεRIα axis in combination with PolyICLC and anti-PD-L1 can activate CD8 T cell and NK cell effector pathways and provide long-lasting tumor-protective benefits against pancreatic cancer.

All animal studies were performed in accordance with the Institutional Animal Care and Use Committee guidelines (Approved IACUC protocol no: 15-123-02FC).

The role of B cells in tumor immunity is poorly understood due to their conflicting roles that augment and suppress anti-tumor responses.

We use two different clones of anti-PD-L1 antibody, 10F9G2 and MIH5, which are rat IgG2b and rat IgG2a, respectively, in conjunction with a mouse anti-mouse 4-1BB antibody.

In vivo and in vitro pharmacokinetic studies revealed that MIH5 clone had better bioavailability than the 10F9G2 clone which is attributed to its higher binding affinity to FcRn.

Our results reveal an underappreciated role of B cells in T-cell targeted cancer immunotherapy.

All experimental animal procedures complied with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, 2011) and were approved by the Pfizer Global Research and Development Institutional Animal Care and Use Committee (IACUC)

Cytotoxicity T cells play central roles in destroying cancer cells in cancer immunotherapy, which is similar to immunological rejection [1].

We conjugated ovalbumin (OVA), a model foreign antigen, to biocompatible hyaluronic acid (HA) via simple reductive amination method.

The OVA contents in the polymeric conjugate were 13.2 wt.% determined by BCA assay.

In this study, we synthesized model foreign antigen-bearing polymeric conjugate.

Although immune checkpoint blockade (ICB) may result in clinical benefit for a subset of cancer patients, multiple mechanisms of resistance can impair optimal response.

Here by using pre-clinical models of melanoma and analysis of clinical samples we sought to characterize the mechanisms of immune suppression associated with the AHR pathway and to evaluate its potential as therapeutic target.

RNAseq analysis of human cancers revealed an up-regulation of Kyn-degrading enzymes in patients responsive to PD-1 blockade.

In summary, our findings demonstrate that targeting the Kyn pathway through AHR inhibition could overcome key suppressive mechanisms and sensitize otherwise immune resistant tumors to PD-1 blockade.

All mice were maintained in microisolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations.

An in-depth understanding of immune escape mechanisms in cancer are likely to lead to innovative advances in immunotherapeutic strategies.

Several pre-clinical syngeneic and transgenic tumor models, an autologous humanized mouse tumor model, and clinical plasma and tumor specimens derived from advanced melanoma patients were utilized to investigate the genetic and cellular alterations of tumors escaping anti-PD-1 antibody immunotherapy.

Using several pre-clinical tumor models, we have determined that granulocytic myeloid-derived suppressor cells (PMN-MDSCs) accumulate within progressing tumors undergoing treatment with anti-PD-1 antibody (ab) immunotherapy.

This work reveals a tumor-intrinsic PD-L1-NLRP3 inflammasome signaling pathway triggered by CD8+ T cell activation that ultimately drives adaptive resistance to anti-PD-1 immunotherapy by promoting the recruitment of PMN-MDSCs to the tumor bed.

This study utilized clinical specimens derived from ongoing tissue acquisition protocols approved by the Institutional Review Boards at Duke University Medical Center (IRB approval number Pro00059349) and Vanderbilt University Medical Center (IRB approval number 100178).

Associations between modifiable factors (e.g., adiposity) and immunotherapeutic efficacy remain uncertain.

BALB/c mice were administered a high-fat diet for 20-weeks.

Both OBR and DIO therapy-treated mice had significant reductions in tumor weight compared to therapy-free controls (66% reduction for OBR;

When controlling for diet, increased adiposity reduced the response rate to combinatorial anti-CTLA-4 based therapy.

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham, approval number 20233.

The cornerstone of the existing treatment of high-grade serous ovarian cancer (HGSOC) is DNA-damaging chemotherapy;

We hypothesize that the immune composition and gene expression signatures of the resulting tumors will vary based on the combination of genetic alterations and the DNA repair proficiency of the transformed cells.

To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP-inhibitor and DNA-damaging-agent sensitivity.

These results reveal how common mutational drivers determine the microenvironment of the tumor and its response to treatment.

Characterization of heme malignancies, particularly in response to therapies, requires detailed monitoring and analysis of changes to patient immune subsets.

ASAP was built on several existing R packages (flowCore, flowPeaks, ggcyto, uwot) and makes use of marker enrichment modeling (MEM) scores for cell subset classification [2].

Results produced by ASAP are highly concordant with those produced by expert manual gating, r2=0.98 (Figure 1).

ASAP enables the automated analysis of flow cytometry data from large studies with precision, accuracy, and reproducibility comparable to the gold standard of expert manual gating.

Orbital tumour board is a multidisciplinary approach towards ophthalmic cancer management, where relevant experts collaborate to manage patients holistically.

Out of a total of 80 patients in the study, 40%(n=32)were female and 60%(n=48) were male.

Nivolumab is a fully human immunoglobulin-G4 (IgG4) monoclonal antibody that selectively inhibits PD-1 activity, and can have various side effects.

Skin biopsies were obtained, revealing interface dermatitis with focal epidermal pallor and necrosis, along with occasional eosinophils.

Our case illustrates the development of DILE with SCLE morphology in a patient without previous history of autoimmune disease with nivolumab.

Consent was obtained from the patient for publication of this abstract.

Retinoblastoma is the most common intraocular malignancy of childhood, accounting for 3 - 4% of all pediatric neoplasms.

Retrospective review of retinoblastoma cases from May 2016 to April 2018 at Aga Khan University Hospital.

At our center we see Retinoblastoma in children with diverse background and majority presented with advanced disease.

The evolving melanoma treatment algorithm has highlighted the role of immune checkpoint inhibitors in treating patients with this cancer.

An online continuing medical education (CME)-certified interactive, text-based activity developed by Medscape Education Oncology and the Society for Immunotherapy of Cancer included 2 patient cases, which served as the foundation for interactive questions.

Participation in education resulted in statistically significant improvements and an extensive educational effect for oncologists (n=62;

This online interactive, case-based, CME-certified educational activity resulted in significant gains in oncologist competency in identifying and selecting appropriate treatments for patients with melanoma in the adjuvant and metastatic setting, and in implementing monitoring and management strategies (Figure 4).

While platinum-based chemotherapy doublets were the first-line (1L) standard of care for metastatic non-small cell lung cancer (mNSCLC), the treatment landscape has recently shifted with the approval of immune checkpoint inhibitors.

Oncologists were recruited via a healthcare research panel to complete a cross-sectional online survey.

Oncologists (N=216) with an average of 16 years in practice participated;

This study provides key insights into the trade-offs that oncologists are willing to make between efficacy and toxicities when selecting 1L treatment for patients with mNSCLC.

The authors acknowledge Oliver Will, PhD, and Bernadette Hallissey, PhD, for their assistance with data analysis

This study was reviewed by Pearl Institutional Review Board (Indianapolis, IN, US) and determined to be exempt.

Providing education and support for caregivers of cancer patients improves physical, psychosocial, and quality of life outcomes for both caregivers and patients.[1-4] As the use of immunotherapy continues to expand, it is important that caregivers have access to education and community resources to support their loved ones in making informed decisions, recognizing side effects, and getting optimal care.

One thousand two hundred thirty-two adults attending FSAC: Immunotherapy psychoeducational workshops nationwide from 2014-2018 completed a post-workshop survey.

Of the 282 caregivers, 54% were spouses/partners, 31% family, and 15% friends.

Results suggest that the FSAC: Immunotherapy program increases caregivers’ perceived knowledge about immunotherapy and their perceived confidence in talking to their doctor about immunotherapy.

These workshops and analysis were funded in part by generous support from AstraZeneca, Bristol-Myers Squibb, and Merck.

This study was conducted under IRB-exempt protocols [category 45 CFR 46.101(b) 2].

Microsatellite stable colorectal cancers are known to harbor low mutation burden, a limited immune infiltration of the tumor and thereby respond poorly to immunotherapy.

Here we describe a case of microsatellite stable metastatic colon cancer with an outstanding anti-cancer response.

Analysis of the liver resection by flow cytometry revealed a large number of immune cells infiltrating the tumor, a phenotype distinct from the other colorectal liver metastases (CRLMs) we analyzed to date.

This case shows that even cancers with low tumor mutation burden can lead to strong immune responses overtime and this knowledge should be considered to establish patient’s treatment plans in the future.

All subjects signed written informed consent approved by the Providence Portland Medical Center Institutional Review Board (IRB protocol no.

Estrogen receptor positive (ER+) breast cancer tumors are generally poorly immune infiltrated tumors as compared to triple negative breast cancer (TNBC).

Fresh surgical tumor specimens were obtained from consented patients.

This work is the first to demonstrate a T cell subset beneficial to survival outcomes of ER+ breast cancer patients.

Fresh tissues were obtained from patients who gave institutional review board (IRB)-approved written informed consent prior to inclusion in the study (City of Hope IRB 05091, IRB 07047, and IRB 14346).

Background: CD8 T cells are a critical component of anti-tumor immunity and enhancing their function can promote tumor elimination.

Wild type (WT) or Gal-3-/- OT-I CD8 T cells were adoptively transferred into WT mice and then stimulated with soluble ovalbumin protein along with an agonist anti-OX40 mAb or rIL-2/IL-2 mAb complexes (IL-2c).

In the current study, we demonstrate that Gal-3-deficient CD8 T cells exhibited no defects in early (36 hrs) activation or proliferation.

Together, these data implicate intracellular Gal-3 as a critical mediator of OX40-mediated CD8 T cell survival and memory formation following antigen exposure.

T-cell retention in epithelial tumors is mediated by transmembrane proteins (CD49a, CD49b, CD103) known as retention integrins (RI)[1].

Peripheral blood mononuclear cells isolated from five human donors were stimulated with mouse anti-CD3 and anti-CD28 for 24 hours.

As shown in Table 1, TGFβ upregulated expression of CD49a and CD103 in TCR stimulated (TCR+) CD4+ cells relative to TCR+ controls (p = 0.02,

When comparing the expression of RI by CD4+ cells to our prior findings in CD8+ cells[5], we noted both common themes and significant differences.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that consists of myeloid progenitor cells and immature granulocytes or polymorphonucleares (PMN), immature macrophages (M), and immature dendritic cells (DCs).

TMAs containing 247 patients with stage II/III NSCLCs (adenocarcinomas, ADC=162;

Using the median density of MDSCs we identified overall higher densities of those cells in SCC than ADC, with significant P value of total granulocytic (CD66b+CD11b+, P=0.016) cells, PMN-MDSCs (CD11b+CD66b+CD33+, P=0.021), and M-MDSCs (CD11b+Agr1+CD14+CD33+, P=0.009, Figure 1 and Table 1).

While SCCs showed the highest densities of MDSCs as compared to ADCs, our data suggest MDSCs are related with clinicopathologic characteristics that can increase the risk of tumor progression, such as solid pattern, advanced stage, smoking status and prognosis.

Current FDA-approved immunotherapies aim to reinvigorate CD8+ T cells, but the contribution of the humoral arm of the immune response in human cancer remains poorly understood [1,2].

We utilized a multi-level approach to categorize B cells in HNSCC patients.

We demonstrated distinct trajectories for B cells in HPV+ and HPV- disease.

Characterization of B cell phenotype and function in HNSCC is important for devising new therapeutic options for patients.

This study was approved by the local Institutional Review Board under protocol UPCI 99-069, and patients provided informed consent.

The interaction between the central nervous system and immune system underscores an intricate neuro-immune network.

We were intrigued by the expression of glutamate receptors (GluRs), particularly ionotropic GluR3 (AMPAR subtype 3, GluA3) and metabotropic GluR1 on T-lymphocytes, which were upregulated following TCR activation.

We found a delayed CD8+T-cell activation exhibited by downregulated CD25, CD69 and CD44 molecules, resulting in impaired proliferation and cytolytic activity against orthotopic mammary tumors expressing a cognate low-avidity antigen in vivo or specific peptide-loaded target cells in vitro.

Data suggest a costimulatory effect of GluRs on T-cell function.

Undifferentiated Pleomorphic Sarcoma (UPS) is the most common STS subtype in older adults.

We aimed to identify tumor infiltrating immune cell subset and tumor specific antigens in UPS using a robust and well-validated multi-parameter flow cytometry platform and determine if there was any correlation between the immune phenotype and other recognized prognostic factors such as tumor size and depth.

All patients undergoing resection of grade 2 or 3 UPS were eligible for inclusion.

From January 1, 2017, to July 31, 2019, 20 resected UPS specimens were collected and successfully immunoprofiled using well-validated multi-parameter flow based cytometry.

We were able to successfully identify the tumor infiltrating immune cell subsets in 20 freshly resected extremity undifferentiated pleomorphic sarcoma specimens.

The study was approved by the UCSF Committee on Human Subject Research, approval number 15-16384

Although immunotherapy with PD-1/PD-L1 antagonists has significantly advanced patient care, the majority of cancer patients currently do not benefit from checkpoint inhibitor therapies.

In vivo genome-wide CRISPR/Cas9 screens were conducted by introducing sgRNA libraries into Cas9-expressing TCR-Tg T cells, followed by adoptive transfer into tumor-bearing hosts.

Our CRISPRomics platform identified clinically active molecules, such as PD-1, and also predicted recent clinical failures.

We describe an Immune-CRISPRomics platform which 1) identified multiple functional targets with therapeutic promise and 2) functionally screened combinations of these targets to reveal the most potent T-cell target combinations for the treatment of PD-1 resistant solid tumors.

In multiple myeloma (MM), marrow infiltrating lymphocytes (MILs) have shown increased anti-tumor reactivity and proliferative capacity compared to their peripheral blood counterparts.

Here, we used multiparametric flow cytometry to perform single-cell analysis on CD8+ T cells from MM patients to identify those subsets that are enriched in the bone marrow (BM) compared to peripheral blood (PB).

The activation and tissue retention marker CD69 identified a population of CD8+ MILs that is virtually absent in PB and displays peculiar phenotypic and functional features compared to its CD69-negative counterpart.

In conclusion, we identified a subset of MILs that appears to display a stem-like/resident phenotype.

The tumor microenvironment (TME) is a complex network, consisting of the tumor, blood vessels, stromal and immune cells, and soluble factors.

The ability of therapeutics to reverse or inhibit T cell exhaustion was performed using primary human cells.

PI enhanced tumour cell killing in a 2D co-culture assay.

We present an immuno-oncology platform to model the TME in human cells which enables a rapid assessment of the immunomodulatory capacity of therapeutics.

The study was approved by HRA NRES South West, Bristol (UK), approval number 15/SW/0029.

Platelets (PLTs) are well-known players during cancer progression.

We investigated the presence of circulating PLT-lymphocytes aggregates in solid and haematological cancers.

Our preliminary results show that, compared to healthy donors, cancer patients have an increased number of PLT-lymphocyte aggregates, specially CD8+ T cells.

These results suggest that binding of PLTs have an impact on T cell activity, which could greatly influence the immune response against tumors.

We have previously demonstrated that high-affinity, leukemia-specific CD8+ T cells are efficiently deleted in leukemia-bearing hosts in a manner that depends on cross-presentation of the antigen by splenic Batf3-lineage dendritic cells [1 ,2].

Here, we generated a T cell receptor transgenic mouse strain (Tg101) that expresses TCR-α and -β chains from a CD8+ T cell clone specific for a native H-2Kb-restricted antigen on C1498 leukemia cells.

In naïve mice, Tg101 thymocytes predominantly develop along the CD8+ lineage, and exist as naïve CD8+ T cells in peripheral lymphoid organs.

Collectively, our results reveal that CD8+ T cell tolerance is effectively generated in leukemia-bearing animals.

The study was approved by The University of Chicago Ethics Board, approval number ACUP 71945.

Regulatory T cells (Tregs) maintain peripheral tolerance;[1] however, in cancer, Tregs dampen anti-tumor immunity, contributing to disease progression.[2,3] Neuropilin-1 (NRP1) is required for intratumoral Treg stability and Treg-specific knockout of NRP1 attenuates tumor growth.[4] Transcriptional analyses revealed that intratumoral NRP1-deficient Foxp3+ Tregs develop an effector phenotype, characterized by interferon-gamma (IFNγ) production and decreased suppression of conventional T cells.[5] Whereas NRP1 is constitutively expressed on mouse Tregs, expression on human Tregs is activation-driven and thus may be modulated by immune processes.

Phenotypic profiling of peripheral blood (PBL) and tumor infiltrating lymphocytes (TILs) from head and neck squamous cell carcinoma (HNSCC), melanoma, non-small cell lung, ovarian, and colorectal cancer patients was conducted by flow cytometry.

NRP1+ Tregs are greatly enriched in cancer patient PBL and TIL across numerous malignancies, and the degree of enrichment negatively correlates with disease-free survival in HNSCC.

NRP1+ Tregs constitute a more suppressive human Treg subset based on their enhanced regulatory phenotype and function.

This study was approved by the local Institutional Review Board under protocol UPCI 99-069, and patients provided informed consent.

We previously demonstrated that patients’ sex is a variable that influences the response to anticancer immunotherapy: in RCTs comparing immunotherapy-containing regimens to standard treatments in several solid tumors, men obtained larger survival benefit than women when treated with anti-CTLA4 or anti-PD-1 drugs as monotherapy, whereas women experienced larger benefit from anti-PD-1 or PD-L1 combined with chemotherapy.[1,2] We hypothesized that such heterogeneity of response is due to differences in the molecular mechanisms that govern anticancer immune-response in men and women.

We analyzed 7 public datasets containing data on genome-wide RNA expression analysis of NSCLC samples.[3-9] We used gene-expression data to estimate the abundance of 64 different cell-types in the microenvironment of each tumor sample, using single sample gene-set enrichment analysis (ssGSEA) according to a previously validated computational algorithm.[10,11] The Enrichment Scores (ES) of the gene-sets specifically associated with each of the 64 different cell-types were first calculated in tumors of men and women and then compared using a multivariable linear model that adjusted for other covariates including patients’ age, stage at diagnosis, tumor-histotype and smoking status.

2590 tumors were analyzed: 1529 tumors (60%) were from men and 1047 (40%) from women.

We found relevant sex-based differences in the cell-type composition of the immune-infiltrate of NSCLC.

The tumor microenvironment (TME) generates high levels of adenosine which binds to A2aR and A2bR receptors on immune cells to inhibit their activity.

CD14+ monocytes were differentiated with IL-4/GM-CSF for 6 days to generate monocyte-derived dendritic cells (moDC).

Using publicly-available gene expression databases, we identified that T cells and other non-myeloid cells predominantly express the A2a adenosine receptor.

Collectively, these studies demonstrate an important role for A2bR in adenosine-mediated immunosuppression and provide a mechanistic rationale for stimulation of anti-tumor immune responses with the dual adenosine receptor antagonist AB928, which is currently undergoing evaluation in several Phase 1/1b trials.

17 HNSCC specimens were processed for scRNAseq with matched peripheral blood leukocytes (PBL) from treatment-naïve HPV+ and HPV- patients.

We identified 30 different TME cell clusters, with 22 clusters formed by immune cells (PBL and TIL) while CD45- non-immune cells formed 8 clusters.

Heterogeneous cell populations of immune, stroma and cancer cells can be found in the TME of HNSCC.

Patients with metastatic osteosarcoma (OS) have 5 year overall survival <25% [1].

Immunohistochemistry (IHC) slides from 66 formalin-fixed paraffin-embedded OS tissue blocks were digitally analyzed.

Digital image analysis of IHC demonstrated significantly higher infiltrating immune infiltrating cells in the pulmonary metastases compared to primary bone tumors, particularly at the tumor-normal lung interface.

Pulmonary metastatic OS lesions are more highly infiltrated with immune cells than primary bone lesions, particularly at the interface.

This study was approved by Johns Hopkins University's Ethics Board, approval number FWA00005752.

We reported that PDL-1 expression on circulating CD8+ T cells was a predictor of bad prognosis and of resistance to CTLA4 blockade in melanoma [1].

We analyzed 132 fresh tumors by flow cytometry: primitive tumor samples of Urothelial Carcinoma (UC;

We found a high variability of tumor infiltrating leukocytes across individuals (from 0% to 90% of CD45+ cells) and across tumor types (median range from 4% for CRC metastasis to 20% for OC and NSCLC primitive tumor samples).

Integrins CD49a and CD49b can mediate retention of lymphocytes in peripheral tissues by binding to collagens type IV and type I, respectively.

To address these hypotheses, CD8 TIL from an implantable breast carcinoma model, BRPKp110, were for expression of CD49a, CD49b and functional markers.

In early stage tumors (d14), T cells were predominantly CD49b single positive (SP) or CD49aCD49b double positive (DP).

Together, our data suggest that in CD49b SP CD8 TIL evolve over time to upregulate CD49a, followed by downregulation of CD49b, as TIL gain an exhausted phenotype.

Anti-tumoral response of Vg9Vd2 T cells requires sensing of phosphoantigens accumulated in malignant cells through binding of butyrophilin 3A(BTN3A).

CRISPR-Cas9-mediated inactivation of BTN2A1/2A2 isoforms was performed in Daudi, K562 and HEK-293T cells.

Degranulation and intracellular IFNg/TNFa (n=6) were abolished in Vg9Vd2 T cells co-cultured with BTN2AKO Daudi, K562 and HEK-293T cells compared to wild-type, in all conditions tested including anti-BTN3 20.1.

Here, we show that BTN2A is mandatory for BTN3A-mediated function in human Vg9Vd2 T cells.

Immune cell infiltration and exhaustion has been a key question within the field of immunotherapy research.

Two sets of 5 serial sections from four different tissue sets (one human tonsil and three different NSCLCs) were stained with the UltiMapper I/O T-reg panel (CD4, CD8, FoxP3, pan-Cytokeratin) on the Leica Biosystems BOND RX autostainer.

Detection of single-marker and multi-marker phenotypes were identified.

The results presented here indicate that the UltiMapper I/O T-reg panel offers robust and reproducible multiplex immunofluorescence data for the detection and characterization of immune cell infiltration and T-cell regulation.

CD8 T cells are a critical part of the immune response to tumors.

To understand how tumor-specific CD8 T cells respond to prostate cancer we have made a prostate cancer model which expresses the viral LCMV glycoprotein (GP) which acts a tumor-specific antigen.

We have data showing that to have a highly infiltrated there must be a population of stem-like CD8 T cells which are capable to differentiating into effector CD8 T cells and supporting the CD8 T cell response against the tumor.

Tumor-specific CD8 T cells do not acquire an effector program after activation and instead gain an undifferentiated but activated phenotype.

DNA vaccines, relative to other vaccine strategies, are an attractive approach for cancer treatment given their safety, easy manipulation, scalability, stability and economical manufacturing.

We used splenic B cells from wild type C57/Bl6 mice, DCs from Flt3L-treated splenocytes from C57/Bl6 mice, CD8 T-cells from OT-1 mice carrying the T-cell receptor specific for the SIINFEKL peptide epitope, and DNA encoding ovalbumin.

We found that B cells are the only APC subtype that could take up DNA and transcribe it to mRNA, but they could not present the antigen themselves to CD8+ T cells.

In our study, seventy-five patients are recruited for a period of 4 years (2013-2017).

In the inflammation group, activation of NC-NFκB proteins found in 82% and 64% of cases while the loss of BAP-1 was observed in 82% of cases.

Our study showed that in an inflammation group loss of BAP-1 showed the synergistic role with the activation of NC-NFκB proteins and are the poor prognostic indicator of overall and metastasis-free survival.

The study was approved by the institutional ethics committee, All India Institute of Medical Sciences (IESC/T-417/2015).

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.

Myeloid-derived suppressor cells (MDSCs) are developed to protect the host, and in the context of immuno-oncology these cells protect tumor cells through suppression of immune cell activity.

Detection of co-expression of multiple markers in the panel and range of expression for HLA-DR was identified.

Given the robustness and high reproducibility of the UltiMapper I/O MDSC panel, the identification and assessment of MDSC cell types in tissue is reliably attainable.

Blockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors;

Texh and were isolated from B16 melanoma by expression of inhibitory receptors (IR) and assayed in tandem in microsuppression assay with progenitor exhausted and CD4+FoxP3+ regulatory T cells.

When sorted directly from tumor, terminally exhausted (PD-1hiTim3+) Texh, but not PD-1 intermediate, progenitor exhausted CD8+ T cells, induce marked suppression of T cell effector responses, comparable to suppression from Foxp3+ Treg cells sorted from the same environment.

Our data support a model that as CTL progress to terminal exhaustion, hypoxic exposure upregulates CD39, providing Texh a mechanism to suppress proinflammatory processes.

Apart from anogenital cancers, high-risk human papillomavirus type 16 (HPV16) can induce oropharyngeal squamous cell carcinomas (OPSCC).

HPV16 status was determined by GP5+/6+ PCR and p16ink4a staining.

Intratumoral HPV16-specific T cells were detected in 5/9 HPV16+ and in 0/4 HPV16- OPSCC.

HPV16+ OPSCC patients respond better to standard therapy due to infiltration of highly activated and functional HPV-specific effector memory T cells that express CD161 and/or Tbet into the tumor.

This study was financially supported by grants from the Dutch Cancer Society (2014–6696 and 2016–10726).

Human T cells represent a highly heterogenous population that can recognize a wide variety of antigens through their T cell receptor.

On the Lightning, BLI’s proprietary light technology was used to load IFNɣ capture beads into NanoPens with volumes less than 1 nL.

Linking complex cellular phenotypes to genotype can be applied to a diverse range of cell interactions and applications including CAR construct screening and validation as well as TCR and antigen discovery and validation.

Colorectal cancers (CRCs) have very low response rates to current immunotherapeutic approaches with the exception of the microsatellite instability high (MSI-H) phenotype.

The biological connectivity of 6 known ICPs with IFNγ and its downstream genes was examined by NGS in 79 stringently procured and processed CRC and their normal, patient matched intestinal epithelium pairs.

In our study, the 6 “classical” ICPs were statistically upregulated and correlated with IFNγ and 183 additional immune related genes in IFNγ positive (FPKM>1) tumors (Figure 1).

The expression of higher abundance and novel ICPRG genes, including IFI30, GBP1 and GBP4 requires further evaluation of protein expression levels in tumors because these genes have the potential to be druggable targets for immunotherapy of CRC as well as other tumors.

In mammals, NO is generated by nitric oxide synthases (NOS 1, 2, 3).

300,000 Renca cells per well were plated in 6 well plates in RPMI media containing 1,000;

Here, we report that decreased availability of L-Arg in Renca cells blocked the expression of NOS2 protein and NO-induction after stimulation with IFNgamma.

The lack of IFNgamma-induced responses could likely be due to the tumor’s ability to develop strategies of tumor escape and define a distinct mechanism by which L-Arg can regulate the activity of its associated NOS2 enzyme.

The requisites for translation in T cells are poorly understood and how translation shapes the anti-tumor efficacy of T cells is unknown.

We used multiple innovative tools to dissect how translation shapes T cell lineages and impacts tumor growth.

Here we demonstrate that memory T cells are primed by metabolic energy sensor AMP-activated protein kinase (AMPK) to undergo diminished translation relative to effector T cells.

Study of modulation of translation for applications in cancer immunotherapy revealed that translation is suppressed in CD8 TILs in multiple human tumor types, and that direct ex vivo pharmacological inhibition of the translation elongation step primes powerful T cell anti-tumor immunity by remodeling metabolic properties of T cells.

Our work elucidates the new finding that paths to translation shape CD8 T cell anti-tumor capability.

The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune suppressive environment.

Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function.

Furthermore, GCN2 inhibition induced tumor specific immunity by increased antigen-specific T cell frequency and increased cytokine production.

The GCN2/eIF2α pathway is activated in immune cells during amino acid deprivation, and this induces a functional suppression of the immune response.

The study was approved by RAPT Therapeutics Ethics Board, approval number FL0002

Regulatory T (Treg) cells are vital for maintaining immune homeostasis and preventing autoimmunity but represent a major barrier to robust cancer immunity as the tumor microenvironment (TME) actively recruits, activates, and promotes their differentiation.

Fluorescent glucose tracers, extracellular flux analysis, isotopic flux analysis, and RNA sequencing were used to determine the metabolic profile of murine lymph node and B16 tumor resident Treg cells.

Here we show that Treg cells, especially tumor derived, do not robustly engage in glycolysis or consume glucose.

Taken together these data support a model in which Treg cells are metabolically flexible to utilize ‘alternative’ metabolites to support their function in nutrient poor environments like that of the tumor.

Animal work was done in accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh (protocol #17071235).

The alteration of epigenetic status by HDACs, regulates chromatin structure and transcription by controlling protein access to DNA through the removal of acetyl groups.

Normal PBMCs isolated from healthy individuals were subjected to HDAC Inhibitors with varying specificities: Panobinostat, Entinostat, Vorinostat and OKI-005 and stimulated with PMA/Ionomycin and protein export was blocked with BFA.

To determine HDACi effects on T-cells, I evaluated cytokine production on bulk CD4 and CD8 T-cells, and observed a decrease in IL-2, consistent with previous literature that HDACi is negatively impacting T-cells overall.

Naïve T-cells comprise a large portion of T-cells from PBMCs, which may explain why previous studies with HDACi show immunosuppressive effects.

Immunotherapy has transformed cancer treatment, providing durable responses for a subset of patients.

Consistent with their anti-proliferative activity, several classes of chemotherapy agents potently inhibited anti-CD3 plus anti-CD28–driven T-cell proliferation.

Many chemotherapeutic agents inhibit T-cell proliferation and IFNγ production, with IC50 values in the range of concentrations achieved at therapeutic exposures.

The protocol was approved by site institutional review boards or independent ethics committees and conducted according to Good Clinical Practice guidelines, per the International Conference on Harmonisation.

The classification of tumor microenvironments based on the presence of tumor infiltrating lymphocytes (TILs) and programmed cell death ligand-1 (PD-L1) expression has been used to predict the efficacy of immune checkpoint blockade in several cancer types.

CD8+ TIL density and tumor PD-L1 expression were assessed by immunohistochemistry using pretreatment biopsies from one-hundred and eighty nine consecutive patients with locally advanced rectal cancer who had preoperative chemoradiotherapy (CRT).

High CD8 TILs showed borderline significant association with better disease-free survival (DFS).

The study was approved by Seoul National University Bundang Hospital's Ethics Board, approval number B-1707-406-306.

Prostate cancer (PCa) shows a modest clinical benefit from immunotherapy.

We evaluated RNA-seq data from a 190 patient cohort comprised of benign prostate (n=29), localized prostate adenocarcinoma (PCa;

Immune cluster analysis revealed a predominantly T-cell depleted immune status across prostate subtypes, where in all de novo NEPC cases belonged exclusively to the immune depleted cluster.

NEPC is characterized by a T-cell depleted tumor microenvironment similar to other metastatic prostate cancer subtypes but with higher PDL1 expression, which is comparable to SCLC.

Network analysis in biology has led to discovery of molecular and genetic interactions, biomarkers of disease, and therapeutic targets.

This gene set consists of 4 major pathways observed in immune rejection of cancerous tissue, th1 signaling, chemokine production, effector function and immune regulation Higher expression of these genes is associated with tumor infiltrating lymphocytes and an active immune engagement, often correlated with prolonged survival.

Consensus clustering of tumor samples based on ICR gene expression divides them into three classes, ICR high, medium and low.

Our aim is to identify key transcription factors (TF) i.e.

Downstream analysis of the master regulators specific to the ICR Low phenotype resulted in identification of the NOTCH signaling pathway, transcriptional regulation of oncogene TP53 and several other cancer related signaling pathways that need to be investigated further to understand mechanisms of poor immune response (ICR Low).

Cryoablation is one of treatment modalities for kidney cancer and is expected to induce strong local and systemic T cell-mediated immune reactions.

In this study, we collected tumor tissues and blood samples from 22 kidney cancer patients, before cryoablation and at 3 months after cryoablation.

TCRB repertoire analysis revealed expansion of certain T cell clones in tumor tissues by cryoablation.

Our findings revealed that cryoablation could induce strong immune reactions in tumors with oligoclonal expansion of anti-tumor T cells, which circulate systemically and induce abscopal effect in distant tumor.

In order to develop therapeutics which drive the immune system to target tumour cells and eliminate tumour growth, sophisticated in vitro and in vivo models are required.

To determine the effect of checkpoint inhibitors, mice bearing a defined population of ovalbumin (OVA)-specific T cells were challenged either with antigen (PD model), or an OVA expressing tumour (EG7 or MC38).

In the PD model, anti-PD-L1 caused robust upregulation of CD25, IFNγ and granzyme B by antigen-specific CD8+ T cells.

These models allow testing of novel therapeutics alongside benchmark reagents.

The study was approved by HRA NRES South West, Bristol (UK), approval number 15/SW/0029.

Multiple myeloma (MM) is characterized by accumulation of malignant plasma cells in the bone marrow (BM) with an associated immunosuppressive BM microenvironment.

Samples were labeled with PE-PD-L1, APC-CD11b, PE-Cy7-CD33, and FITC-HLA-DR (Invitrogen™ eBioscience) to detect MDSCs using no-lyse no-wash methods.

PD-L1 expression was found dramatically increased after stimulation (n=33 subjects, 94.3%) ranging from 2 to 650 times.

PD-L1 reactivity appears to result from complex interactions that can only be detected with minimal sample perturbation.

All procedures were performed in accordance with the internal protocols of the laboratory, which were authorized by the HGTiP’s Ethical Committee, in accordance with current Spanish legislation, by the Departament de Medi Ambient i Habitatge (file #1899) of the Autonomous Government of Catalonia (Generalitat de Catalunya).

Immunotherapy has proven to be a powerful anti-tumor therapy by harnessing the body’s own immune system to target and kill tumor cells.

In this study, we employed a highly sensitive and specific RNAscope in situ hybridization (ISH) duplex assay to directly visualize the tissue distribution of cells expressing COL4A1, COL11A1, LOXL2, and TGFB1 in relation to tumor infiltrating CTLs in non-small cell lung carcinoma (NSCLC).

We observed COL4A1 expression in both tumor and tumor-associated stromal cells in different samples.

Taken together, these results demonstrate that the RNAscope assay provides a powerful approach to directly examine the interactions between tumor, ECM, and T cell immune infiltration and offers advantages over immunohistochemistry (IHC) for identifying the cellular sources of secreted proteins such as ECM components in the TME.

DPX-Survivac is a novel lipid-based formulation designed to elicit de novo cytotoxic T cell response to survivin-expressing tumours.

Pre-treatment and longitudinally collected on-treatment PBMCs were used to assess survivin-specific T cell response by ex vivo IFN-γ ELISPOT and in vitro MHC multimer staining.

Treatment with DPX-Survivac induced systemic survivin-specific T cell response in nearly all evaluable subjects as assessed by ELISPOT.

RNA-profiling of immune cells within the tumour micro environment of subjects demonstrating clinical response revealed on-treatment overexpression of genes related to T cell activation and cytolysis as well as enrichment of B cell and NK cell specific signatures, suggesting the strong potential of treatment to induce tumor-infiltration and activation of cytotoxic cells.

DPX-Survivac combinational therapy induces robust and sustained survivin-specific responses and promotes T cell infiltration of tumours, without a loss in functionality.

The study was approved by Dalhousie University's research ethics board, approval number 2018-4659

Tumor progression is accompanied by extensive remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness.

Using 3D collagen gels of varying density, we identified that T cells respond to their ECM environment and that T cell proliferation is significantly reduced in a high-density matrix, while cancer cell proliferation is unaffected.

Our study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.

With continued growth in development and approval of biologic drugs and cell therapies, comes a need for more robust and reliable cell-based assays and analysis systems.

These methods are demonstrated with single-cell analysis models compatible with flow cytometry and image-based analysis.

Penetration and potency of immune effector cells can be evaluated using whole-spheroid imaging.

Loss of Interleukin (IL)-15 expression by colon tumors strongly correlates with classification as microsatellite stable, decreased tumor infiltrating T cells, increased metastasis, and poor responses to immunotherapy.

After one round of CRISPR/Cas9 deletion of IL-15, we have generated MC-38 cells that lack one IL-15 allele (i.e IL-15+/- MC-38 cells) and express decreased levels of soluble IL-15 complexes.

Surprisingly, deletion of one IL-15 allele was sufficient to affect tumor growth as IL-15+/- MC-38 tumors grew faster than Wt MC-38 tumors but had similar growth kinetics in vitro.

These results demonstrate that even partially decreasing the levels of IL-15 in the TME negatively impacts the numbers of CD8 T cells and NK cells and promotes differentiation of putative MDSCs.

All animal procedures were conducted in accordance with the animal care and use protocols (00000851-RN02) approved by the IACUC at the UT MD Anderson Cancer Center.

During the last years, significant advancement has been made in the clinical application of cancer immunotherapies.

Mixed lymphocyte reaction assays using both innate cells and lymphoid cells mimic a real physiological T cell response and are widely used for the potency screening of candidate therapeutics.

All these in vitro T cell assays are optimized and fine-tuned for the screening of certain types of molecules.

Early evaluation of the effectiveness of candidate therapeutics and combination therapies can be assessed using mouse models and in vitro bioassays with mouse or human immune cells.

The increasing interest in the tumour microenvironment leads to focus on new bioassays to represent all the players of the cancer immune response.

Macrophages possess important active and regulatory functions in both innate and adaptive immune responses.

Although distinguished classification and in vitro generation and polarization of M1- and M2-like macrophages is challenging, in vitro assays can be a first step to screen the effect of the test molecules on the phenotype and function of the macrophages.

The use of the bioassays contributes to a better understanding of the tumour microenvironment and the steps needed to generate an anti-tumour response by the immune system will help to assess the functional potential of new drugs, design clinical trials and ultimately discover relevant biomarkers.

Transforming growth factor beta (TGFβ) is a multipotent cytokine capable of promoting tumors in part through immunosuppression.

Based on our previous work, we initiated a Phase II trial of a TGFβ type I receptor (TGFβRI or ALK5) inhibitor in combination with neoadjuvant chemoradiation in patients with locally advanced rectal adenocarcinoma (NCT02688712).

No improvement in tumor growth or radiation response was seen in animals with Treg or macrophage specific ALK5 deletion.

These results indicate that TGFβ signaling represses CXCR3-dependent trafficking of CD8+ T cells into tumors and can be reversed to improve tumor immunity alone and in combination with radiation therapy.

This clinical study was approved by Providence Health &

Rituximab is widely used to treat B-cell non-Hodgkin lymphomas (NHL).

Seventy-five patients with B-cell NHL [follicular lymphoma or marginal zone lymphoma] were monitored for immune phenotype and NK cell function in peripheral blood, as well as 54 healthy volunteers.

NK cell degranulation responses were not significantly different between healthy donors or pretreatment samples from NHL patients.

Our results provide evidence that a treatment course of rituximab can potentiate degranulation activity of peripheral blood NK cells under natural cytotoxicity conditions for at least 3 months after the end of therapy.

We thank the FCCC Biosample Repository for coordinating patient consenting and sample acquisition and support from the FCCC Keystone Program, FCCC Boards of Associates, and NIH grants CA083859 (KSC) and CA06927 (FCCC).

The study was approved by the Fox Chase Cancer Center Ethics Board, approval numbers 99-802 and 99-839

Elevated levels of myeloid-derived suppressor cells (MDSCs) have been associated with a blunted response to PD-(L)1 inhibitors in vivo and suppressive myeloid cells have been shown to inhibit T cell responses in vitro.

Recombinant and endogenous ARG1 from human granulocytic lysates were used to determine AB474 potency.

Using sorted immune cells from healthy donor PBMC, we determined that PI3Kγ expression is highest in monocytes, whereas PI3Kδ expression is higher in lymphocytes.

Myeloid cells are associated with blunted responses to checkpoint protein blockade.

The interest in the therapeutic potentials of the innate immune system is growing as we gained more knowledge of role of the innate immune system in fighting cancer.

Using our novel electroporation technology, we have found that the innate immunity cells can all be modified by protein expression plasmid vectors or CRISPR-Cas9 mediated gene editing.

Primary NK cells, primary bone marrow monocytes and peripheral blood monocytes need relatively high voltage for electroporation.

Our capability to modify all of the major types of the innate immunity cells by electroporation is a significant step that could contribute to harnessing the power of the innate immunity system for immunotherapy of cancer.

IRAK-M has an important role in tightly controlling innate immune responsiveness to preserve immune homeostasis by acting as a negative feedback regulator of TLR/IL-1R signaling pathway.

Optimization of IRAK-M degraders have conducted by applying RaPPIDS(TM) which is a proprietary divergent degrader synthetic platform and identified multiple preclinical candidates within a year.

We demonstrated a dose- and time-dependent degradation of IRAK-M protein in THP1 human monocytic leukemia cells with degrader treatment in a proteasome-dependent manner.

In summary, we demonstrated IRAK-M degrader as a novel and promising immuno-modulatory therapeutic modality and the great potential of shifting the balance between tolerance and immunity by releasing immunosuppressive network activity, leading to a more favorable tumor microenvironment to enhance host immunity.

Tumor infiltrating myeloid cells, such as Myeloid Derived Suppressor Cells (MDSCs) and Tumor Associated Macrophages (TAMs), are important mediators of immune evasion in cancer, and are associated with worse overall survival and progression free survival in various types of solid tumors.

In this work, we use various adoptive transfer models to generate bone marrow specific inflammasome knockout mice and utilized a B16 melanoma model to assess changes in tumor growth.

We first performed single cell sequencing of Head and Neck Squamous Cell Carcinoma (HNSCC) tumors, and we found IL-1β expression is restricted to the tumor infiltrating myeloid cell populations.

This work will have broad implications across all solid tumor types and will allow for the understanding of how myeloid-specific production of IL-1β promotes tumor growth.

Recent clinical trials in brain tumor immunotherapy have gathered tremendous attention while at the same time establishing a favourable safety profile and preliminary efficacy.

TAMC can control both Bregs and Tregs via microvesicle production.

Glioma TAMCs utilize microvesicles to remotely promote Tregs and Bregs survival and immunosuppressive functions.

Among the various immune cells recruited to the tumor microenvironment (TME), tumor-associated macrophages (TAMs) are particularly abundant and play a central role at all stages of cancer progression.

Here we assessed the presence, abundance and localization of TAMs subsets within the CRC microenvironment by using the Brightplex® solution, an automated sequential chromogenic multiplex assay.

Briefly, a tissue section was sequentially stained, digitized, unstained and re-stained with antibodies targeting the seven markers.

Deciphering the heterogeneity and plasticity of TAMs populations in CRC could help to identify new reliable prognostic markers to improve patient’s stratification and design more individualized therapeutic approaches.

The SIRPα-CD47 axis represents a myeloid checkpoint in cancer.

Multiple functional assays were performed to ascertain the mechanism of action of AL008 and to benchmark activity against anti-CD47/anti-SIRPα antibodies in the clinic.

AL008 is a non-competitive inhibitor of SIRPα that triggers SIRPα internalization and degradation from the cell surface and simultaneously activates FcγR.

AL008 potentiates the anti-tumor effector functions of macrophages and dendritic cells in vitro, as well as in vivo models.

All in vivo studies were reviewed and approved by Alector Institutional Animal Care and Use Committee (IACUC).

V-set and Immunoglobulin Domain Containing 4 (VSIG4), also known as Complement Receptor of the Immunoglobulin superfamily (CRIg), is a cell surface receptor structurally related to the B7 family of immune regulatory proteins.

Using our new VSIG4 (CRIg) antibody (clone NLA14), we demonstrate that Large Peritoneal Macrophages (LPM) in mouse consist of two distinct subsets, the VSIG4+ LPM and VSIG4- LPM.

VSIG4+ LPM co-express Arginase 1 more frequently than the VSIG4- LPM, particularly in C57BL/6 mice.

Characterization of VSIG4+ LPM might provide an opportunity to better understand the immunoregulatory role of VSIG4 in tumor infiltrating macrophages.

IL-17A is a key pro-inflammatory cytokine indicated in multiple pathologies including tumor progression via suppression of anti-tumor immunity.

Human cutaneous squamous cell carcinoma were collected from patients undergoing surgery to remove skin tumors that were diagnosed as SCCs by a dermatopathologist.

We detect CD45+Lin-(CD3-CD14-CD19-CD2-)IL-17A+ cells in the cutaneous squamous cell carcinomas (cSCCs) by flow cytometry of the cell suspensions prepared from tumor tissues.

Taken together, we found a population of lineage negative IL-17A producing cells present in the cSCCs, which share the “CD45+Lin-” features with innate lymphoid cells.

Resistance to the current generation of immunotherapies is mediated by complex relations between stromal, cancer and immune cells found within the tumor microenvironment (TME).

Single cell RNA sequencing (scRNAseq) was performed from head and neck squamous cell carcinoma (HNSCC) specimens (n=17) with matched blood from treatment-naïve patients.

Major cell type clusters (immune, epithelial, fibroblast and endothelial cells) were identified.

Our data suggest that in the setting of HNSCC, TAMs are the major contributors of ICL in the HNSCC TME.

Recent reports suggest that tissue resident memory cells (TRM) may play an important role in anti-tumor immunity.

Naïve CD8+ T-cells from human peripheral blood were activated in normal cell culture conditions (20% O2) or hypoxia (2% O2) and then cultured in the presence of rhTGF-β1.

We demonstrate that differentiation of human CD8+ T-cells in hypoxia and TGF-β1 in vitro led to the development of a TRM phenotype, characterized by an increase in CD69+CD103+ cells expressing CD49a, CD101, and PD-1 – hallmarks of human TRM (n=7, pRM phenotype cells than the additive effects of either condition alone (n=4, pRM and TILRM gene signatures in CD69+CD103+ cells generated in hypoxia and TGF-β1 (Figure 1).

Our findings identify a previously unreported cue for TRM differentiation and enable a facile means of generating human TRM phenotype cells in vitro with the potential for application to multiple existing Adoptive Cell Therapy (ACT) modalities.

This study was approved by University of Texas MD Anderson Cancer Center’s Institutional Review Board, approval number PA14-0105.

Severe cytokine release syndrome (CRS) results in an increased incidence of infections after T-cell engaging therapies.

We evaluated 78 consecutive adult haploHCT recipients at our center for the development of CRS, graded by Lee et al.

Overall, 81% patients developed infections (63/78) within 180 days.

These findings suggest that the severity of CRS developing after haploHCT using PTCy-platform is associated with an increased frequency of bacterial infections, including BSI and CLABSI, and may lead to increased mortality.

Metastatic melanoma patients successfully treated with combination immunotherapy using checkpoint blockade often display pre-existing antitumor immunity at time of initial treatment.

To generate optimal anti-tumor immune responses in melanoma patients, we previously developed a dendritic cell (DC)-based adenovirus-engineered vaccine encoding three common melanoma-associated antigens: Tyrosinase, MART-1, and MAGE-A6.

Patient (matured and adenovirally-transduced) monocyte-derived DC expressed reduced levels of cell surface ICOSL (a costimulatory molecule) vs.

Reduced levels of ICOSL on patient vaccine DC may limit their ability to prime anti-tumor immune responses in vitro (and in vivo).

This study utilized the UPMC Hillman Cancer Center’s Immunologic Monitoring and Cellular Products Laboratory shared facility, supported in part by award P30 CA047904.

The study was approved by the University of Pittsburgh's Ethics Board, approval number IRB12010416.

Early stage myeloid-derived suppressor cells (eMDSCs) are a newly identified immunosuppressive cell subtype, but less is known about the generation of eMDSCs yet.

We constructed IL-6 knockdown 4T1 mammary tumor-bearing BALB/C models and myeloid lineage specific SOCS3 knockout C57BL/6 model.

We identified a subpopulation of CD11b+Gr-1-F4/80-MHCII- eMDSCs in situ in IL-6 knockdown 4T1 mammary tumor-bearing mice models, which displayed more potent suppression on T cell immunity than conventional CD11b+Gr-1+ MDSCs.

Tumor derived IL-6 activated the JAK/STAT3/ miR155-5p pathway in myeloid precursors by inducing SOCS3 deficiency, which consequently downregulated C/EBPβ and PU.1, induced myeloid differentiation arrest, and promoted the development of eMDSCs.

This work was supported by National Natural Science Foundation of China (grant numbers: 81872143, 81702280, 81472473, 81272360) and Key Project of Tianjin Health and Family Planning Commission (16KG126).

ATOR-1017 is a Fcγ-receptor (FcγR) crosslinking dependent agonistic IgG4 antibody targeting the costimulatory receptor 4 1BB.

Human 4-1BB knock-in transgenic mice with established murine colon carcinoma MC38 tumors were used to demonstrate anti-tumor efficacy and immune cell infiltration with FACS after systemic treatment with ATOR-1017.

ATOR-1017 reduced tumor growth and improved survival dose dependently in human 4-1BB knock-in transgenic mice with established tumors.

ATOR-1017 induced a potent tumor-directed immune response, leading to an efficient tumor eradication and survival and development of a long-lasting immunological memory.

The study was approved by Institutional Animal Care and Use Committee (IACUC) of China, approval number 10278L001.

PD-1/PD-L1-targeting monoclonal antibodies (mAbs) have shown impressive responses in a wide range of tumors, although only in a subset of patients, leaving room for improvement.

Surrogate mbsAb-PD-L1x4-1BB induced significant expansion of adoptively transferred OT-1 T-cells upon ovalbumin vaccination in proof-of-concept studies in vivo.

Dual targeting of PD-L1 and 4-1BB with an Fc-silenced bsAb combines immune checkpoint blockade and conditional T-cell co-stimulation in one molecule.

Cynomolgus monkey studies were performed at Charles River Laboratories, Tranent, UK, in accordance with the EU legislations described in Directive 2010/63/EU.

Claudin18.2 (CLDN18.2), a gastric epithelial junction protein, has been discovered as a potential therapeutic target for stomach cancers.

CLDN18.2 specific clones were obtained by Abpro’s proprietary platform [1].

Out of 7 hybridomas, 2 clones, ABP150-1 and ABP150-2 were finalized based on relative binding affinity and specificity to CLDN18.2.

Lead CLDN18.2 clones, ABP150-1 and ABP150-2 as T-cell engagers showed CLDN18.2 specific binding and functionally weak CD3 binding (in the absence of target) resulting in strong TDCC response only in the presence of CLDN18.2 tumors.

Interleukin-12 (IL-12) is a particularly compelling target for cytokine therapy, as it showed promise in preclinical tests.

LbL IL-12 NPs with poly-L-arginine and poly-L-glutamic acid layers (PLE-IL-12-NPs) were able to efficiently load and deliver active cytokine.

Upon TCR stimulation via tumor-specific antigen presentation, CD8+ cells upregulate the IL-10 receptor (IL-10R) and become responsive to this cytokine, leading to localized T effector cell (Teff) proliferation and tumor infiltration along with the release of IFN and cytolytic products.

The Synthorx synthetic biology platform expands the genetic alphabet (EGA) by one base pair that generates new codons for site-specific incorporation of novel amino acids at any position within the polypeptide, which enables the design of biotherapeutics with improved drug properties.

Analytical characterization of purified IL-10 Synthorins showed that they are homogeneous, and dimeric pegylated variants.

We have demonstrated that our synthetic biology platform can generate functional, pegylated Synthorin IL-10 variants that retain the folding and structural stability of native IL-10.

IL-15, distinctly from IL-2, induces proliferation and survival of CD8+ T memory (Tmem) cells, activates natural killer (NK) cells, and enhances antibody-driven cellular toxicity without the expansion of immunosuppressive regulatory CD4+ T cells (Treg).

We applied the Synthorx Expanded Genetic Alphabet platform to the rational design of pegylated IL-15 compounds with extended half-life and differential interaction with the IL-15Rα and IL-2/15Rβ.

THOR-924 is a pegylated IL-15 Synthorin with similar binding affinity to IL-15Rα and IL-2/15Rβ, and comparable signaling potency, relative to native IL-15.

IL-15 Synthorins are biochemically differentiated forms of IL-15 with extended half-life and optimized pharmacologic parameters for balanced engagement of IL-15R vs IL-2/15R inducing proliferation and cytotoxicity of CD8 T and NK cells in vivo without significant activation of immune-suppressive Treg cells or toxicities associated with dosing of native IL-15.

T-SIGn virus vectors for cancer gene therapy are transgene-modified variants of enadenotucirev (EnAd), an Ad11p/Ad3 chimeric group B adenovirus, which retain all the functional and epithelial tumor-selectivity properties of EnAd while also mediating the selective expression of transgenes by infected tumor cells.

T-SIGn virus constructs were generated to express either the p35 and p40 chains of human IL-12 separately or as a linked single chain molecule using a short linker sequence.

ELISA assays measuring levels of the full IL-12 p70 molecule or the specific p40 chain in infected tumor cell supernatants showed that the virus encoding separate IL-12 chains produced p40 very effectively but very little p70 was detected.

In this study, we have shown that a functionally active single chain variant of human IL-12 can be effectively produced by tumor cells infected with a T-SIGn virus encoding this transgene payload.

Interleukin-2 (IL-2) has been the first effective cancer immunotherapy to treat metastatic melanoma and renal cell carcinoma for more than three decades.

To overcome these limitations, we developed a novel immunocytokine that is constituted of a high affinity bivalent blocking anti-PD-1 antibody and a heterodimeric silent Fc-part fused to a monomeric IL-2 variant (IL2v).

IL2v binding to CD25 is abolished, and therefore the binding to Tregs and endothelial cells is reduced.

PD1-IL2v is a differentiated immune cell-targeted IL-2v which promotes an effective and long-term anti-tumor immune-response by delivering IL-2v to PD-1+ tumor-specific T cells

Lymphocyte counts in the peripheral immune system and tumor are correlated with positive clinical outcomes in PD-(L)1 immunotherapy.

PDL1 binding affinity and antagonist function of L1I7 were evaluated by BiaCore and PD1/PDL1 cell-based functional assay, respectively.

L1I7 series of immunocytokine maintained PDL1 binding and antagonist function when compared with PDL1 mAb.

The novel immunocytokine L1I7 is designed to target poor and non-responders to PD-(L)1 treatment and even PD-(L)1 treatment relapsed patients.

Immunotherapy has become a powerful strategy for cancer treatment.

We have developed an ultra-pH sensitive nanoparticle (ONM-400) with similar pH sensitivity to ONM-100, which is being tested in a Phase 2 clinical trial for intraoperative imaging of tumors.

ONM-400 nanoparticles were able to encapsulate IL-2 with high loading.

The ONM-400 nanoparticles can efficiently encapsulate IL-2 and alter its pharmacokinetics in vivo.

All animal experiments were reviewed and approved by, and performed in accordance with, the UT Southwestern Institutional Animal Care and Use Committee under Animal Protocol Number: 2015-101065.

Efforts to modify IL-2 for immuno-oncology applications have focused on alterations that reduce interaction with the Rα subunit of the receptor complex via mutation, chemical modification, or complexation with antibodies or the Rα-ectodomain.

Peptides were selected from recombinant peptide libraries to identify molecules binding simultaneously to the β and γc subunits of the IL-2 receptor.

We report the discovery and optimization of small synthetic peptides, unrelated to IL-2, that simultaneously bind IL-2Rβ and γc subunits to induce IL-2R signaling.

PK-enhanced MK1169 is an attractive alternative to engineered IL-2 variants.

The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)

NKTR-255 is a polymer-conjugated IL-15 pathway agonist that retains binding affinity to the alpha subunit of the IL-15 receptor and exhibits reduced clearance.

NKTR-255 dose dependent enhancement of NK cell cytotoxicity as a single agent or in combination with cetuximab was evaluated in vitro in co-cultures of purified human NK cells with HCT-116 or FaDu tumor cells.

NKTR-255 pre-treatment of NK cells led to enhanced cytotoxic activity against HCT-116 and FaDu cells in vitro.

NKTR-255 treatment of NK cells enhanced ADCC activity against tumor cells in vitro and in vivo efficacy of ADCC inducing antibodies in human solid tumor xenograft models.

All procedures in this study were reviewed and approved by Nektar Therapetuics Institutional Animal Care and Use Committee.

Interleukin-2 (IL2) is known to support effector T cell differentiation, proliferation and survival.

We designed nucleoside-modified, RNA encoded, extended half-life IL2 and IL7, formulated as nanoparticles for intravenous delivery.

Using nanoparticle formulated, nucleoside-modified RNA coding for extended half-life IL2 and IL7, we demonstrated that IL2 and IL7 each increases the number and frequency of antigen-specific CD8+ T cells in combination with an antigen-specific RNA vaccine compared to the vaccine alone.

Based on the complementary mode of action and the promising preclinical data, clinical evaluation will be pursued.

Experimental group sizes were approved by the regulatory authorities for animal welfare after being defined to balance statistical power, feasibility and ethical aspects.

Therapies through systemic injection of recombinant immunomodulatory cytokines have not been successful due to low therapeutic index.

These well-defined pDNA/lPEI nanoparticles, delivered systemically, were well-tolerated in mice and accomplished significant reduction in levels of acute inflammatory cytokines compared to an equivalent CpG-containing plasmid.

Increased survival in both an orthotopic and a metastatic model of lung cancer using clinically relevant nanoparticles raises the possibility of translation to human therapy.

This study is partially supported by a research contract from Cancer Targeting Systems, Inc.

All animal experiments were performed in accordance with protocols approved by Animal Care and Use Committees of the Johns Hopkins University and of the Pennsylvania State University.

IL-15 is a cytokine that activates and provides survival benefit to T and NK cells and has great potential for the treatment of cancer.

For cellular receptor binding, IL-15Ralpha-overexpressing HEK-293 cells were incubated with rhIL-15, NKTR-255 or IL-15 superagonist to measure surface binding of the cytokines by flow cytometry.

NKTR-255 binds to surface IL-15Ralpha on the receptor-overexpressing HEK-293 cells.

NKTR-255 is a novel IL-15Ralpha dependent molecule that provides enhanced PK and PD properties relative to the native IL-15 cytokine while IL-15 superagonist is a direct engager to IL-2Rbeta complex.

Bempegaldesleukin (NKTR-214) is a first-in-class, CD122-preferential interleukin-2 (IL2) pathway agonist being investigated for its potential ability to leverage the clinically validated IL2 pathway and selectively stimulate an immune response, without overactivating the immune system.

To generate a model of bulky unresectable disease, C57BL/6 mice were inoculated with B78 melanoma cells on the right flank and tumors were allowed to grow for ~7 weeks (average tumor size ~800mm3).

In very large B78 tumors (~800mm3), [RT]+[NKTR-214]+[α-CTLA-4] caused greater tumor regression than [RT] +[NTKR-214].

Our preliminary results suggest that [RT]+[NKTR-214]+[α-CTLA-4] has the ability to mount a T cell dependent anti-tumor response capable of regressing large unresectable tumors and disseminated, heterogeneous metastatic disease in murine melanoma models.

Ovarian cancer represents an area of great unmet clinical need, with the 5-year survival rate for high grade ovarian cancer at just 25% for those with advanced disease and with only ~ 10% of patients responding to checkpoint inhibition or other immune therapeutics [1].

PLE-IL-12-NPs offer a simple approach to ameliorate the toxicity of IL-12.

IL-2 therapy has been approved for the use in certain cancers since 1989.

Tumour reactive HLA-A2+ CD4+ and CD8+ T cells were generated by co-culture with growth-arrested A375 cells, supplemented with IL-2 and IL-7.

KY1043 induced T-cell-mediated killing of A375 tumour cells in vitro, with a higher potency compared to non-targeted immunocytokine or anti-PD-L1 alone, and comparable to that of anti-PD-L1 and equimolar amounts of recombinant native IL-2.

KY1043 induces potent T cell activation and can direct highly effective tumour killing in vitro and in vivo.

We thank the Technology and Animal Sciences teams for support of knock-in mice and cell line generation, and the Antibody Discovery team for identification of the parental anti-PD-L1 antibody

Interleukin-2 (IL2) supports the differentiation, proliferation, survival and effector functions of T cells [1–4].

We designed a nucleoside-modified RNA encoded IL2 variant with the goal of an extended half-life, reduced Treg bias and potent stimulation of effector T cells and NK cells.

Characterization of the IL2 variant in vitro validated potent proliferation of CD8+ T cells, NK cells and a reduced Treg bias.

We demonstrate substantial therapeutic activity of a RNA encoded, nanoparticle formulated IL2 variant in monotherapy or in combination with cancer immunotherapy.

Experimental group sizes were approved by the regulatory authorities for animal welfare after being defined to balance statistical power, feasibility and ethical aspects.

Recombinant human interleukin-2 (IL-2) promotes the proliferation of activated T cells and has long been used in vivo to stimulate and maintain the growth of effector T cells and increasingly, to expand T cells in vitro (including genetically engineered CAR-T cells) for adoptive cell therapy in patients with cancer.

CyTOF (cytometry by time-of-flight mass spectrometry): antibody panel designed by Dana Farber Cancer Institute.

The research focused on three major T cell subset populations (Treg, Tcon and CD8 T cells).

The toxicity associated with high dose IL-2 has led many to seek alternatives that can achieve positive therapeutic outcomes while minimizing negative side effects.

Tumor microenvironment (TME) often harbors epigenetic alterations mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs)[1, 2].

In the TC-1 mouse tumor model, entinostat (3mg/kg) was administered with a tumor-specific vaccine (E7-peptide;

We found that entinostat, in presence of optimal CD8 cell priming using tumor specific E7-peptide vaccine, significantly delayed the tumor growth and prolonged the mice survival.

Overall, our study strongly supports the key role of epigenetic factors in regulating CD8+ T-cell effector and/or memory generation, and metabolic fitness.

Tertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation, including tumors in some cases.

Recently, pre-clinical studies have shown that low doses (well below maximum tolerate dose) of antiangiogenic agents normalize the tumor vasculature, leading us to hypothesize that treating tumors with low doses of these vascular normalizing (VN) therapies will improve immune cell infiltration and TLS formation within the tumor microenvironment (TME).

To test this hypothesis, tumor-bearing mice were treated intratumorally with VN agents.

We observed that the VN agents Dasatinib, Aduro, Bevacizumab, and agonist anti-TNFR1 antibody each induced global changes in the TME that are potentially supportive of immune cell infiltration and TLS formation.

VN agents induce global changes in immune cell infiltration and TLS-promoting factors in the TME.

The thorough understanding of the immune homeostatic mechanisms has led to the development of target-based immunotherapies.

In vivo, ITF3756 was as effective as an anti PD-1 antibody and their combination additionally reduced tumor growth.

This study shows in vivo and in vitro mechanisms whereby ITF3756 increases the immune response and constitutes a robust basis for the rational use of selective HDAC6 inhibitors either alone or in combination for the effective induction of antitumor immune response.

All the animal procedures (including housing, health monitoring, restrain, dosing, etc) and ethical revision were performed according to the current Italian legislation (Legislative Decree March 4th, 2014 n.

Lung cancer is the world's most common malignancy (>2 million new cases &

Because RB94 is active against human but not mouse tumor cells, we have utilized human NSCLC cell lines (H292 &

Intravenous injection of scL-RB94 markedly inhibited growth of NSCLC xenografts (Figure 2) by inducing immunogenic cell death (ICD) (Figure 3).

scL-RB94 renders NSCLC tumors more immunologically 'hot' by triggering ICD and enhancing innate immunity via pleiotropic effects on multiple immune-relevant genes (Figure 8, Box A &

All animal studies was performed under an IACUC-approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals in the AAALAC-accredited animal facility at Georgetown University, which adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council .

Acute myeloid leukemia (AML) remains difficult to treat due to mutational heterogeneity and the development of resistance to treatment.

AML murine model Mice expressing FLT3-ITD under the endogenous FLT3 promotor were crossed to mice with the Tet2 gene flanked by LoxP sites and CRE recombinase under control of the LysM promotor.

In the mouse model, trametinib increases T cell viability and restores T cell proliferation.

This work was supported by grant U54CA224019 (E.F.L., S.K.M.) from the National Cancer Institute, grant U41HG003751 (G.W.) from the National Human Genome Research Institute, and by the Oregon Clinical and Translational Research Institute (OCTRI) grant TL1TR002371 (K.B.M.) from the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH).

All mouse experiments were performed in accordance with IACUC protocol #IP00000907.

Regulation of mitochondrial metabolism is crucial for immune cell differentiation and function.

Consistent with the hypothesis, in a syngeneic mouse model, BPM31510 increased cytotoxic T cell frequency and cytotoxic T cell/regulatory T cell ratio in tumors.

Collectively, the results identify and characterize immune-modulatory activity of BPM31510, particularly in the T cell compartment, specifically on the regulation of T cell exhaustion and immunosuppression in supporting an anti-cancer effect.

Localized prostate cancer (PCa) treated with high dose rate brachytherapy (HDRBT) has an excellent cure rate in low, but not in high grade group tumours.

In this study we used ultrasound guided core biopsies collected from 24 men prior to, and 14 days post-HDRBT (10Gy).

Our study showed PCa has a heterogeneous immune context including a pre-existing TIS, which is further increased by HDRBT.

This study was approved by the Peter MacCallum Cancer Centre Human Research Ethics Committee, approval number 14/182

Beta-blockers have been associated with anti-tumorigenic effects, potentially by reducing adrenergic-mediated stress responses.

We retrospectively reviewed the health records of 104 patients who were treated at Northwestern University between January 2014 through August 2018 with immune checkpoint inhibitors for non-small cell lung cancer (NSCLC).

Among 104 patients treated with immune checkpoint inhibitors for NSCLC, 27 of them were concomitantly prescribed beta-blockers.

Beta-blocker use of at least 1 year was associated with improved PFS among patients treated with immune checkpoint inhibitors for NSCLC in our cohort of patients.

In prostate cancer (PCa), androgen receptor (AR) signaling regulates the cell cycle and mitigates replication stress via upregulation of CDC6 and the TopB1-ATR-Chk1 pathway, the key axis in replication stress response (RSR)[1].

We used PCa cell lines derived from ras+myc—induced mouse PCa tumors (RM-1, RM-9 and RM-1-BM) to analyze ATRi (BAY1895344 or VX-970) effects on ATR-driven survival/proliferation, DNA damage, cGAS-STING signaling, and chemokine expression in comparison to olaparib.

Treatment of the PCa cell lines with ATRis (BAY1895344 or VX-970) or a PARP inhibitor (olaparib) demonstrated dose-dependent (0.125-8μM) growth suppression and cytosolic DNA accumulation, while ATRis showed greater growth suppression than olaparib.

ATR inhibition induces S-phase specific DNA damage, accumulation of cytosolic DNA, activation of downstream cGAS-STING signaling, and suppression of cancer growth in immunocompetent PCa models.

We would like to acknowledge members of the Bayer-MD Anderson Cancer Center Alliance, including but not limited to Dr. Antje Wengner, Dr. Dominik Mumberg.

The study, including animal work, was approved by MD Anderson Cancer Center's board, approval number #885 and #889.

Radiation-induced immunostimulation is of great interest as a strategy to reinvigorate or augment an immunotherapy-induced tumor-specific response.

Using the orthotopic 4T1 breast cancer mouse model, we evaluated the immunomodulatory effects of very low (0.4 Gy), low (1 Gy) and high (4 Gy) doses of CiRT (290 MeV/u, SOBP) delivered to mammary tumors at the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan compared to BEDs (RBE 2) of PhRT (SARRP, Xstrahl;

At low doses, CiRT was lymphocyte sparing effect compared to PhRT.

We demonstrate that at low doses, lymphocytes differ in their sensitivity to CiRT compared to PhRT -- CiRT being more lymphocyte-sparing.

The use of immune stimulatory agents as systemic drugs to augment adjuvantation can be problematic due to elevated risks of toxicity and non-specific immune responses.

To determine if systemic administration of 7HP349 in combination with known vaccines would increase adjuvantation, 7HP349 was co-administered with T.

In the CT26 model, 7HP349 monotherapy significantly delayed tumor growth (Table 1).

7HP349 as systemic drug may facilitate immune priming against a variety of antigens by stabilizing αLβ2/ICAM-1 and α4β1/VCAM-1 interactions, with a high safety margin.

An important component of research is ensuring rigor and reproducibility of work using animal models.

We injected C57BL/6 mice with syngeneic B78 melanoma cells in the flank region.

Physical examination demonstrated that ID-implanted tumors were mobile upon palpation, while SQ-implanted tumors became fixed to the underlying fascia.

Intratumoral activation of innate immune signaling pathways is a promising approach to overcome the immunosuppressive tumor microenvironment and induce or restore anti-tumor immunity.

Mice were injected subcutaneously with the syngeneic colon carcinoma cell line CT26 or the B cell lymphoma cell line A20 and established tumors were treated intratumorally with CV8102 alone or in combination with systemic anti-PD-1 antibodies.

Intratumoral administration of CV8102 led to a dose-dependent anti-tumoral response in syngeneic mouse models.

Our results demonstrate that intratumoral CV8102 delivery is a promising approach for local cancer immunotherapy.

4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T and NK cells, by enhancing proliferation, survival, cytolytic activity and IFN-γ production.

ALG.APV-527 was built using the ADAPTIR™ platform with binding domains to 4-1BB and 5T4 generated using the ALLIGATOR-GOLD® human scFv library and subsequently optimized to increase binding affinity, function, stability and manufacturability.

The discovery that the tumor immune microenvironment can be used as biomarker of cancer progression and the advent of immunotherapies are revolutionizing cancer treatments.

Here we investigate a tumor antigen-agnostic intratumoral approach that harnesses preexisting antiviral T cell responses.

Intratumoral injection of mCMV MHC-I and MHC-II restricted peptides led to the expansion of CD8 and CD4 mCMV-specific T cells in situ and in blood.

Our results provide a proof-of-concept for a new generation of tumor antigen-agnostic immunotherapies based on pre-existing antiviral T cells that is potentially applicable to multiple tumor types.

Agonist antibodies that target co-stimulatory receptors, including targets such as CD28, CD40, CD137, and OX40, have demonstrated impressive efficacy in preclinical cancer models.

To contain the activity of agonist antibodies to the tumor microenvironment (TME), we have fused a collagen binding domain (CBD) to the Fc region of several agonist antibodies targeting a suite of co-stimulatory receptors.

Imaging studies revealed that antibody-CBD fusions were retained significantly longer in the TME compared to their non-CBD counterparts and experienced little to no detectable systemic dissemination.

Intratumorally administered antibody-CBD fusions represent a new class of therapeutics with improved therapeutic indices compared to agonist antibodies that have already entered the clinic.

Intra-tumoral immunotherapy has emerged as an important modality of delivering anti-tumor immunomodulators.

To test the capacity of pTNF_EAK in activating antigen presenting cells, BALB/c mice were injected with 100 mcg of the peptide in each footpad.

Preliminary results indicated that pTNF_EAK exhibited self-assembling characteristics based on Congo red staining (data not shown).

pTNF_EAK appears to upregulate the functions of antigen-presenting cells in draining lymph nodes in vivo.

The study was approved by Duquesne University's IACUC, protocol number: 1806-04

Exhaustion and short persistence are factors that limit the efficacy of endogenously generated or adoptively transferred effector T-cells.

Mice bearing TC-1 tumors (a mouse lung epithelial cell-line expressing human papillomavirus-specific E7-peptide) were treated with MEKi (selumetinib) in conjunction with tumor-specific E7-peptide vaccine, followed by determination of tumor growth rates and mice survival, and estimation of immune responses in the TME.

We found that MEKi treatment of tumor-bearing mice in conjugation with tumor-specific E7-vaccine resulted in a significant reduction in tumor growth and enhancement of mice survival.

Our data provide a novel strategy to reprogram CD8 cells into a self-regenerating stem cell phenotype with higher metabolic fitness and superior effector functions.

Talimogene laherparepvec (T-VEC) is an oncolytic herpes simplex virus, type 1 (HSV-1) encoding granulocyte-macrophage colony stimulating factor (GM-CSF) and is approved for the treatment of melanoma.

Human melanoma cell lines were plated in 96-well plates (104 cells per well) and treated with T-VEC (MOI 0.001-1.0).

We initially interrogated multiple human melanoma cell lines and found differential sensitivity to T-VEC-mediated oncolysis.

Intracellular STING was inversely associated with T-VEC-mediated lysis and suggests that STING may be a predictive biomarker for T-VEC responses.

Oncolytic virotherapy is an attractive immunotherapeutic approach as it can induce robust immune infiltration in immunologically cold tumors.

Infection and flow cytometric analyses of T cell populations were performed using both in vitro and in vivo modeling systems.

Analysis of the immune infiltrate of treated tumor-bearing mice revealed that Texh and Treg in the tumor were actively infected by virus.

These data suggest that the mechanism of action of oncolytic vaccinia also includes the direct ability of the virus to infect immune populations in the tumor.

Oncolytic virus (OV) is an emerging class of cancer treatment which has received increasing attention.

The functional outcome of interaction between circulating immune cells, OVV, and cancer cells was evaluated quantitatively and qualitatively using a trans-well co-culture system and analyzed by fluorescence-activated cell sorting.

Time to 90% lethality after intranasal administration of Western Reserve (107 pfu) vaccinia virus was significantly prolonged by inhibition of OV-DMCs, 30 days+;

OV-DMCs play a critical role in the pharmacodynamics of OVV therapy in a variety of preclinical tumor models.

This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (HA16C0013) and National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2015R1A5A2009656).

1.Park BH, Hwang T, Liu TC, Sze DY, Kim JS, Kwon HC, Oh SY, Han SY, Yoon JH, Hong SH et al: Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial.

Immune checkpoint blockade has revolutionized cancer therapy;

We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) tumors derived from luciferase-expressing B16-Red-FLuc cells in s.c./s.c.

Intratumoral injection of Delta-24-RGDOX induced an in situ antovaccination of the treated melanoma, resulting in systemic immune activity against the disseminated tumors.

Vaccinia virus (VACV) has been intensively used as oncolytic virus for the treatment of various types of cancers over the last years.

We here report the identification of M2L as a strong binder to both CD80, and CD86 co-stimulatory receptors.

Finally, the TD oncolytic backbone might be interesting for the development of our invirIO™ platform.

The purine nucleoside adenosine dampens innate and adaptive immune responses under various inflammatory conditions [1].

SRF617 displays single-digit nanomolar inhibition of CD39 enzymatic activity and binds tightly to CD39 on cells.

These studies demonstrate that SRF617 blocks CD39 enzymatic activity, thereby reversing the anti-inflammatory/immunosuppressive TME caused by reduced extracellular ATP and the generation of adenosine.

We are investigating low dose mTRT as an immunomodulator in combination with in situ tumor vaccines (ISV).

We generated syngeneic B78 melanoma flank tumors in C57BL/6 mice.

All three staining protocols involving cryopreservation performed similarly to freshly stained cells.

We demonstrate that fixation and cryopreservation after labeling closely reproduces the analyses of freshly dissociated tumors.

Melanoma is currently the fifth most common cancer in men and sixth most common in women with an estimate of 7230 deaths from the disease in 2019 in the United States.

499 melanoma cells, derived from unirradiated B16-F10 tumors resistant to treatment with anti-CTLA-4 and radiation, were implanted on both flanks of C57BL6 mice.

Dual immunotherapy with 4-1BB agonist and anti-PD-1 significantly increases overall survival, whereas there is no benefit from treatment with either antibody alone.

Overcoming resistance to immunotherapy crucially depends on cells of the innate and adaptive immune system especially the macrophages and CD8+ T cells.

Extracellular adenosine, acting predominantly through the A2A receptor (A2AR), mediates immunosuppression which includes suppression of Th1 responses and cell-mediated cytotoxicity as well as increasing the activity of Tregs and MDSC.

MCA205, CT26, A20, and EMT6 tumor cells were subcutaneously implanted into immunocompetent syngeneic mice.

The adenosine concentrations in individual tumors ranged from 2 to 50uM across different models, while adenosine levels in the non-tumor region were always less than 1uM.

These data demonstrate that the adenosine concentrations vary in the tumor microenvironment of different syngeneic tumor models and that EOS100850/1 is able to inhibit the A2AR induced immunosuppression and therefore tumor growth in combination with chemotherapy regardless of the adenosine concentration in the TME.

We would like to thank the CRO Explicyte for performing the microdialysis experiments.

Liposomal drug delivery systems have historically focused on tumor accumulating properties.

Weakly cationic PEGylated Oxaliplatin (L-OHP) liposomes (R2) were formulated (POPC/cholesterol/DOTAP/chol-PEG/Chol, (52.5/36/7.5/4)) (size 120 nm, polydispersity 0.1, charge +10 mV).

The in vitro half-maximal inhibitory concentration (IC50) of CT26 cancer cells was comparable between R2 liposomes and free L-OHP (2.2 and 2.0 μM).

Here we report the potent anti-cancer effect of weakly cationic PEGylated L-OHP liposomes that depletes suppressive populations and eradicates CT26 tumors.

Tumor-targeted superantigens (TTS) such as Naptumomab Estafenatox (Nap) are fusion proteins that consist of genetically engineered Superantigens (Sag) linked to Fragment antigen binding (Fab) moieties directed to tumor-associated antigens.

The combination of Nap with PD-L1 inhibitor (durvalumab) was tested in vitro against high (MDA-MB 231) and low (RKO) 5T4-expressing cancer cell lines in the presence of human PBMCs.

Combination of Nap with durvalumab had synergistic anti-tumor effect against both high and low 5T4-expressing cancer cell lines.

Our studies show that combination of CPI with STR overcomes the limited effect of CPI monotherapy regardless of tumor antigen expression level.

The study was approved by of the Institutional Animal Care and Use Committee (IACUC) of Tel Aviv University, approval number 01-18-008

The generation of a robust antigen-presenting cell (APC) response at the site of a tumor is central to effective cancer immunotherapy.

Female BalbC mice were inoculated orthotopically in the mammary fat pad with 5x10^5 4T1 TNBC cells.

Immuno-NPs carrying both STING/TLR4 agonists mediated a robust synergistic production of IFNβ from macrophages that was 10-fold increased from NPs carrying either agonist alone (Fig.

Nanoparticle-mediated co-delivery of synergistic STING/TLR4 agonists drives a significant therapeutic outcome in the treatment of TNBC and prevention of metastasis in the 4T1 orthotopic model.

Ectonucleotide pyrophosphatase 1 (ENPP1) was identified as the extracellular hydrolase for cGAMP, the natural ligand for STING [1,2].

ANG-1084 and 1623 have IC50 of <0.5 nM against human ENPP1 with cGAMP as substrate at pH7.6, and are selective against hENPP2/3, PDEs, CEREP44 and stable in human and mouse microsomes.

Novel selective and potent small molecule inhibitors were designed to inhibit extracellular ENPP1.

1.Li, L et al., Hydrolysis of 2’3’-cGAMP by ENPP1 and design of nonhydrolyzable analogs.

The stimulator of interferon genes (STING)-pathway is a critical component of the adaptive immune response against tumors.

Data show that low to mid-range doses caused injected tumor collapse and the activation and expansion of tumor-specific CD8 T cells.

These data demonstrate that the induction of anti-tumor CD8 T cells is inhibited by TNF-alpha during overstimulation of the STING pathway, suggesting that intervention in TNF-alpha signaling may broaden the therapeutic window of STING agonists.

All animals were used according to protocols approved by Institutional Animal Use Committee of Aduro Biotech.

Stimulator of Interferon Genes (STING) is a major player in the initiation of robust innate immune activation leading to initiation and enhancement of a tumor-specific adaptive immunity.

Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies.

Selvita’s agonists bind to all tested recombinant STING proteins: human, mouse, rat and monkey.

Selvita’s STING agonists activate STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity.

STING pathway is a major innate immune sensing mechanism for the detection of immunogenic tumors.

Using methylation microarray analysis, we identified human melanoma cell lines with aberrant STING promoter methylation across a panel of 16 cell lines and subjected them to treatment with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5AZADC).

We identified 6 STING-negative human melanoma cell lines with high levels of STING promoter methylation.

We provide evidence that DNA methylation is a major contributor to the suppression of STING signaling in melanoma.

The cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway is becoming an important point of focus in the expanding landscape of next-generation immunotherapies.

cGAS and STING expression were simultaneously detected in melanoma tissues using our novel fluorescent nanoparticle IHC approach (Quanticell™).

Our results show that the Quanticell assay format enables a more objective and quantitative scoring of target proteins compared to standard IHC assay formats.

We show that our technique quantitatively measures cGAS and STING expression in cancer tissue biopsies with high sensitivity.

STING is a ubiquitously expressed innate immune sensor which is essential for the production of host defence-related proteins including type I interferons and proinflammatory cytokines that promote the recruitment and effector activity of innate and adaptive immune cells [1].

CTx has developed potent drug-like small molecule STING agonist compounds that show direct STING binding.

CTx has a mature STING agonist program that has successfully produced direct STING activators that can promote primary DC function and elicit immune-mediated anti-tumor responses in mouse models of solid cancer.

Previous work from our laboratory has shown that tumors activate the STING pathway in antigen-presenting cells.

In vitro experiments were performed using macrophages and bone marrow-derived dendritic cells stimulated with the STING agonist DMXAA alone or in combination with several TLR agonists.

Each innate immune agonist was titrated to identify the dose inducing maximal IFN-β expression.

TLR activation by LPS synergized with STING signaling to augment IFN-β production beyond the level of either agonist alone.

Activation of the STING pathway by direct intra-tumoral administration of synthetic cyclic dinucleotide (CDN) agonists has shown anti-tumor activity in animal models and early stage human clinical trials.

Exosomes were engineered to overexpress PTGFRN, an exosome surface protein, and loaded ex vivo with a proprietary CDN.

In vivo pharmacodynamics (PD) and anti-tumor responses were demonstrated in a B16F10 syngeneic mouse model and ex vivo histo-culture of human tumor explants (head and neck squamous cell carcinoma [HNSCC] and non-small cell lung cancer [NSCLC] patients) was utilized to evaluate PD after intra-tumoral administration.

ExoSTING™ was preferentially taken up by differentiated macrophages and dendritic cells.

ExoSTING™ is an engineered exosome therapeutic candidate that specifically targets the STING pathway in APCs in the tumor microenvironment, resulting in enhanced PD effects, avoidance of T cell uptake and enhanced anti-tumor immune responses compared to free CDN.

The study was approved by Codiak BioSciences' internal IACUC committee, approval number CB2017-001.

Cyclic dinucleotides (CDN) – agonists of Stimulator of IFN genes – can initiate potent anti-tumor immunity due in part to activation of antigen presenting cells [1].

We employed a surgical resection model of metastatic 4T1 mammary carcinoma to examine the effects of surgery and lung metastasis on CDN efficacy.

In mice bearing established 4T1-luciferase tumors, administration of three CDN doses results in no cures if surgical resection is not performed.

Our findings suggest that CDN monotherapy is less effective in the recurrent, orthotopic model of 4T1 mammary carcinoma than in flank tumors, but combination with systemic immunotherapy can improve immune control of both primary and metastatic disease.

All mouse experiments were approved by MIT’s Committee on Animal Care, protocol# 0717-076-20.

Stimulator of interferon genes (STING) is a master regulator of interferon (IFN)-mediated immune responses, and the cGAS-STING pathway plays an essential role in detection of aberrant cytosolic DNA fragments.

TTI-10001 was administrated as a single dose (oral, i.v., or i.t.) to MC38 tumor-bearing mice and plasma or tumor samples were collected for pharmacokinetic (PK)/pharmacodynamic (PD) analysis.

Mice dosed with TTI-10001 demonstrated favorable drug exposure with excellent oral bioavailability (85%) and a long tumor retention time (T1/2: ~ 4-8h) following all three routes of administration.

TTI-10001, a novel non-CDN small molecule STING agonist, is well tolerated in vivo and displays an excellent PK/PD profile that results in potent anti-tumor activity by i.t., oral, and i.v.

All mouse experiments were approved by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on Animal Care (University of Toronto approved protocol # 20011874).

E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular localization or degradation by the ubiquitin proteasome system.

We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations.

Furthermore, the compounds increased antigen recall responses in human PBMCs.

These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology.

While ICPIs (immune checkpoint inhibitors) have fundamentally changed the practice of cancer therapy for tumors arising from many different tissues, ways to increase both response rate and durability are critically needed.

Key assays in our testing cascade include: •

While TLR7 agonists having appropriate potency and specificity were discovered early in our program, systemic oral delivery even with an optimized prodrug approach was relatively poor.

From our original starting point - a relatively weak TLR7 agonist with no oral bioavailability - we have invented a novel series of molecules that are designed to be dosed QOD continuously over a 24-month period to appropriately engage innate immunity at a level that is well-tolerated by the patient while increasing treatment response rate and durability.

TLR7 agonists boost immune responses in the tumor microenvironment (TME), primarily through dendritic cell (DC) engagement, enhancement of antigen presentation and T cell co-stimulation.

TLR7 agonists and several liposomal formulations were screened for optimal T cell tethering and release, measured by HPLC.

ACT with Deep TLR Primed PMEL T cells inhibited tumor growth significantly more than ACT of PMEL T cells alone or combined with systemically delivered TLR agonists.

Deep TLR Primed T cells display superior efficacy, PD, and safety compared to T cells alone or co-administered with systemic TLR agonists.

Expression of T-cell checkpoint receptors occurs following T-cell activation during exhaustion, and ligation of these receptors can impair T-cell function and anti-tumor immunity [1].

Splenocytes were isolated from OT-1 mice and stimulated with chicken ovalbumin peptide (SIINFEKL) in the presence of different combinations of TLR agonists for 4 days.

Activation of CD8+ T cells in the presence of specific TLR ligands resulted in decreases in expression of PD-1 and/or CD160 and increases in CTLA-4.

Multiple TLR agonists can modulate the expression of T-cell checkpoint receptors, notably PD-1 and CTLA-4.

Although immune checkpoint blockade therapy (ICT) contributed remarkably to elevated survival rate and durable tumor remission for the past decade, several limitations of ICT such as that only partial patients have corresponded to those therapeutic interventions have appeared.

For in vitro studies, bone marrow-derived macrophages (BMDMs) were prepared as previously reported.

The polymeric nanocarrier (TR-NP) to target M2-like TAMs was successfully prepared by the layer-by-layer assembly, resulting in 145 nm of hydrodynamic diameter.

In this study, we designed the polymer nanocarrier that targets TAMs and repolarizes them toward M1-like macrophages.

Difficultly translating therapeutic vaccination success from murine models to the clinical treatment of cancer has highlighted the need for vaccination strategies that may be more broadly applicable.

Efficacy studies were conducted in poorly immunogenic orthotopic murine tumor models of EMT6 breast cancer and B16F10 melanoma.

Tumor retention studies tracking fluorescently labeled pTLR7-tAbs showed pTLR7-tAbs are retained within the tumor microenvironment (TME) 3-fold longer than isotype control antibody-pTLR7 constructs.

Together, these data support the use of our pTLR7-tAb platform for in situ vaccination and as a therapeutic tool for enhancing anticancer immunity.

All studies with animals were carried out in accordance with procedures approved by the Institutional Animal Care and Use Committee at the University of Chicago.

Depending on the origin of the primary lesion, patients with distant metastases of aggressive solid tumors have traditionally been deemed incurable.

Two liver metastases were treated with combination immunotherapy suspended in montanide, an oil adjuvant.

No suspicious uptake was noted anywhere in the body on the 12-week post-procedure PET/CT.

Intratumoral injection allows a much higher proportion of immunotherapy to directly interact with the tumor microenvironment compared to intravenous injection.

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.

Local delivery of pattern recognition receptor agonists (PRRAs) to the tumor microenvironment (TME) stimulates innate immune sensors such as toll-like receptors (TLR) and can enhance antigen uptake and presentation, induce proinflammatory immune cell recruitment, and reverse tumor-associated immunosuppression (1, 2).

Given the need for safe and effective intratumoral therapies, we developed a sustained-release TransCon™ (refers to “transient conjugation”) TLR7/8 Agonist prodrug by conjugating resiquimod to a hydrogel via a TransCon linker to provide consistent intratumoral release of unmodified resiquimod.

In vivo pharmacokinetics in rodents, following local injection of TransCon TLR7/8 Agonist, showed long-term resiquimod release over several weeks with minimal systemic exposure compared to an equivalent dose of unconjugated resiquimod.

These data provide strong evidence that a single dose of TransCon TLR7/8 Agonist can mediate long term local release of unmodified resiquimod with minimal systemic exposure and systemic pro-inflammatory induction compared to an equivalent dose of unconjugated resiquimod.

Analysis of current data with cancer vaccines suggests that the lack of efficacy is likely due to their two primary design challenges: 1.

PASCal operates by 3 moduls: (1) validated epitope database containing 108 true HLA-epitope pairs (2) Expression frequency-based shared tumor antigen database established for 19 indications based on >96,000 tumor biopsies.

(3) Validated algorithm for the identification of immunogenic peptides by the selection of personal epitopes (PEPIs) binding to multiple autologous HLA alleles.[1,2] Using PASCal, a library of 3,286 immunogenic 20mer peptides derived from 184 antigens associated with 19 cancer indications -based on 16,000 subjects’ HLA genotype (both class I&II alleles) was compiled.

Personal cancer vaccines were selected to fulfill the following criteria: 12 immunogenic peptides derived from 12 different tumor-specific antigens frequently expressed in the patient’s disease type, with the expected number of expressed antigens on the patient’s tumor cells of at least 3 (by statistical estimation).

PEPIs outperform reported immunogenicity of mutated neoantigen-based personal vaccines and induced unprecedented immune responses in cancer patients.

This study was performed in accordance to the Declaration of Helsinki (amendment 37).

Signed informed consent were obtained from each patient, including allowance of publication.

Neoantigens are mutated tumor antigens that make attractive targets for immunotherapy as they are recognized as foreign by the immune system.

ATLAS screening of the B16F10 melanoma mutanome was performed, consisting of whole exome sequencing, non-synonymous mutation library construction, and screening of splenic CD8+ T cells from tumor-bearing C57BL/6 mice.

ATLAS screening of B16F10 melanoma identified 66 stimulatory and 57 inhibitory neoantigens, the majority of which were not predicted by peptide binding affinity.

Currently, the only method to reliably and comprehensively identify neoantigen targets and rationally omit potentially deleterious antigens is ATLAS screening using autologous cells.

Animals were housed within a USDA registered, AAALAC accredited and OLAW assured (#A4591-01) animal facility in accordance with the Institutional Animal Care and Use Committee (IACUC).

Androgen deprivation therapy is the primary treatment for recurrent and metastatic prostate cancer.

We implanted the androgen-responsive mouse prostate tumor line MycCaP into 6 week old male FVB mice.

Mice treated with AR vaccine before androgen deprivation (AR →ADT) had slower tumor growth compared to the control plasmid (pTVG4 →ADT) but not when vaccinated after ADT (ADT→Vx).

These results have implications on the scheduling of androgen deprivation with immunotherapy for the treatment of prostate cancer, and correspond to similar results observed in human clinical trials with anti-tumor vaccines.

Recent advances in immunotherapy have confirmed that adaptive immune responses can recognize and eliminate cancer cells.

We created a de novo pan-cancer transcriptome assembly with RNA-seq reads from 31 cancer types obtained from TCGA.

Our de novo assembly revealed the presence of approximately 100 melanoma-specific transcripts encoding over 2,000 potential antigens (ORFs).

We have identified a number of novel melanoma-specific antigens that are shared among patients.

The results presented here are based in part upon data generated by The Cancer Genome Atlas (TCGA) Research Network (http://cancergenome.nih.gov/);

All work involving the use of human tissue was approved by the NHS Health Research Authority South Central - Oxford A Research Ethics Committee (reference number 04/Q1604/21), and North West - Haydock Research Ethics Committee (reference number 19/NW/0216).

Diffuse intrinsic pontine glioma (DIPG) is one of the most lethal pediatric brain tumors.

We developed a novel C57BL/6-syngeneic DIPG model by culturing cells from a Sleeping Beauty de novo DIPG induced in a neonatal mouse using a K27M-mutated histone 3.3 plasmid and other oncogenic plasmids.

Flow cytometry analysis showed that SB-DIPG-11 cells express both MHC I (H-2Kb/H-2Db) and EphA2 on their surface.

Compared to control, combination therapy proved the most effective, significantly prolonging the survival of tumor-bearing mice.

The strong involvement of neoantigens-driven CD8+ T-cells is considered a substantial element of immune-mediated tumor rejection.

We perform a benchmarking study of the available methods for vaccine design.

For each of the vaccine design tools, we assess the enrichment of immunogenic neoepitopes within the suggested vaccine compositions (e.g., top 20 peptides) using early-enrichment metrics [4].

The presented work provides insights into the vaccine design process by quantitatively and qualitatively comparing available tools for the selection of both cancer vaccine composition and targets for adoptive cell therapies.

Immunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed.

These findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC.

Patients were studied following informed consent, and with Institutional Board Review (IRB) (HSR-IRB 13498 and 15781, respectively) and FDA approval.

Written informed consent was obtained from patients for publication of this abstract and any accompanying images.

Recent advances in whole exome sequencing have produced a renewed interest in therapeutic cancer vaccines targeting tumor-specific neoantigens.

The CT26 mutanome was evaluated with Ancer™, an innovative and automated neoantigen prediction platform that combines proprietary machine learning-based MHC class I and MHC class II neo-epitope identification tools with removal of inhibitory regulatory T cell (Treg) epitopes for optimal personalized cancer vaccine design.

Average tumor volumes in CT26-Ancer™ immunized mice were significantly reduced by 45% (unpaired t test, p = 0.0156) and 38% (unpaired t test, p = 0.0291) at days 21 and 25 post implantation, respectively, compared to vehicle (poly-ICLC) immunized tumor-bearing animals (Figure 2).

The results presented here highlight the efficiency of the Ancer™ platform in selecting highly immunogenic neoantigen targets and that these responses are able to control tumor growth in a peptide formulation.

ChIP-seq, RNA-seq, and exome-seq was conducted on murine tumor cell lines treated with the EZH2 inhibitor PF-06821497.

In this study, we have identified a common mechanism of EZH2-mediated epigenetic suppression of genes involved in tumor immune recognition.

Our data illustrate the potential for targeting EZH2 to increase the immunogenicity of tumors through relieving epigenetic suppression of tumor antigen presentation machinery and silenced neoantigens.

The NCI PREVENT Cancer Preclinical Drug Development Program (PREVENT) is a peer-reviewed program that supports the preclinical development of innovative cancer preventive interventions towards clinical applications (https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical).

PREVENT utilizes NCI contract resources to operate its agent development pipeline, which consists of three key stages: early proof of concept phase, secondary testing phase, and advanced preclinical development phase.

Since the Program’s inception, a total of 21 cancer preventive vaccine projects have been selected for support.

While the expanded knowledge base in tumor immunology has uncovered the potential hurdles for developing effective cancer immunoprevention strategies, it has also helped better understand the key determinants, including the selection of optimal target antigens, vaccine delivery strategies and schedules, immune biomarkers for vaccine immunogenicity, immune durability, and immune correlates of antitumor protection.

Induction of optimal anti-tumour immune response is challenging as tumour antigens are self-antigens and antigen-specific receptors against self-antigens are negatively selected in the body.

As solid tumour models, A375 melanoma or U87-MG glioblastoma cells were subcutaneously engrafted into CD34 humanised NCG.

We observed reduced tumour growth in both A375 and U87-MG tumour models in KLH vaccinated mice where KLH served as vaccine and i.t.

These findings support the new concept that tumour independent antigens can be used to develop vaccines against solid tumours.

Cancer vaccines have traditionally succeeded in preventing viral-induced malignancies, such as papilloma virus-associated cervical cancer and hepatitis B-associated hepatocellular carcinoma.

The CT26 mutanome was evaluated with Ancer™, an innovative and automated neoepitope prediction platform that combines proprietary machine learning-based MHC class I and MHC class II neoepitope identification tools with removal of inhibitory regulatory T cell (Treg) epitopes for optimal personalized cancer vaccine design.

We report that prophylactic vaccination with poly-neoepitope UNITE vaccine successfully induced IFNγ-producing Th1 cells, with complete rejection of CT26 tumors observed in 50% of mice.

Therefore, targeting multiple mutations encoding the best set of CD4 and CD8 neoepitopes, as predicted by Ancer™, and using the UNITE platform may solve critical problems in current cancer immunotherapy development.

Subunit vaccines targeting tumor antigens have shown limited capacity for expanding cytotoxic T-cells against tumors in the clinic.

C57BL/6J mice received two or more doses of benchmark or amphiphile-modified vaccines, comprised of mKRAS peptide and CpG adjuvant, subcutaneously injected into the tail base in two-week intervals.

We show robust immune responses that yield strong activation against all common mutations in the mKRAS protein compared to low or undetectable responses generated by soluble or benchmark treatments.

By targeting immunogens directly and precisely to the LNs, the Amphiphile platform can significantly amplify the potency of subunit vaccines.

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia strain that belongs to the poxvirus family.

To identify vaccinia viral inhibitors of the cGAS/STING pathway, we performed an unbiased screen of 70 vaccinia viral early genes in HEK293 T cells using a dual-luciferase system.

Whereas MVA infection of bone marrow-derived DCs (BMDCs) induces cGAS degradation in a proteasome-dependent manner, recombinant MVA with deletion of the E5R gene (MVA∆E5R) infection fails to do so.

In this study, we discovered that the vaccinia E5R gene encodes a dominant cGAS inhibitor.

The study was approved by the Committee on the Ethics of Animal Experiments of Sloan Kettering Cancer Institute, approval number 19-01-002.

Systemic immunity is regulated by interactions of commensal bacteria with immune cells in the gut.

The clinical efficacy of EDP1503 was investigated in subcutaneously implanted isograft tumor models as well an intravenous lung metastasis model.

EDP1503 increases expression of costimulatory molecules on CD11c+ dendritic cells (DCs) within the mesenteric LNs with an accompanying increase in proinflammatory CD103+ DCs within tumor draining lymph nodes.

Together, these data clearly demonstrate the ability of an orally delivered non-colonizing monoclonal microbe to enhance innate and adaptive anti-tumor immunity and substantiates the rationale for ongoing clinical trials.

Bacillus Calmette-Guérin (BCG) is commonly used in the treatment of non-muscle invasive urothelial cancer (bladder cancer;

Human monocyte-derived macrophages and freshly resected human bladder cancer specimens were cultured ex vivo in the absence or presence of BCG, indomethacin (COX-1/2 blocker), celecoxib (selective COX-2 blocker) or selective EP4 blocker (ARY-007).

We observed that the BCG-driven-induction of MDSC- and Treg-attracting chemokines in human macrophages and whole BlCa explants was correlated with its induction of COX-2, the key enzyme controlling the PGE2 biosynthesis.

We observed that PGE2 represents a nodal point controlling the balance between desirable/immunostimulatory and undesirable/immunosuppressive components of BCG activity and that interference with PGE2 synthesis or it’s signaling improves the magnitude and character of the BCG-orchestrated immune response in BlCa TME.

Immune checkpoint blocking therapies (ICB) such as anti-PD1 antibodies have revolutionized the treatment of melanoma and lung cancer, leading to clinical benefit in approximately 50% and 19% of patients, respectively [1,2].

We established a cohort of melanoma and lung cancer cell lines HLA- and antigen-matched to tumor-specific T cells using the MART-1 TCR system [8, 9].

Several AXL-positive melanoma and lung cancer models showed a dose-dependent cooperative sensitivity to combined treatment with EnaV and tumor-specific T cells.

Our findings demonstrate that EnaV and immunotherapy cooperatively exert a strong anti-tumor response in therapy-refractory melanoma and lung cancer models.

The collection and use of human tissue was approved by the Medical Ethical Review Board of the Antoni van Leeuwenhoek.

Cancer immunotherapies using checkpoint inhibitor antibodies (CPI) show considerable antitumor response in the clinic.

We show that PlGF-2123-144-conjugated CPI localized to the injection site for long time, and limiting the CPI exposure to the body and toxicity including diabetes induction risk (AB).

Our data suggest that the tumor-matrix binding localized CPI technology can improve both safety and efficacy of CPI therapy, with high translational promise.

1.Ishihara et al., Sci Transl Med 9, eaan0401 (2017).

STimulator of Interferon Genes (STING) has emerged as an innate immune pathway capable of inducing anti-tumor immune activity through activation of antigen presenting cells and production of type I interferon, leading to T-cell priming and activation.

We generated novel STING ADCs by developing a conjugation platform which consists of a STING agonist incorporated into a chemical scaffold for bioconjugation, designed to provide optimal drug-like properties.

The STING ADCs achieved a 100-fold higher potency than the corresponding free agonist in in vitro assays using THP-1 reporter cells.

Importantly, a single intravenous administration of the STING ADC led to a significant increase in immune cell infiltration and tumor cell death within 72 hours and a significant increase in tumor-localized inflammatory cytokines within 12 hours, while levels of systemic cytokines remained relatively low.

We have developed a STING ADC platform and demonstrated target-dependent anti-tumor immune responses both in vitro and in vivo for two therapeutic targets.

Animal studies were conducted following the recommendations of the Guide for Care and Use of Laboratory Animals and in compliance with the Institutional Animal Care and Use Committees of Translational Drug Discovery and Charles River Discovery Services.

While checkpoint inhibitors have found stunning success as single agent therapies, their clinical efficacy only extends to a subset of patients across multiple cancers.

Previous work from our laboratory devised a strategy for dramatically improving systemic administration of an immune agonist of Toll-like receptor 9 (TLR9) known as CpG Oligodexoynucleotide (ODN) whose anti-tumor efficacy has been traditionally limited to intra- and peri-tumoral injections.[1,2] Through antibody-guided delivery of conjugated CpG ODN to the tumor microenvironment (TME), TLR9 activation promotes dendritic cell maturation thus upregulating cross-presentation of TME antigens.

Here we describe our latest multi-targeted approach which tackles immune suppression by overcoming mechanisms of acquired resistance to immunotherapy and synergizing therapeutic responses.

Tumor growth inhibition was calculated by using the geometric mean of relative tumor volumes.

By transitioning to bioluminescence imaging techniques of tumor cells, we overcome the limitations of traditional tumor volume measurement by caliper and capture a fuller picture of both early therapeutic response events and metastatic disease.

All animals were treated humanely and in accordance with the guidelines of the USC’s Institutional Animal Care and Use Committee under protocol 20265.

Regulatory T cells (Tregs) play an important role in the establishment and progression of tumors and are considered a major obstacle to tumor eradication by immunotherapies [1].

Sur301 is an antibody-drug conjugate (ADC) composed of PC61, a rat monoclonal antibody directed against mouse CD25, conjugated to a pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug-to-antibody ratio of 2 [4].

In order to characterize the mode of action of sur301, we analysed the immunophenotype of Tregs and Teffs isolated from tumors, blood and spleens of mice bearing established MC38 tumors following a single dose of sur301 either alone or in combination with an anti-PD-1 antibody.

In conclusion, these new data show that sur301 is able to mediate potent Tregs depletion without affecting Teffs.

Major histocompatibility complex class I chain-related protein A (MICA) is a stress-induced ligand for the activating receptor NKG2D on natural killer (NK) cells.

When the proteins were added into the co-culture of NK cells (NK-92 cell line) and breast cancer cells (T47D cell line), the cytotoxicity was measured using a lactate dehydrogenase (LDH)-based assay, the granzyme B released by NK-92 cells was detected using ELISA, and the apoptosis of the T47D cells was examined using a caspase 3/7 luminescence assay.

We successfully generated HEK293 stable clones to produce MICA-G129R, MICA and G129R proteins.

The chimeric protein MIC-G129R can bridge NK cells and breast cancer cells, and induce the cytotoxicity of NK cells to kill the breast cancer cells.

Checkpoint inhibitors have transformed cancer treatment in certain patient subgroups.

DSP107 binds to CD47 and 4-1BB with high affinity (1.2 nM and 0.7 nM, respectively).

DSP107 is a bifunctional, fusion protein with a unique mechanism of action.

With reference to the study in h41BB-KI-C57BL/6 mice, the protocol and any amendment(s) or procedures involving the care and use of animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio prior to execution.

Strong scientific rationale and preclinical data support the combination of CD27 costimulation with PD-1 blockade.

We developed novel human antibodies to CD27 and PD-L1 from which the best pair was selected based on activity and manufacturability to engineer into a whole IgG-scFv bispecific antibody format.

CDX-527 was found to be a potent PD-1 inhibitor and a stronger CD27 agonist than anti-CD27 monoclonal antibodies.

CDX-527 effectively combines PD-1 blockade and CD27 costimulation into one molecule that is more potent than combination of the parental antibodies.

Bispecific T cell redirecting antibodies (T-BsAbs) targeting tumor cells and T cells are an increasingly popular form of cancer immunotherapy.

Our novel T-BsAbs targeting various solid and liquid tumor indications cause efficient tumor cell lysis, but with minimal cytokine release in respective in vitro tumor cell and T cell co-culture models.

This next generation of T-BsAbs thus shows promise as an effective therapy against multiple cancer indications.

Animal studies were done in accordance with IACUC protocol # AUP-17-0331-M-2

Bi-specific T cell Engager (BiTE®) antibody constructs are synthetic cancer immunotherapies aimed at inducing polyclonal anti-tumor T cell responses by re-directing T cells to specific surface antigens expressed on tumors.

We have engineered a knock-in mouse expressing a chimeric mouse/human CD3e receptor recognized by BiTE® antibody constructs from the endogenous mouse CD3e genomic locus to elucidate the pharmacokinetic, pharmacodynamic and efficacy parameters governing BiTE® activity in preclinical models.

We have evaluated tumor types with varying baseline T cell densities to understand the relationship between T cell density and BiTE® activity in a tumor.

These studies will allow defining key mechanisms that may limit BiTE® efficacy in solid tumors and informing optimal therapeutic combination strategies capable of maximizing the potential of BiTE® therapy in solid tumors.

All in vivo work was conducted under an IACUC approved protocol and in an AAALAC accredited facility.

Recently, the role of TOX as a key transcription factor guiding T cells towards exhaustion have shed light on strategies to reprogram tumor infiltrating lymphocytes.

Human TIGIT-Fc-OX40L and TIGIT-Fc-LIGHT (and the murine surrogates) were purified, and target binding assessed by ELISA, Octet, and cell-surface binding assays.

Each respective domain of TIGIT-Fc-OX40L and TIGIT-Fc-LIGHT bound human and cyno PVR, OX40, LTbR, and HVEM with low nanomolar affinity.

The addition of OX40L or LIGHT as co-stimulatory molecules dramatically improved the efficacy of checkpoint blockade to TIGIT both alone and in combination with PD-1 or CTLA-4 blockade.

Agents targeting mutant EGFR in NSCLC have become standard of care but acquired resistance invariably develops due to new mutations in EGFR and activation of compensatory pathways such as cMET.

To examine this hypothesis, we performed a comprehensive assessment of the Fc effector functions of JNJ-372, including effects on EGFR and cMet levels, downstream signal transduction, and role in mediating anti-tumor activity.

Despite observing potent anti-tumor activity of JNJ-372 in EGFR mutant xenograft models, only modest anti-proliferative effects were observed in NSCLC cell lines in vitro.

Collectively, these observations represent a novel Fc-dependent mechanism of action of JNJ-372 and support the continued clinical development in patients with aberrant EGFR and cMET signaling.

Previous studies examining vaccination against guanylyl cyclase C (GUCY2C) in colon cancer patients revealed pre-existing immunity to the adenovirus serotype 5 (Ad5) vaccine vector as a barrier to effective vaccination in patients, which was confirmed in animal models [1].

The immunogenicity and antitumor efficacy, safety, and biodistribution of the Ad5.F35 vaccine (Ad5.F35-GUCY2C-S1) was evaluated in mouse models of metastatic colorectal cancer.

Like the previous Ad5 vaccination, and Ad5.F35 induced CD8+ T cell responses in adenovirus-naïve BALB/c mice.

These studies demonstrate that a chimeric adenoviral vector composed of Ad5 with the Ad35 fiber is an advantageous alternative to Ad5 alone for vaccinating human populations.

The authors thank the Center for Cell and Gene Therapy, Baylor College of Medicine for assistance in Ad5.F35 vaccine manufacturing.

No sensitive or identifiable patient data is included in this abstract.

Immune mobilising TCRs against cancer (ImmTAC) molecules are soluble bispecific fusion proteins comprised of affinity‐enhanced TCR and anti-CD3.

pHLA epitope numbers were quantitated using a novel semi-automated single molecule pHLA counting technique (derived from [1]) and HLA levels determined by flow cytometry.

Activation of T cells, induced by ImmTAC co-incubated with 70 different tumour cell lines, correlated tightly with pHLA presentation as predicted by gene expression (n=70, R2=0.74) and with single molecule surface pHLA numbers based on TCR labelling (n=19, R2=0.86).

The number of pHLA per tumour cell could be increased by incubation with T cells and ImmTAC, presumably due to release of T cell derived inflammatory cytokines [2], or by incubation alone with IFNβ (~3-fold, p In patients treated with tebentafusp, comparison of on-treatment changes in tumour biopsies from partial response vs progressive disease patients, found HLA-associated antigen processing gene expression to be significantly enriched (p=0.006).

Using a state-of-the-art single molecule surface pHLA detection technology, T cell activation by ImmTAC is highly sensitive and correlates with pHLA number.

Checkpoint inhibitor (CPI) immunotherapy has achieved remarkable success in the clinic, yet its efficacy in poorly immunogenic, immune-excluded tumors has been modest.

B16F10 melanoma is a poorly immunogenic and checkpoint-unresponsive tumor model.

Collectively, our data show that CBD-IL-12 can safely cure large, established tumors without antigen-specific approach, greatly increasing the therapeutic index of IL-12.

All animal experiments performed in this work were approved by the Institutional Animal Care and Use Committee of the University of Chicago.

Immune-related adverse events (irAEs) related to immune checkpoint inhibitors (ICI) may target any organ and originate from autoreactive T cells injuring host tissues.

We designed a prospective study at Loyola University Medical Center to document clinical profiles of pts diagnosed locally advanced or metastatic solid tumors.

Pt enrollment and data collection began in March 2017 and ended May 2019.

Our results do show a similar incidence of irAEs in our population (42.4%) as compared to other studies [2,3].

The study was approved by the Loyola University Medical Center institutional Review Board, approval number LU209364.

Immune checkpoint inhibitors have been approved in over 10 different types of malignancies.

Data was obtained using a retrospective chart review of Ochsner patients with metastatic cancer who received at least one cycle of immunotherapy (Nivolumab, Pembrolizumab, Atezolizumab, or Durvulamab) with no other drug combination between October 1st, 2014 and February 1st, 2019.

Our results suggest that the presence of IrAE may represent a potential predictive indicator for treatment response to immune checkpoint inhibitors.

Radiotherapy (RT) in patients with primary head and neck cancer (HNC) and those receiving immune checkpoint inhibitors (ICI) for any cancer, are independent risk factors for developing hypothyroidism.

Data were obtained from a commercial de-identified database (Explorys, IBM, Inc.) that integrates electronic health records from 26 major integrated U.S. healthcare systems from 1999 to July 2019.

We recorded 221,760 patients with an active diagnosis (within the last 5 years) of primary HNC, and 547,570 patients with an active diagnosis of a primary lung, gastrointestinal, skin, melanoma or urinary cancer in the database.

The risk of developing hypothyroidism after ICI is higher in patients with HNC compared to other primary cancers.

Trials conducted for ICI efficacy have largely included the young and healthy, while elderly patients have been excluded or under-represented.[1] In our clinic’s real-world population of predominantly elderly men, we aimed to compare the rate of clinically significant side effects and hospitalizations.

This is a retrospective observational study approved by the local IRB including all patients (n=147) who received ICI for non-hematologic malignancies from 2013-2019 at VA Loma Linda Healthcare System.

There were 73 patients in elderly cohort and 74 in younger cohort.

Elderly patients in our cohort did not experience more frequent grade 3-5 AEs or hospitalization, but more elderly patients discontinued treatment due to AEs.

Treatment with immune checkpoint inhibitors (ICIs) can result in immune-related adverse events (irAEs).

This is the first study to our knowledge that quantitates the incidence of developing irAEs with ICI conditioned on time elapsed without development.

The study was approved by Dana Faber Cancer Institutution’s Ethics Board, approval number 02-021 /17-000.

Immune checkpoint inhibition (ICI) against PD-1/PD-L1 has shown remarkable efficacy in many solid tumors.

We characterized immune features in relapsed/refractory acute myeloid leukemia (AML) patients who developed irAEs during treatment with pembrolizumab and decitabine (PD-AML, NCT02996474).

We first queried for the presence of circulating anti-thyroid autoantibodies.

We performed a detailed characterization of immunological and molecular features accompanying the development of irAEs during anti-PD-1 ICI and identified specific T cell clonotypes that may have contributed to anti-PD1-induced irAEs in relapsed/refractory AML patients.

The clinical success of checkpoint inhibitors targeting CTLA-4 and PD-1/PD-L1 alone or in combination has paved the way for the development of new immunotherapy options and combination therapies.

We recently described a model whereby transient depletion of T regulatory cells (Tregs) using the FoxP3-GFP-DTR mice, prior to treatment with immune modulating antibodies, increased physical and biochemical immune related adverse events (irAEs) similar to what is observed in humans.

In this study, we demonstrated how Treg depletion can model clinical irAES and investigate methods to alleviate the toxicity induced by agonistic anti-CD40 and anti-CD137.

This study further highlights how T-reg depletion can be used to rapidly model clinically observed irAES and investigate methods to alleviate the toxicity of potent immunotherapies

Although antibodies and CAR-T cell therapies have been successfully used for cancer treatment, they can have significant adverse effects such as cytokine release syndrome (CRS).

We have developed a rapid, sensitive and reproducible in vivo humanized mouse model for quantitating CRS.

At day 5 of PBMC engraftment, levels of human CD45+ cells ranged between 10 to 15% CD45+ human cells in peripheral blood consisting of approximately 70% CD3 T cells and 25% CD56 NK cells.

We have developed a rapid, sensitive and reproducible novel in vivo PBMC humanized mouse model that is able to differentiate human PBMC donors based on individual safety response to single agent and combination therapeutics of immune checkpoint inhibitors and possibly bispecific antibody and CAR-T therapy.

The study(#16000) was approved by The Jackson Laboratory IACUC committee on 3/12/19 and expires 3/12/22

Severe neurological and endocrine toxicities are well recognized adverse events of immune checkpoint inhibitors (ICI).

We studied the case of a patient who developed fatal combined neurological and endocrine toxicity while on treatment with dual immune checkpoint blockade for his metastatic sarcomatoid mesothelioma.

Incubation with patient's plasma resulted in upregulation of multiple DC activation markers, most prominently CD25 and CD11b.

This is the first clinical report of an association between ICI-related neuroendocrine toxicity and elevation of IL17A, a Th17-produced cytokine involved in numerous autoimmune disorders of the central nervous system.

The study was approved by the IEO Institutution’s Ethics Board through its procedure for observational studies, approval number 2062.

The combination of the immune checkpoint inhibitors (ICI) anti-CTLA-4 (ipilimumab) and anti-PD-1 (nivolumab) has become a mainstay of treatment for select patients with metastatic melanoma.

Here we report a case of simultaneous T cell-mediated red cell aplasia and antibody-mediated auto-immune hemolytic anemia (AIHA) in a patient receiving combined ICI therapy for metastatic melanoma.

This report describes a rare case of two concurrent hematologic irAEs in a patient treated with combined ICI therapy: 1) auto-antibody mediated AIHA and, 2) CD8+ T cell-mediated red cell aplasia.

Institutional Review Board protocol is not required at The University of Chicago for a single case report.

Individual patient consent was not applicable as no information in this report can be categorized as identifiable.

Colitis a common immune related adverse events (irAEs) associated with immune checkpoint inhibitor (ICIs) therapy with severe toxicity occurring in up to 25% of patients receiving therapy[1].

As part of an established observational study peripheral PBMCs were isolated from patients presenting with colitis secondary to ICI therapy for malignant melanoma.

This is preliminary data however the differential expression of Cd45+CD3+CD8+ α4β7 at the initiation of toxicity highlights its potential to predict steroid sensitivity.

Dr Anna Olsson-Brown is a MRC Clinical Research Fellow based at the University of Liverpool and is supported by the North West England Medical Research Counical Fellowship Scheme in Clinical Pharmacology and Theraputics which is funded by the medical research council (Award Ref MR/N025989/1) Roche Pharma, Eli Lily and company limited, UCB Pharma, Novartis, the University of Liverpool and the University of Manachester.

Patient samples and clinical data were collected under the observational study ‘A Mechanistic Investigation into Drug and Chemical Induced Hypersensitivity Reactions’ - REC Ref: 12/NW/0525 UKCRN ID: 13348

Data and samples taken as part of the ‘A Mechanistic Investigation into Drug and Chemical Induced Hypersensitivity Reactions’ - REC Ref: 12/NW/0525 UKCRN ID: 13348 and consent gained for entry into the trial.

Protein therapeutics represent a significant proportion of the currently available therapies for a broad spectrum of diseases and is one of the fastest growing markets.

One major hurdle in the therapeutic development is the risk for unwanted immunogenicity which can lead to decreased efficacy and safety concerns.

Often used as a first step is an In silico T cell epitope prediction algorithm such as NetMHCpan which can be used to assess and compare the immunogenic potential of the lead candidates and guide de-immunization strategies.

Another important step is the compilation of the different datasets and translation of the results into a comprehensive risk management plan, allowing further de-risking of test candidates and selection of the best candidates to move forward into humans.

The gut microbiome impacts the efficacy of immune checkpoint inhibitor (ICI) therapy and the development of ICI-mediated diarrhea and/or colitis (IMDC).

Patients who received ICI therapy from January 2016 to January 2018 were examined retrospectively.

Whereas antibiotic therapy appeared to be protective against IMDC onset, use of antibiotics, especially those with anaerobic activity, after ICI therapy was associated with increased risk of severe IMDC.

Written informed consent was waived for this study given retrospective study.

Immune checkpoint inhibitor (ICI) therapy is commonly used in the treatment of advanced malignancy.

We performed a retrospective analysis of patients treated with ICI therapy at five MedStar Health hospitals from January 2011 to April 2018.

Of the 113 patients the most common cancers were melanoma (69) and non-small cell lung cancer (21).

Our findings suggests that the incidence of OI is low (<

The study was approved by MedStar Georgetown University Hospital Ethics Board, approval number 2017-0559.

Understanding of the immunobiology of checkpoint inhibitors (CPI) induced immune related toxicities, such as immune related enterocolitis, and how these compare to the immune signatures in tumors could lead to the development of strategies that de-couple autoimmunity from anti-tumor immunity.

Total RNA from patient-matched colitis and normal colon FFPE tissue from patients [n=23] receiving CPI were profiled with the 770 gene NanoString nCounter PanCancer Immune Profiling Panel (NanoPCIP).

In patients with colitis, the highest significantly upregulated differentially expressed gene (DEG) in inflamed colon tissue encoded for IL-6 (+24.1) followed by genes for neutrophil and monocyte chemotactic molecules.

Our human-tissue data demonstrates that IL-6-mediated-inflammation may be more prevalent in colitis than in responding-tumors, and in mice blocking IL-6 enhances CPI anti-tumor response.

The protocol was approved by The University of Texas MD Anderson IRB board protocol # PA19-0427

Immune checkpoint inhibitors (ICIs) have dramatically altered the therapeutic landscape across oncology.

To use the online tool, HCPs enter the organ system affected and the grade or severity of the symptom along with their planned management strategy.

Between February and June 2019, 737 HCPs entered 972 unique case scenarios into the tool.

These data suggest that many HCPs are challenged to optimally manage irAEs associated with ICI and are not managing their patients in concordance with the NCCN guidelines.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment over the past decade.

We identified a total of 250 patients treated with either nivolumab, pembrolizumab, or ipilimumab/nivolumab combination therapy found to have a random cortisol level less than 3 or morning cortisol less than 5 during or following therapy.

184 patients met criteria for new onset AI on ICI therapy.

As ICIs are being used with increasing frequency, it is crucial for health care providers to recognize the characteristics of immune-mediated adrenal insufficiency and initiate the appropriate workup, treatment, and referrals.

UCSD Biorepository Protocol (HRPP# 090401) UCSD ImmunoScape Immune Monitoring Protocol (HRPP# 150348) activated 3/12/15

Artificial intelligence deep learning networks are being increasingly applied to resolve complex pattern recognition challenges in quantitative digital pathology.

Exemplar TMA serial sections of gastric adenocarcinomas were IHC labelled for pan-Cytokeratin and CD3, using DAB chromogen and counterstained with hematoxylin.

Tumor and stroma were accurately identified across the serial sections using a single deep learning algorithm.

The exemplar data demonstrated the application of a single classifier, across IHC images, enabling the identification of tumor and stroma, irrespective of marker.

Glioblastomas make up more than 60% of adult primary brain tumors and carry a median survival of less than 15 months despite aggressive standard therapy.

Primary glioblastoma tumors obtained in accordance with the Declaration of Helsinki and with institutional IRB approval (#131870, #030372, #181970) were disaggregated into single-cell suspensions.

On the basis of tumor contact with the V-SVZ, the Citrus and FlowSOM clustering algorithms computationally identified consequential distinctions in the abundance of five T cell, macrophage, and microglia subsets among glioblastomas.

Single-cell mass cytometry in conjunction with the Citrus and FlowSOM clustering tools identified key differences in immune cell abundance between V-SVZ contacting and non-contacting glioblastomas.

This study was approved by the Institutional Review Board at Vanderbilt University, approval #131870, #030372, and #181970.

The current work-up for diagnosis, gene mutation, and programmed death ligand-1 (PD-L1) testing in metastatic NSCLC can exhaust an entire tumor specimen.

Whole slide (40x-magnified) H&E images of lung adenocarcinoma from TCGA were used to train a computational model to infer TMB and PD-L1 status.

In a 'hold-one-TCGA-site-out' approach using 40 images, the overall accuracy of the first model layer in classifying pathologist-confirmed histologic features was 98-99%.

Biomarker inference from digital H&E images has the potential to be highly accurate and to supplement or replace certain tissue-based tests.

Assessing PD-L1 immunohistochemistry (IHC) expression may play a role in identifying patients likely to benefit from anti–PD-1/PD-L1 therapies in some advanced cancers, including urothelial carcinoma (UC).

The PathAI platform showed significantly stronger correlation with pathologist consensus scores compared with scores generated by individual pathologists for quantifying PD-L1 positivity of lymphocytes (r-squared=0.744 vs 0.598) and macrophages (r-squared=0.68 vs 0.287).

We validated performance of the PathAI platform for automated assessment of PD-L1 expression on tumor and immune cells and demonstrated that the AI-predictors perform similar to or better than pathologist-based scoring in all cell types tested, and especially for immune cells where manual correlation is low.

The protocol was approved by site institutional review boards or independent ethics committees and conducted according to Good Clinical Practice guidelines, per the International Conference on Harmonisation.

Neoantigens can be targeted with cancer immunotherapies, including cell therapy and immunization, but most neoantigens are private to each patient, necessitating fully personalized treatments.

We pursued an immunopeptidomics strategy to identify which recurrent mutations in cancer lead to neoantigen peptides presented on the surface of tumor cells.

16 neoantigens (distinct HLA/mutation pairs) derived from 10 driver mutations were detected by MS in 27 tumors and 3 single allele cell lines.

We analyzed immunopeptidomes of human tumors and cell lines and validated 15 novel shared neoantigens, the largest set to-date.

Advances in high throughput measurement technologies (-omics data) have made it possible, and increasingly affordable, to generate high complexity, high volume data for oncology research.

We applied a reverse inferencing approach that systematically interrogates RNAseq measurements from tumor and control biopsies against our library of cause and effect gene networks curated from published experiments.

In LUAD tumor cells, we detected a pattern of gene signatures which indicated a tumor stem cell-like phenotype characterized by predicted decreases in the activity of pro-differentiation factors FOXP2 and PHOX2B and an increased response to hypoxia.

Our in silico analysis of lung cancer patient biopsies generated hypotheses implicating stem cell signaling in tumors, and a further stratification of this signal based on patient pack year burden.

Kinetics of bispecific therapeutic antibody bound with its targets (trimer complex) is more complex than simple monospecific antibody bound with one target.

Developed mathematical model consist of: (1) physiologically-based pharmacokinetic (PBPK) model of blinatumomab including binding to target receptors – CD3 on T cells and CD19 on leukemic cells;

Model was able to reproduce clinical PK data on blinatumomab without fitting.

Developed model demonstrates capability of QSP modeling to reproduce treatment efficacy clinical data on the basis of in vitro data.

Testing for target receptor occupancy in blood can be assessed in clinical studies.

General structure of developed PBPK/RO model is similar to published minimal PBPK models, but there are some differences.

Model is able to reproduce clinical PK data on various doses and regimens of anti-PD1 Abs without fitting and data on PD1 occupancy in blood as well as PD1 occupancy in tumor after treatment with nivolumab [1].

Developed model is able to predict PK of anti-PD1 Abs and PD1 occupancy in blood and tumor without fitting of clinical data.

Since precision medicine is nascent field, majority of its requirements in respect of tools and technologies are in early development.

Data sharing and security is the most debated issue.

Cloud analytics is the most promising solution that helps in analyzing and aggregating data coming from different clinical assays faster enabling quicker diagnosis.

Icore is the faster, cheaper and smarter way of analyzing, aggregating and visualizing the data.

Multiplexed imaging is a rapidly growing field that promises to substantially increase the number of proteins that can be imaged simultaneously.

We have developed a novel convolutional neural network to perform whole-cell segmentation from multiplexed imaging data.

By combining our novel imaging platform with new computational tools, we are able to achieve extremely accurate segmentation of whole cells in tissue.

We have developed a robust and accurate approach to whole-cell segmentation in human tissues.

The objective of this model-based meta-analysis is to characterize the exposure-response (overall response rate [ORR]) across approved anti-PD(L)1 agents (nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab) to quantify the effect of drug, patient and trial characteristics on ORR, and assess the-relationship between ORR and efficacy outcomes such as 6-month progression-free survival (PFS) and 12-month overall survival (OS).

Clinical data from 56 trials in approximately 11,000 subjects, and approximately 45 trials in 9,000 subjects across approved anti-PD(L)1s was included for the exposure-response and correlation analyses, respectively.

The model-based meta-analysis of exposure-response shows similar ORR depending on tumor type, across anti-PD(L)1s at the approved clinical doses.

Tumor purity (TP) estimation is routinely performed for whole exome sequencing (WES) analysis to ensure signal is derived from cancer cells and not from other cells in the tumor microenvironment.

In this study, we utilize artificial-intelligence (AI)-based image analysis to estimate TP, and further benchmark two methodologies of TP estimation on WES against pathologist and AI estimates across 1509 pretreatment samples from patients with melanoma, non-small cell lung cancer, small cell lung cancer, or urothelial carcinoma enrolled in clinical trials of nivolumab.

To assess TP by WES, paired tumor-normal samples were processed by Sentieon [2] or Strelka [3] somatic variant callers with subsequent SciClone [4] tumor heterogeneity analysis.

For 280 samples whereby pathologist TP estimates were available, we found Pearson correlations of r=0.32 and r=0.276 for Sentieon and Strelka, respectively.

We demonstrated that AI-based image analysis can provide a more accurate assessment of TP compared with visual assessment by pathologist for all tumor types tested.

The trial protocols were approved by site institutional review boards or independent ethics committees and conducted according to Good Clinical Practice guidelines, per the International Conference on Harmonisation.

Patients with primary melanoma are at a high risk for recurrence, creating a need for biomarkers.

PCF analysis of qmIF images finds that non-recurrent patients have higher degrees of clustering between tumor cells and CD8+ T Cells as well as tumor cells and HLA-DR+ macrophages (p=0.004).

Our DNN model incorporating features as well as raw image analysis produces an accurate prediction of DMR in H&E images of early stage melanoma, a readily available clinical pathology resource.

Traditionally, omics data analysis of biologic samples has been conducted in a single-level manner.

For the 3D-EX platform, tumor organoids were processed from fresh tumor tissue from NSCLC cancer patients obtained with IRB consent.

Here we present the results from the integrative analyses of multiomic data generated from the ex vivo treatment of 11 fresh tumor samples in the ex vivo assays.

Our results demonstrated that multiple types of omics data obtained from Nilogen’s comprehensive 3D ex vivo drug testing platforms can reveal cellular mechanisms that are active in individual tumors and may classify them into subtypes for response to drug treatment.

Despite the remarkable success, only a subset of patients can benefit from the anti-PD1 therapy.

We hypothesized that aberrations of any gene close to MHC class I genes in a gene network are likely to deregulate MHC I pathway and affect tumor response to anti-PD1.

Our approach successfully identified genes (e.g.

These results suggest that our approach is an effective method to identify genes/pathways associated with anti-PD1 and can be employed to screen potential drugs for combination regimens with anti-PD1.

TIM-3 is an inhibitory immune checkpoint protein currently under clinical investigation.

The computationally generated TIM-3 feedback network (Figure 1) comprises 60 nodes, 132 interactions and 855 possible FBLs (412 positive vs 443 negative).

The cellular components involved in the TIM-3 FBLs are potential targets of drug combination and biomarkers for anti-TIM-3 therapies.

Abstracts published on IDO1, PD-1, PD-L1, anti-PD1/PD-L1 were downloaded from PubMed, and analyzed by text mining.

The IDO1/anti-PD-1 network consists of 79 nodes, 227 interactions with 1277 possible regulatory paths from IDO1 to anti-PD-1.

As the network analyses revealed, inhibition of IDO1 could either up- or down-regulate PD-L1, and enhance or reduce efficacy of anti-PD-1.

Despite the remarkable success of cancer immunotherapies, a significant proportion of patients fail to respond.

Here, we provide evidence demonstrating that the compositions of human T cells circulating in peripheral blood are altered by physical fitness.

These results suggest that distinct T cell populations may be uniquely sensitive to both negative (age, obesity, and lack of physical activity) and positive (exercise) physiological inputs.

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.

Immunotherapy has been paradigm-shifting for melanoma, but durable responses only occur in a subset of patients.

Melanoma patient samples were profiled by Seahorse analysis in parallel to flow cytometric analysis of tumor infiltrating lymphocytes (TIL).

Analysis of melanoma patient biopsies showed striking heterogeneity in the metabolism of tumor cells.

Our data suggest that the metabolic makeup of the tumor microenvironment, driven through deregulated oxidative or glycolytic metabolism of the tumor cell, determines whether T cells have a permissive microenvironment for effective immunotherapy, and that inhibiting tumor cell metabolism may be an attractive strategy to improve the efficacy of immunotherapy.

The gut microbiome has been reported to impact the immune response to checkpoint blockade [1].

Plasma samples (pre-treatment, week 2, week 8 and end of study) from patients enrolled in a randomized phase 2 study in >=2nd line advanced gastric or gastroesophageal junction patients [2] were extracted for analysis on the LC/MS/MS and Polar LC platforms.

Metabolic profiling of human plasma detected 1364 metabolites.

The association of microbial indoles and host-derived tryptophan catabolites with benefit suggests the activation of immunosuppressive mechanisms in non-responders and those who did not experience clinical benefit.

The authors gratefully acknowledge the patients and their families who participated in this study.

This study was approved by the institutional review board or independent ethics committee appropriate for each site.

The gut microbiota has emerged as an important player in cancer pathology and increased evidence supports its influence on clinical response to immune checkpoint inhibitor (ICI) therapy.

Using our multi-technology meta-analysis of published datasets characterizing the baseline microbiome from melanoma patients treated with anti-PD-1, we have identified gut microbiota strains differentially abundant in responders versus non-responders that are concordant across multiple cohorts.

Selected microbiome-derived peptide binders were evaluated for activity in cell-based assays using isolated human primary T cells, dendritic cells, and macrophages.

Collectively, our results demonstrate the potential of Second Genome’s discovery platform to identify and characterize novel immunomodulatory factors produced by the gut microbiota.

Efficacy of immune checkpoint inhibitor (ICI) therapy is associated with the composition of the gut microbiome, but the mechanism remains to be elucidated.

We used IFNg production in colonic T cells as a readout for VE800 anti-cancer efficacy.

Our data suggest that bacterial density drives VE800 efficacy for induction of IFNg+ CD8 T cells.

Our data suggests VE800 induces IFNg+ CD8+ T cell induction and provides the necessary microbial component to promote anti-tumor immunity in combination with dual checkpoint inhibitor therapy in the multiple tumor models.

The development of lung cancer is closely associated with chronic inflammation, but the source of such inflammation has not been clearly defined and the contribution of specific cellular and molecular component of the immune system is yet to be elucidated.

We interrogate the immune-microbiota interaction in lung cancer using an autochthonous genetically engineered mouse model of lung adenocarcinoma driven by oncogenic Kras and loss of p53.

We found lung cancer development was associated with increased total bacterial load and reduced bacterial diversity.

Our study provides strong evidence that local microbiota play a critical role in promoting lung cancer development by provoking tumor-associated inflammation.

Recent years have brought the breakthrough to the immune checkpoint therapy (ICT) research with the discovery of the role of gut microbiota (GM) in the treatment response rate.

We carried out a classification study to identify responders to anti-PD1 treatment for metastatic melanoma, using shotgun metagenomic sequencing data from three published cohorts.

In analyzed cohorts we detected a function-associated signal and used it for R/NR classification.

Presented work advocates for the importance of function-oriented microbiome research in ICT.

The gut microbiota has emerged as a promising biomarker to assess the efficacy of immune-modulatory drugs in the CTLA-4 or PD-1 pathways.

Our improved assay includes customized primers to multiple hypervariable regions of the 16S region.

We designed a multiplexed Ion AmpliSeqTM assay including 16S targets and species-specific markers targeting important microbial species.

Using the Ion AmpliSeq™ technology, we have created a highly sensitive and specific assay for robust characterization of microbiota.

The microbiota has an impact on different aspects of cancer immunology.

Mice were treated with antibiotics (ABX) for two weeks and vaccinated with a plasmid DNA encoding MC38 neoantigens delivered by electroporation (EP) [1].

Unexpectedly, ABX treatment allowed a statistically significant reduction of tumor growth in mice vaccinated with DNA-EP.

Clostridiaceae have been associated with the production of specific short-chain fatty acids (SCFA) mainly butyrate and with the induction of T -reg.

The gut microbiome has emerged as a therapeutic approach to immune-stimulation treatment of solid tumours[1,2,3].

Lung cancer is the leading cause of cancer-related mortality worldwide.

Here we used advanced imaging techniques, such as histo-cytometry [1], Clearing-enhanced 3D (Ce3D) microscopy [2] and multiplex FISH, together with single-cell RNA sequencing and high-dimensional unsupervised spatial-temporal analysis, to study the tumor-immune microenvironment in an autochthonous genetically engineered mouse model of lung adenocarcinoma, which is driven by an activating mutation of Kras and a loss of function mutation of p53.

These studies have revealed a role for local dysbiosis in tumor progression, a connection to interleukin-17 secreting gamma-delta T cells in the immune response to the tumor, and the presence in the tumor micro-environment of an unexpectedly heterogeneous population of neutrophils, a cell type known to have adverse effects in lung adenocarcinoma.

Together, these findings are beginning to produce a more complete picture of the three-way interaction among malignant cells, the immune system, and the microbiota in driving tumor progression, with implications for possible interventions to decrease tumor support and facilitate useful anti-tumor immunity.

In the past decade, numerous studies of microbiota have shown the tremendous impact of this symbiont community on human health.

Mice harboring a humanized gut microbiome were sacrificed, and colon tissue collected.

We have optimized and validated a panel of ~ 20 antibodies either targeting cell type makers presented in the small intestine and colon, including immune cell (CD3, CD4 etc), intestinal epithelial cell (DCLK-1etc ), or targeting host functional marker including histone modification (H3K27me3, H3K27ac), inflammatory response (MPO, pSTAT3 etc) or mucus secretion (UEA-1).

We introduce microMIBI, a multiplexed imaging technique that combines bacteria-specific ISH with host specific protein detection, with potentially >40 parameters (Figure 1).

We thank Matt Newgren for tireless technical support on the MIBI instrument, Rachel Finck, Xiao-Jun Ma and Bingqing Zhang for helpful discussions.

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images.

Antigen presenting cells (APCs) process tumor antigens and facilitate recruitment of tumor-specific T cells to generate a robust anti-tumor immune response.

For human in vitro assessment of ISACs, primary PBMCs or myeloid APCs were isolated from human healthy donor blood and activation was measured by flow cytometry, cytokine-bead array, and ELISA-based methods.

In vitro co-cultures with human primary leukocytes and HER2-positive cancer cell lines revealed that the anti-HER2 ISAC potently activates APCs, leading to increased co-stimulatory molecule expression and secretion of pro-inflammatory cytokines.

These data provide a strong rationale for pursuing an anti-HER2 TLR7/8 ISAC into the clinic for the potential treatment of HER2-positive cancers.

Unleashing the adaptive immune response from checkpoint blockade has shown therapeutic efficacy in oncology, although in a subset of patients.

We used solid-phase ELISA and cell binding assays to characterize target binding and blocking by our antibodies.

Here we present novel discovery-stage antibodies that either bind human SIRPα variant 1 or both variant 1 and variant 2, the two most common variants in the human population.

Our novel anti-SIRP antibodies induce promising and differentiated in vitro single agent and combination phagocytosis and show no adverse effects on T cell functionality.

All human material was purchased from Astarte Biologics, INC., 22122 – 20th Ave SE, H-150, Bothell, WA 98021.

Despite clinical success with bispecific antibodies (bsAbs) targeting hematological malignancies (e.g., blinatumomab, a CD19xCD3 bsAb), efficacy in solid tumor indications remains a significant challenge.

COBRA molecules are designed to bind to target antigen, which may be expressed on both tumor and normal cells, yet not engage T-cells unless exposed to a proteolytic microenvironment, which is common in tumors but not in normal healthy tissues.

Here we report the novel design of the COBRA molecule MVC-101 and demonstrate its ability to engage CD3 and Epidermal Growth Factor Receptor (EGFR) to elicit potent cytotoxic activity in vitro and in vivo (Figure 2, 3).

In T-cell dependent cellular cytotoxicity (TDCC) assays, we demonstrate that protease dependent activation increases the potency of MVC-101, yielding a therapeutic with sub-picomolar potency.

The binding assay was measured by testing the ability of DZD2269 to replace radio-labelled ligand for each adenosine receptors.

DZD2269 is a potent, selective A2aR antagonist, which effectively reverses high adenosine induced immunosuppression.

The ability of tumor cells to evade the immune system (immune escape) as well as their acquired resistance to anti-cancer drugs constitute important barriers to the successful management of cancer.

PD-1 mRNA knockdown was assessed by qRT-PCR on total cellular RNA using Taqman probe sets.

These findings support the hypothesis that local PD-1 mRNA silencing by Ph-762 is a viable approach for overcoming tumor-induced immunosuppression resulting in enhanced T cell functionality and tumor cell kill which warrants further investigation in patients.

Despite efficacy of current immunotherapy in specific cancers, most tumors still develop resistance by exploiting different immune evasion pathways.

Syngeneic mouse tumor models, CT26Ep12.1 (colon) and 4T1 (breast) expressing EpCAM receptor were intravenously treated with saline, 1x10^9 EpCAM targeted EDVs (Ep-EDV), or 1x10^9 EpCAM targeted nanocells loaded with PNU-159682 (Ep-EDV-D682).

Immune cells isolated from CT26Ep12.1 and 4T1 mice treated with Ep-EDV-682 showed a significant increase in M1/M2 macrophages, activation of NK cells concurrently with a predominantly Th1 cytokine response.

Targeted nanocells carrying a super-cytotoxic payload are capable of a dual attack on the tumor in syngeneic mouse models of colon and breast cancer.

Mouse experiments were approved by the EnGeneIC Animal Ethics committee, under the following approval numbers, AEC 1/2016, AEC 14/2016, AEC 15/2016, AEC 11/2017, and AEC 05/2019.

Accumulating clinical data suggest that infiltration of solid tumors with immunosuppressive myeloid cells is associated with a lower likelihood of response to T cell checkpoint immunotherapy.

To study the role of ILT3 in these aspects of immunity, we created antibodies that are highly potent and selective for ILT3 over other ILT family members.

These antibodies effectively antagonized polarization by IL-10 to enhance dendritic cell activation state and enable T cell priming.

These data support the testing of anti-ILT3 antibodies in humans for the treatment of cancer.

All mice were maintained under specific pathogen-free conditions in accordance with Merck Research Laboratories (MRL) Institutional Animal Care and Use Committee (IACUC) regulations and were performed in accordance with guidelines in approved protocol P12217.

Target engagement (TE) and translational pharmacodynamics (PD) biomarkers are critical in drug discovery and development.

Hu-NSG mice with subcutaneous SK-MEL-5 or Panc 08.13 tumors were treated with an anti-human ILT3 mAb.

TE assessed by ILT3 occupancy on the surface of CD14+ myeloid cells and by plasma total sILT3 levels was detected after single dose administration of the anti-ILT3 mAb ranging from 0.003 to 20 mg/kg.

High levels of ILT3 receptor occupancy in the tumor were required for the upregulation of immune activation markers in the humanized tumor model.

Regulatory T cells (Treg) critically maintain immuno-suppression in the tumor microenvironment, representing an attractive immuno-oncology target.

We have discoverd a clinical candidate AZD8701, a next-generation antisense oligonucleotide inhibitor of FOXP3 (utilizing the Ionis Gen 2.5 cEt-modified ASO platform).

AZD8701 treatment knocked down FOXP3 in primary human Tregs via free uptake (IC50 65nM), which was also associated with modulation of known FOXP3 target genes including 25-50% reduction in CTLA4, ICOS, CCR8 and GITR.

In summary, AZD8701 represents a first-in-class clinical candidate to target Tregs in cancer in a highly selective manner, and may provide therapeutic benefit to patients either as a monotherapy or in combination with immune checkpoint blockade.

Current immunotherapies have shown promise in treating multiple cancer types but success remains elusive for cancers like pancreatic, the majority of colorectal cancers as well as cholangiocarcinoma.

Significantly higher levels of galectin-9 were detected across patient blood/tissue samples (n >100 patients) compared with healthy controls.

LYT-200, a first-in-class human antibody that inhibits tumor-permitting activity of Galectin-9 was developed.

Tumour-associated peptide-human leukocyte antigen complexes (pHLA) represent the largest pool of cell surface expressed cancer-specific epitopes, making them attractive targets for cancer therapies.

Using structural, biochemical, functional and computational approaches, we assessed the selectivity of two novel classes of T cell redirecting pHLA-targeting bispecifics based on TCR-mimic antibodies or high-affinity TCRs.

We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side-chains.

These findings extend our understanding of the basic principles that underpin pHLA-selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.

This poster illustrates an exciting new way to characterize cell populations far beyond traditional methods.

25,000 cells were isolated using Celsingle 250K Slide on the Genesis System and sample preparation protocol was performed.

We observed signal from all 60 antibodies across the population of thousands of cells analyzed.

These data highlight the simplicity and feasibility of multiplexing 60 antibodies in a single run with the Genesis System.

Only 40% of the patients with metastatic melanoma respond to FDA-approved immune-checkpoint blockade therapies.

Murine melanoma cell lines: B16F10, B16F1, SM1 and D4M were cultured in DMEM-based media for 24 hours and cell-free supernatant was quantified for VIP concentration via VIP-specific competitive Enzyme Immunoassay (EIA).

All melanoma cell lines secrete low levels of VIP (Figure 1B).

In preclinical melanoma models, pharmacological antagonism of VIP-signaling significantly increased survival rates (Figure 4) by activating adaptive T cell immunity.

The tumor microenvironment (TME) has multiple mechanisms of immunosuppression, one being recruitment of arginase (ARG) expressing myeloid cells.

Human CD8+ T cell and NK cell proliferation was evaluated in various concentrations of L-arginine by measuring CellTrace Violet (CTV) dilution in response to stimulation (anti-CD3/anti-CD28 stimulation for CD8’s;

Human CD8+ T cells and NK cells showed decreased proliferation with decreased L-arginine concentration.

Our pre-clinical data demonstrates that an ARG inhibitor in combination with a checkpoint inhibitor can reverse tumor immunosuppression leading to strong immune activation and anti-tumor responses, suggesting arginase inhibitors could provide an opportunity to increase activity of checkpoint inhibitors clinically.

Cancer immunotherapies have demonstrated remarkable clinical benefits and durable responses even for late-stage cancers.

Using our common light chain discovery platform we have developed a novel class of multi-specific antibodies that bind to one or two antigens at the surface of tumor cells and engage both CD16 and NKp30 activating receptors on NK and gamma deltaT cells.

Unlike conventional monoclonal antibodies that require high levels of target antigen for efficacy, our TAAxNKp30 engagers can potently activate NK and gamma delta T cells to kill tumor cells that express high, medium and low levels of tumor antigen.

Compass NKp30 engagers are highly differentiated from monoclonal antibodies for their capability to activate different subsets of NK and gamma delta T cells and induce killing of tumor cells expressing low levels of tumor associated antigen.

NEO-201 is an IgG1 mAb reactive against many different carcinomas, but not reactive against most normal tissues.

PBMCs were collected from 5 normal donors and used for phenotypic and functional analysis.

The % of NEO-201+ cells in the population of CD4+CD25highCD127negFoxP3+CD15s+CCR4+Tregs ranged from 60%-80%.

NEO-201 reacts against human Tregs and can be used as a novel marker to identify and to purify Tregs.

In diverse human and murine cancers, tumor necrosis factor receptor 2 (TNFR2) is heavily expressed in the tumor microenvironment on regulatory T cells (Tregs) and myeloid-derived suppressor cells.

We analyzed TNFR2 expression in humans using mean fluorescent intensity (MFI) of TNFR2 on Tregs, T effector (Teff) cells and primary tumor cells from subjects with lymphoma vs controls and analyzed over 1000 human cell lines from different primary tumor sites.

The TNFR2 antagonists killed tumor-residing Tregs and cancer cells expressing the oncogene in vitro.

Dominant TNFR2 antagonists kill both tumor-residing Tregs and cancer cells expressing the oncogene in human specimens but not normal circulating cells.

Oncolytic viruses (OVs) represent a new class of therapeutic agents for the treatment of cancers that poorly respond to immune checkpoint inhibitors.

ONCR-177 is a highly engineered recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumor indications.

In in vitro co-culture studies, DCs loaded with ONCR-177 oncolysates were superior in stimulating MART1+ tumor antigen-specific T cells compared to lysates from the unarmed vector.

In conclusion, ONCR-177/mONCR-171 potently activates local and systemic anti-tumor immune responses that result in durable responses, extended survival, and the elicitation of protective immunity.

Whole blood was collected from healthy donors as described in the ethical protocol/amendment IXP-001_V3 (Belgium;

Photoimmunotherapy (PIT) is an investigational cancer treatment platform that utilizes an antibody-dye (IRDye® 700DX, IR700) conjugate and illumination with a non-thermal, red-light laser for selective cell killing.

Immunocompetent mice inoculated with CT26 tumors were treated with CD25 PIT alone or in combination with anti-PD1 treatment.

Single tumor bearing mice treated with saline, anti-PD1 antibodies, CD25 conjugate without PIT, CD25 PIT alone, or in combination with anti-PD1 antibodies, resulted in complete response (CR) rates of 0% (0/15), 8.3% (1/12), 0% (0/15), 50.0% (5/10), and 73.9% (17/23), respectively.

Depletion of intratumoral Tregs by CD25 PIT resulted in the local activation of CD8+ T-cell mediated anti-cancer activity, and a systemic tumor specific immune memory response.

All animal studies conducted followed the guidelines put out by the 'Guide to the Care and Use of Laboratory Animals' by the National Research Council.

Stimulating existing anti-cancer immune responses by activating T cell costimulatory receptors of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) is potentially the next significant stage of cancer immunotherapy.

To understand factors contributing to toxicity, different CD137 agonist antibodies were compared for their ability to activate T cells in the presence or absence of secondary crosslinking antibodies, used to mimic the effect of FcgR-mediated crosslinking in vivo.

The CD137 antibodies tested showed varying levels of in vitro potency in stimulating T cells and certain antibodies were able to induce crosslink-independent activation.

CD137 agonist antibodies associated with increased liver inflammation were found to be crosslink-independent, suggesting this factor may contribute to their hepatotoxicity risk.

Over the last decade, various new therapies have been developed to promote anti-tumor immunity.

We report here the design, production and characterization of a new generation of trifunctional NK cell engagers (NKCEs) consisting of mAb fragments targeting the activating NK cell receptor NKp46 together with a Tumor Antigen (TA) and an Fc fragment, to promote antibody-dependent cell-mediated cytotoxicity (ADCC) via the activating receptor CD16 expressed on NK cells.

Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen.

Trifunctional NKCEs targeting CD19, CD20 or EGFR as tumor antigens triggered tumor killing by human primary NK cells in vitro.

Notch receptor-ligand interactions are an important regulation governing T-cell-mediated immune responses and are highly contextual.

We engineered monomeric soluble JAG1 (N-EGF3, sJAG1) and multimeric clustered DLL1 constructs and evaluated their immunological effects in lung and pancreatic cancer murine models.

Treating tumor-bearing mice with monovalent sJAG1 and multivalent DLL1 elicit antigen-specific T-cells, significantly reduce Treg frequency in spleen and lymph nodes, improve anti-tumor immunity, attenuate tumor growth, and increase IFN-γ production by splenocytes and improve mice survival compared to untreated mice.

Data demonstrate efficacy of functional Notch ligand-based constructs in reducing exhaustion and improving anti-tumor T-cell responses.

The animal studies were performed at The Ohio State University Wexner Medical Center in compliance with the protocol approved by the Institutional Animal Care and Use Committee regulations.

The checkpoint inhibitor (CPI) class of immunotherapeutic drugs demonstrates a key role for active T cells in anti-tumor immune responses and positive clinical outcomes in cancer treatment.

Our IRC™ technology was used to generate unbiased and virtually error-free, natively paired heavy and light chain sequences of antibodies expressed by individual B cells isolated from patient samples.

We expressed plasmablast-sourced antibodies that, in part, had evidence of progressive affinity maturation and class switching and then performed in vitro and in vivo studies to further characterize each antibody for their (i) target identity, (ii) activity profile in vitro (iii) ability to cause tumor growth inhibition, regression and immunologic memory in vivo in a panel of model systems and (iv) safety profile in non-human primates.

Using our methodology we now report on ATRC-101, a fully human IgG1/kappa engineered version of an antibody and identified by IRC™ from a plasmablast B cell isolated from a patient with NSCLC adenocarcinoma who had been treated, in part, with checkpoint inhibitor therapy.

Based on robust in vitro and in vivo data ATRC 101 is now being advanced to the clinic for evaluation in solid tissue malignancies.

We acknowledge the significant effort and contributions of our colleagues from the histology, in vivo pharmacology, in vitro pharmacology and target identification groups as well as our consultants.

TAA/CD3 bispecific antibodies are highly effective at depleting cancer cells and have achieved encouraging results in the clinical setting.

We produced a novel HER2/PD-L1/HSA tri-specific scMATCH3 (ND030) and ND022 to evaluate the con-tribution of the αPD-L1 domain to tumor cell-depletion and to confirm that PD-L1-blockade is restrict-ed to the cytolytic synapse.

Both ND030 and ND022 strictly inhibit PD-1-signaling on T cells when such cells are co-cultured with a PD-L1+HER2+ cancer cell-line, but not on HER2− cells, confirming cytolytic synapse-restricted PD-L1-binding.

ND022 is a next-generation tetra-specific molecule with exquisite antitumoral efficacy in HER2+ cancer models.

OX40 (CD134, TNFRSF4) is a type I transmembrane cell surface protein member of the TNF receptor superfamily transiently expressed on recently activated T cells, present at a low level on memory CD4 T cells, and is expressed on activated and intratumoral T regulatory cells.

ABBV-368 was selected based on the best bioactivity in these assays.

ABBV-368 is an anti-OX40 agonist with unique signaling activity that is efficacious in human cell in vitro and in vivo models and represents a promising clinical candidate currently being evaluated in several ongoing Phase I clinical trials (NCT03071757;

We characterised three newly isolated TCRs specific for the cancer testis antigen NY-ESO-1 a.a 157-165 presented on HLA-A*02:01.

We show that all three TCRs have different peptide specificity profiles, enabling recognition of distinct off-target peptides.

We demonstrate that TCRs targeting the same antigen can achieve the necessary specificity to achieve self-tolerance despite recognising distinct peptide repertoires.

4-1BB (CD137/TNFRSF9) is an inducible costimulatory receptor belonging to the TNF receptor superfamily.

Unlike traditional biologic approaches, the small size (~2 kDa) and tunable pharmacokinetic (PK) parameters of Bicycles enable superior tumor penetration and allow exploration into the relationship between pulsatile dosing and 4-1BB activation while de-risking hepatoxicity concerns due to a differentiated renal clearance mechanism combined with tumor-localized activation.

We envisioned clustering and activation of 4-1BB could be achieved by conjugating a 4-1BB binding Bicycle to a tumor antigen targeting Bicycle.

Nectin-4/PVRL4 is highly expressed in numerous tumors, including bladder, pancreatic, and lung.

These data indicate that the biological effect of the bispecific Bicycle agonist persists beyond the detection of the molecule in systemic circulation and suggests that continuous coverage of the target may not be necessary when activating 4-1BB with low molecular weight and highly tumor penetrant Bicycles.

Cryopreserved human dissociate tumor cells (DTCs) and PBMCs were purchased from approved vendors that follow stringent Institutional Review Board (IRB) and Ethics Committee compliance.

Promising clinical activity has been observed with multiple CD3 bispecific antibodies (bsAbs) in hematological malignancies.

DuoBody-CD3x5T4 is an Fc-silenced IgG1 bispecific antibody that was obtained by controlled Fab-arm exchange of a humanized CD3ε and a human 5T4 monoclonal antibody.

The expression of 5T4 in different tumor types was confirmed by flow cytometry across tumor cell lines, and immunohistochemistry in TMAs for patient-derived samples.

DuoBody-CD3x5T4 is a bispecific antibody that induces CD4+ and CD8+ T-cell activation, cytokine production and T-cell-mediated cytotoxicity of 5T4-expressing tumor cells in vitro and in vivo.

The CDX animal experiments performed are in compliance with the Dutch animal protection law (WoD) translated from the directives (2010/63/EU) and are approved by the Ethical committee of Utrecht.

Tumor cell-extrinsic mechanisms, such as exclusion of cytotoxic T lymphocytes (CTL) and immunosuppression by tumor associated macrophages, myeloid-derived suppressor cells, and regulatory T cells, contribute to immunotherapy primary resistance.

SBT6050 activation and reprograming of myeloid cells was evaluated in vitro using human myeloid cell:HER2-expressing tumor cell co-culture assays with flow cytometry and MSD-based ELISA read-outs.

SBT6050 potently activates human myeloid cells to produce pro-inflammatory and TH1 priming cytokines such as IL-12p40 and IFN-gamma in the presence of HER2-expressing tumor cells.

These data show the potential for SBT6050 to reprogram an immunosuppressive TME and support the clinical evaluation of SBT6050 as a monotherapy and in combination with CPI in HER2-expressing tumors, including those with low pre-existing CTL infiltrate.

The promise of IL-12 as a treatment for cancer has never been fulfilled despite numerous clinical trials.

IL-12 was displayed on the surface of exosomes by genetic fusion to PTGFRN.

The exoIL-12 constructs retained equivalent potency in vitro to rIL-12 as demonstrated by IFNγ-release using primary human PBMCs or murine splenocytes.

ExoIL-12 is a novel cancer therapeutic candidate with tumor-retained pharmacological activity and superior potency as compared to the rIL-12.

The study was approved by Codiak BioSciences' internal IACUC committee, approval number CB2017-001.

KEYNOTE-045 (ClinicalTrials.gov identifier: NCT02256436) demonstrated statistically and clinically meaningful improvements in overall survival (OS) in 2L mUC subjects treated with pembrolizumab versus investigator’s choice of vinflunine, docetaxel, or paclitaxel (median survival, 10.1 versus 7.3 months;

To synthesize RCT evidence and indirectly compare the relative treatment effects of pembrolizumab versus atezolizumab, fixed-effect Bayesian network meta-analysis (NMA) was conducted to determine the relative efficacy of both treatments.

Pembrolizumab consistently showed favorable OS benefit over atezolizumab in both analytical approaches (NMA method: HR [95% CrI] = 0.83 [0.63-1.07];

These results suggest that pembrolizumab offers a better probability of prolonging OS compared to atezolizumab as 2L therapy for mUC patients.

CD3ε is one of the key signaling subunits of the CD3 co-receptor of the T cell receptor (TCR) complex.

To expedite the in vivo interrogation of human CD3-based therapeutics, Biocytogen generated the B-hCD3ε mice where the extracellular domain of human CD3ε replaces that of the mouse counterpart.

B-hCD3ε mice responded well to anti-PD-1-mediated tumor suppression and exhibited T cell depletion by an anti-human CD3 antibody.

Taken together, Biocytogen’s B-hCD3ε mice are validated and exhibit expected T cell profile, robust in vitro activity, and efficacious in vivo response to anti-human CD3 antibody and anti-human CD3 bispecific antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory T cell signaling axis by sharing common ligands CD80 and CD86, which regulate antigen-specific T cell immunity.

In vivo, ADG116 alone exhibits robust dose-dependent single agent anti-tumor activity in established tumors and/or increases overall survival in syngeneic mouse models of liver, breast, lung and colon cancers, as well as induces durable antigen-specific memory immunity that protect animals from re-challenged tumor cells.

The activity of ADG116 to enhance activation and inflammatory cytokine release of primed T cells in vitro and specifically deplete intra-tumoral Treg cells in vivo, is consistent with the MOA by which ADG116 acts through two distinct mechanisms: 1) functions as a checkpoint inhibitor to directly enhance effector T cell activation, and 2) functions as an ADCC targeting antibody to alleviate immunosuppression in tumor microenvironment through specific Treg depletion in tumors but not in peripheral tissues.

Disialoganglioside GD2 is abundantly expressed on neuroblastoma (NB) with little expression on normal cells, thus is a suitable target for immunotherapy.

Between 2012 and 2014, six high-risk NB patients (2-8 y);

Vaccinations were well tolerated with mild transient local reactions only.

CD39-CD73-adenosine pathway plays an important immuno-suppressive role within the tumor microenvironment (TME) as adenosine receptor signaling suppresses effector T cells and enhances immune inhibitory effects of Tregs and MDSCs.

We have generated a CD39 antibody, ES002, by traditional hybridoma technology using human CD39 overexpressing HEK293 cells as immunogen, and the antibody was subsequently humanized through complementarity determining region grafting.

ES002, a humanized anti-hCD39 antibody, binds to human and cynomolgus CD39 with sub-nanomolar binding affinity and effectively blocks CD39 ATPase activity.

We have successfully developed a potentially best-in-class anti-CD39 therapeutic antibody with strong efficacy in tumor growth inhibition.

Altered metabolism is an important mechanism by which tumors evade detection and destruction by the immune system.

IL4I1 enzymatic activity was measured using an HRP-coupled enzyme assay.

Interrogation of The Cancer Genome Atlas (TCGA) revealed high expression of IL4I1 in multiple solid tumor types and correlated with reduced overall patient survival (HR = 1.5, p = 4.4 x 10-15).

These results indicate that small-molecule inhibition of IL4I1 may represent a novel strategy for cancer immuno-therapy.

Guanylyl Cyclase C (GUCY2C) is a regulator of intestinal homeostasis and is expressed in more than 90% of colorectal cancer (CRC), as well as in other gastrointestinal malignancies [1].

GUCY2C-CD3 mediated activity was evaluated in vivo in several cell line-and patient derived-xenograft models of CRC, using adoptive transfer of human T cells in established subcutaneous and orthotopic tumors.

GUCY2C-CD3 bispecific showed preferential biodistribution to GUCY2C expressing xenograft tumors compared to normal tissue.

Our preclinical data demonstrate that a GUCY2C-CD3 bispecific can selectively target colorectal tumors, including those with KRAS or BRAF mutations, which are difficult to treat with currently approved therapies.

All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.

Although immune checkpoint inhibitors (CPI) targeting the CTLA-4 and PD-1/PD-L1 pathways have demonstrated significant clinical activity in many cancers, many patients fail to respond, or they develop acquired resistance due at least in part to insufficient anti-tumor T cell activation and/or exhaustion.

X-ray crystallography and cell-based reporter assays were used to demonstrate the simultaneous binding of PD-L1 and CD28 to the single CD80 IgV domain of ALPN-202.

The CD80 vIgD:PD-L1 crystal structure was elucidated at a resolution of 3.15 Å, revealing a non-overlapping binding interface distinct from the previously published CD28:CD80 interaction (Figure 2).

The CD80 IgV domain utilizes separate, non-competing epitopes to bind CD28 and PD-L1.

Animal experiments and euthanasia protocols performed in this study were approved by the Alpine Immune Sciences Institutional Animal Care and Use Committee (IACUC protocol IR 17-01)

4-1BB (CD137) is a member of TNF superfamily involved in the activation of several immune cells including T cells and NK cells.

Tumor targeting EphA2/4-1BB Bicycles are chemically synthesized using our proprietary Bicycle platform and the molecules are tested for activity in various in vitro assays and relevant animal models.

The unique ability of EphA2/4-1BB dual targeting Bicycles to precisely and potently activate immune cells in tumors without systemic immune activation is very promising and provides us a rationale for developing first-in-class Bicycles to target EphA2+ cancers.

Monoclonal antibody therapies have transformed cancer treatment.

Immune modulating antibodies modify immune cell function regardless of tumor antigen expression while direct targeting antibodies bind directly to antigens on tumor cells and mediate their anti-tumor functions through inhibiting signaling, targeting toxins or by additionally interacting with immune cells or complement through their Fc domains.

Each antibody was tested for its ability to bind to multiple different MUC1+ cancer cells and to interact in co-cultures with NK cells, macrophages, and complement to mediate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).

Several of the antibodies could mediate ADCC and ADCP, although none of the antibodies mediated an appreciable amount of CDC.

Fully human MUC1 IgG1 antibodies generated in healthy individuals at risk for cancer were capable of mediating immune effector functions such as ADCC and ADCP and are candidates for further testing as human therapeutics.

The immunosuppressive role of adenosine attracts attention of many researchers as an interesting target for anticancer therapy.

We are demonstrating how our inhibitors restore adenosine depleted immune functionality in the series of functional in vitro assays in primary cells.

By head-to-head comparison of inhouse developed dual A2A/A2B antagonists to compounds currently pursued in clinical development we have demonstrated the best-in-class characteristics of our molecules.

New class of dual A2A/A2B adenosine receptor antagonists combines two crucial activities which addresses distinct, cell type mediated mode of action of adenosine related immunosupression.

Transgene-enhanced oncolytic adenoviruses (OVs) represent a promising novel therapeutic option for the treatment of cancer especially for patients with chemotherapy- and immunotherapy-resistant, advanced, aggressive, and systemic disease.

In 129S1 immunocompetent mice bearing ADS-12 tumors (mouse lung carcinoma), BETA-PRIME was injected intratumorally and intravenously at the following dose levels in groups of 25 mice each (Table 1) Group Dosed Article Route Dose Level (per animal) Term Group 1 None - 0 Untreated Group 2 Vehicle IT Vehicle 0.1mL IT Vehicle Group 3 Vehicle IV Vehicle 0.2mL IV Vehicle (high volume) Group 4 BETA-PRIME IT 5E8 VP /0.1 m IT Low Dose Group 5 BETA-PRIME IT 5E9 VP /0.1 mL IT Medium Dose Group 6 BETA-PRIME IT 5E10 VP /0.1 mL IT High Dose Group 7 BETA-PRIME IV 1E11 VP /0.2 mL IV High Dose Group 8 Vehicle IV 0.1 mL IV Vehicle (low volume) Group 9 BETA-PRIME IV 5E9 VP /0.1 mL IV Low Dose Soluble TGF-beta trap was detected in the serum using ELISA

single viral injection resulted in long-term TGF-beta trap expression of over a week in the serum from all doses of intravenous and intratumoral groups while in vehicle treated mice TGF-beta trap was not detected. In the high dose IV group the TGF-beta trap was detected at concentrations of 210-585 ng/ml while in high-dose intratumoral mice circulating TGFb trap was detected at concentrations of 4-13 ng/ml.

Despite local injection of BETA PRIME, systemic levels of the TGF-beta trap transgene were observed, which demonstrates that the virus follows the “think globally act locally” dictum and may in part account for evidence of regression of non-injected, distant tumor lesions.

Chemerin (RARRES2) is an innate leukocyte chemoattractant known to recruit cells that express its chemotactic receptor (CMKLR1) along its concentration gradient.

No significant difference in in vitro cell proliferation, nor differences in expression of CMKLR1, MHC class I, CD-1d, CD44, or PD-L1 levels was found between control or chemerin-expressing lines.

Here we show for the first time that modulation of chemerin, an innate leukocyte chemoattractant, can result in significant prostate tumor suppression using an immune competent preclinical model.

The study was approved by Washington University's IACUC institutional review board, approval number 20170174.

The proportion of patients who benefit from checkpoint inhibitor (CPI) therapy is modest, and additional immune pathways need to be targeted to improve overall patient outcomes.

We developed a lead anti-TREM2 mAb, termed PY314, that was designed to deplete TREM2-positive TAMs.

The PY314 mAb binds with high affinity to human, cynomolgus and mouse TREM2.

Collectively, the available preclinical and nonclinical data support PY314 immunotherapy, alone or in combination with a CPI, in cancer patients who are resistant or refractory to CPI therapies, to improve both the overall response rates as well as the durability of responses.

Targeting and engineering γδ T cells has recently emerged as an orthogonal therapeutic approach in oncology with capacity to modulate both innate and adaptive immune properties.

We determined the amino acid sequences of tumor specific δ1-TCR chains from primary PDA, CRC and gastric cancer samples.

Because the tumor-derived δ1 chains showed diverse CDR3 sequences, we developed antibodies that bind diverse δ1 TCRs.

We have defined a novel therapeutic immuno-oncology strategy and translated it to develop a lead clinical candidate anti-δ1 monoclonal antibody.

~40% of newly diagnosed lung cancer patients have tumors that have metastasized and non-small cell lung cancer (NSCLC) has a poor prognosis.

Solid tissue biopsy samples were dissociated into single cells and stained with a total 26 immune, and cancer markers (aneuploidy) that were then analyzed with high-parameter flow cytometry.

Correlations in CTC number and the frequency of immune PD-1+ lymphocytes with the PD-1+CD103+CD4+ T lymphocytes which are a biomarker for T- regulation cells, are proximal to the tumor.

Profiling multiple immune and cancer markers on cancer samples with multi- parametric flow cytometry yielded protein expression information at the single cell level.

Human papilloma virus (HPV)-driven cancers cause significant morbidity and mortality worldwide.

Human E7-specific T cells (Astarte Biologics, Bothell, WA) and human PBMCs were tested to demonstrate cellular activity and specificity of CUE-101, while in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice.

The mutated IL-2 domains of CUE-101 exhibit reduced functional activity relative to wildtype IL-2.

Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E7(11-20)-specific population of cytotoxic CD8+ T cells in vitro and in vivo.

All animal studies were approved under SmartLabs Institutional Animal Care and Use Committee protocol MIL-100 and were performed in compliance with federal guidelines.

Engineered Toxin Bodies (ETBs) are comprised of a deimmunized Shiga-like toxin-A subunit (DI-SLTA) genetically fused to an antibody-based targeting domain.

In vitro activity of ETBs was measured by cell viability and kinetic imaging of labeled target cells in an effector T-cell co-culture assay.

ETBs targeting PD-L1 showed potent and specific activity across human tumor cell lines at picomolar concentrations in vitro.

The potent and direct activity of ETB mediated tumor cell destruction is demonstrated across tumor cell lines from multiple indications, on PD-L1 positive immune cells ex-vivo, and by tumor growth and survival benefits in PDX models.

IL-27, a heterodimeric member of the IL-12/IL-23 cytokine family, consists of two subunits, IL-27p28 and Epstein-Barr virus-induced gene 3 (EBI3).

To explore the therapeutic potential of IL-27 blockade, SRF388, a first-in-class, fully human anti IL-27 monoclonal antibody that binds to the p28 subunit of IL-27 with high affinity, was developed.

SRF388 prevented IL-27 from binding to IL-27RA, inhibited IL-27-induced STAT1 phosphorylation in human cell lines and primary T cells, and blocked IL-27-responsive immunoregulatory receptor expression in human PBMCs.

Collectively, these data demonstrate that IL-27 exerts multiple immunosuppressive effects and that SRF388, a first-in-class antibody targeting IL-27, can potentiate antitumor responses in cancers that rely on IL-27-mediated immune escape.

The human GITR/GITR-L complex was co-expressed, purified, crystallized, and structure solved by an initial molecular replacement solution using monomeric ELR1 as the search model (PDB: 3WVT).

We described the first crystal structure of the human GITR/GITR-L complex at a resolution of 2.85 Å which revealed an unusual receptor-mediated non-covalent dimer of two GITR-GITR-L trimeric assemblies forming a hexagonal network.

Our data suggests that GITR-L or bivalent antibody-mediated GITR agonism on T cells is critically dependent on the receptor’s structural conformational geometry.

There is a critical, unmet clinical need for effective therapies for osteosarcoma, a rare bone cancer that primarily affects adolescents and young adults.

Dogs with spontaneous osteosarcoma were enrolled in a veterinary clinical trial.

28 dogs were enrolled and treated on the study.

Neoadjuvant VSV treatment is well-tolerated, and shows preliminary evidence of biological activity and clinical efficacy.

Adaptive immunity to cancer cells has been shown to form a crucial part of cancer immunotherapy.

Peripheral blood memory B cells were immortalized using AIMM’s immmortalisation technology (ectopic Bcl-6 and Bcl-xL expression) [2] and analyzed for the presence of tumor-reactive B cells.

The AT1412 antibody was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes.

Taken together, applying AIMM’s proprietary B-cell immortalization technology [2] we isolated a tumor-selective anti-CD9 antibody (AT1412).

The protocol for the melanoma clinical study was approved by the Medical Ethics Committee of the Leiden University Medical Center and conducted in accordance with the Declaration of Helsinki.

Heat Biologics is focused on developing and optimizing a next generation cellular vaccine platform that incorporates a tumor antigen chaperone (gp96-Ig) with costimulation into a single tumor cell line overexpressing a host of cancer associated neoantigens.

As a model, mouse surrogates of each human product (mHS-110 and mHS-130) were generated to activate and expand adoptively transferred OVA-specific T cells (OT-1) responding to tumor challenge with B16F10 cell line over expressing OVA.

Combining mHS-110/gp96-Ig with mHS-130/OX40L-Ig at a ratio of 1 to 1.3, i.e, 339 ng/10^6 cells/24 hrs of gp96-Ig to 441 ng/10^6 cells/24 hrs of OX40L-Ig provided the optimal dose combination for long-term survival and expansion of tumor specific CD8+ T-cells, tumor infiltrating lymphocytes (TILs) and greatest tumor growth inhibition (TGI).

Best dose group combination mHS-110/gp96-Ig to mHS-130/OX40L-Ig was at a ratio of 1 to 1.3, at a dose rate of 339 ng/10^6 cells/24 hrs of gp96-Ig to 441 ng/10^6 cells/24 hrs of OX40L-Ig, with a NOEL of 38 ng gp96-Ig to 50 ng OX40L, a MABEL of 113 ng gp96-Ig to 147 ng OX40L-Ig.

As clinical outcomes improve for immunotherapeutic regimens based on PD-1/L1 blockade, the proportion of cancer patients who develop resistance to PD-1/L1 blockade will increase.

Human and mouse variants of CD86-Fc-NKG2a were produced and characterized using a variety of biochemical assays to determine the correct molecular weight, subunit composition and binding affinity;

The NKG2a domain bound recombinant and cell-expressed HLA-E with high affinity.

The data presented here demonstrates the feasibility and function of a dual fusion protein, designed to competitively inhibit the NKG2a/HLA-E NK checkpoint pathway, while providing costimulation via CD86.

Despite the profound clinical impact that immune checkpoint inhibitors (ICIs) have had, they benefit only a fraction of cancer patients due in part to the multitude of immunosuppressive networks within the tumor microenvironment (TME).

Using a combination of functional genomics and bio-selection strategies, we generated a novel Vaccinia Virus (VACV) backbone, denoted as SKV, having augmented oncolytic activity, improved tumor selectivity and less immunosuppression compared to wild-type VACV.

SKV generates more potent anti-tumor effects and shows enhanced synergy with ICIs compared to that of other clinical VACV candidates.

Our new best-in-class recombinant VACV robustly stimulates systemic anti-tumor immunity and has a substantially improved pre-clinical safety profile compared to other VACV clinical candidates.

To improve the proportion of patients who benefit from checkpoint inhibitor (CPI) therapy additional immune pathways likely need to be targeted.

Human whole blood, peripheral blood mononuclear cells, isolated myeloid cells, or dissociated tumor cells were treated with monoclonal antibodies (mAbs) and analyzed by flow cytometry and Meso Scale Discovery.

We identified Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) as a target on TAMs, TANs, and MDSCs, and developed anti-human and anti-mouse TREM1 mAbs, termed PY159 and PY159m, respectively.

The available preclinical and nonclinical data support PY159 immunotherapy, alone or in combination with a CPI, in cancer patients who are resistant or refractory to CPI therapies, to improve both the overall response rates as well as the durability of responses.

Many peptide vaccines have failed in clinical trials due to insufficient efficacy.

We immunized HLA-A2, -A24 and -A31 KI mice with TAS0313, and evaluated the epitope-specific CTL induction in lymph nodes using an interferon-γ ELISPOT assay.

We demonstrated that TAS0313 can enhance CTL induction and antitumor effects in vivo.

The interferons, including IFNα/IFNβ (type I) and IFNγ (type II) are essential mediators of anti-cancer immunity.

The VH and VL regions from antibody 2B8 recognizing human CD20 were engineered in recombinant form with human IgG1 constant regions, and fused at the C-terminus with mIFNgamma.

Anti-hCD20-mIFNγ suppressed the in vitro proliferation of 38C13-huCD20 lymphoma cells by up to 70% (at 1 nM).

Mechanistic studies showed that depletion of natural killer (NK) cells (using anti-asialo-GM1) significantly abrogated tumor clearance (p=0.01), while depletion of macrophages (clodronate liposomes) had lesser, borderline effects (p= 0.05) (Figure 2), and depletion of complement (cobra venom factor) or T cells (CD4+ or CD8+) had no significant effects on tumor eradication.

Anti-hCD20-mIFNγ fusion protein has in vitro and in vivo efficacy in a syngeneic, immunocompetent model of B cell lymphoma, with NK cells and possibly macrophages implicated in the mechanism(s) of tumor eradication.

Antibody-based immunotherapies have demonstrated improved anti-tumor responses and success in the clinic.

Target protein inhibition was assayed using HEK293 cells overexpressing constructs comprised of signal sequences fused to a luciferase reporter.

We identified a series of compounds that selectively block Sec61-dependent cotranslational translocation of select immune checkpoint proteins.

Small molecules targeting Sec61-dependent cotranslational translocation provide a new approach for blocking multiple immune checkpoint proteins with a single agent.

Glutamine is an essential amino acid for rapidly proliferating cancer cells, thus depriving the same fuel from immune cells and contributing to tumor immune evasion.

Anti-tumor and immunomodulatory effects of DRP-104 were evaluated in vitro using biochemical, cellular and functional assays.

DRP-104 and its active moiety DON showed glutamine dependent inhibition of cancer cell growth in vitro, correlating with broad glutamine pathway inhibition.

DRP-104 is a novel, broad acting glutamine antagonist with potent single agent anti-tumor activity.

Prior research demonstrates tumours with low neoantigen burden respond poorly to T-cell checkpoint blockade, with neoantigen-specific T-cell responses important to tumour regression [1].

Optimised lead series potently inhibit ERAP1 across key species and ERAP1 haplotypes.

We have identified potential first in class, potent and species cross reactive small-molecule inhibitors of ERAP1 that modify the immune peptidome creating neoantigens.

Certain Epidermal growth factor receptor (EGFR) mutations are targets of tyrosine kinase inhibitors (TKIs) and predictive biomarkers of clinical response to TKI therapy.

The immunogenic T cell epitopes of EGFR exon-20 insertions coding peptide were predicted using multiple public bioinformatic algorithms referring on their expression level and major histocompatibility complex (MHC) class I-binding capacity.

Ninety-one types of insertions in EGFR exon-20 were identified, among which two types occurred with considerable high mutation frequencies: V769_D770insASV (19.02%) and D770_N771insSVD (19.2%).

E-ASV could induce cytotoxic T lymphocyte (CTL) responses in vitro, and EGFR exon-20 insertion-based neoantigen peptide might become an alternative immunotherapeutic approach for NSCLC.

This work was supported by National Natural Science Foundation of China (grant numbers: 81872143, 81702280, 81472473, 81272360) and Key Project of Tianjin Health and Family Planning Commission (16KG126).

CD38 is a transmembrane protein with relatively low expression levels on immune cells and some non-hematological tissues.

To determine potential mechanisms that may affect the efficacy of isatuximab, we conducted in vitro studies using a panel of CD38 expressing MM and DLBCL cell lines, as well as primary MM cells from patients.

PD-1 expression on PBMCs was induced shortly after co-culture with MM cells.

Our observations demonstrate that ADCC is a key mechanism for the antitumor activity of isatuximab and provides a rationale supporting the testing of isatuximab in combination with anti-TGFβ or anti-PD1 antibodies to determine whether isatuximab-mediated killing of CD38-expressing tumor cells is improved.

Emerging evidence suggests that combining radiotherapy (RT) and immunotherapy (IT) may enhance outcomes for metastatic non-small cell lung cancer (NSCLC) patients.

Patients diagnosed with stage IV NSCLC between 2004-2016 treated with IT alone or RT+IT were identified in the National Cancer Database.

Early-incorporation IT utilization increased from 0.3% in 2010 to 13.2% in 2016 among patients with stage IV NSCLC (p<0.0001).

The Institutional Review Board deemed this study exempt from review given the de-identified nature of the data.

Ovarian cancer is generally asymptomatic until the advanced stages.

The c-MET/ HGF pathway is often studied in the context of its tumorigenic properties, but the focus of this research was to study its potential to regulate T cell immune responses in ovarian cancer, with the future goal of using this molecule in combination therapy design.

c-MET inhibitor Capmatinib treatment of mice resulted in an average ascites volume of 4.34 ml (Figure 1), and vehicle treatment 9.2 ml (>

With these anti-tumor responses, the performance of Capmatinib for ovarian cancer treatment is promising.

Studies were conducted with the approval of our IACUC board (protocol LU 205167).

Among the factors that influence response to immunotherapy, germline genetics has been linked to outcome [1,2].

Mice obtained from Envigo, Taconic, and Jackson labs were injected subcutaneously with various C57BL/6-derived cell lines including the melanoma line B16.F10.SIY (B16.SIY) expressing a model antigen to quantify tumor-specific immune response [5].

Tumors grew significantly faster in mice purchased from the vendor Envigo compared to mice acquired from either Jackson or Taconic, and showed a significantly impaired antitumor CD8+ T cell response.

Our results indicate that mice from Envigo likely harbor a germline variant that impairs their anti-tumor immune response.

Immunological destruction of tumors is a multistep, coordinated process that can be targeted at several critical points along the cancer immunity cycle to elicit tumor rejection, and to prevent emergence of cancer.

The Immuno-Oncology Translational Network (IOTN) was created in 2018 to improve the efficacy, durability, and safety of immunotherapy across the spectrum of human cancers, and develop immunoprevention approaches that will prevent cancers before they occur [2].

The IOTN currently consists of 12 research groups spanning immunoprevention, novel therapeutic targets, cell-based therapies, neoantigen and TCR approaches, multimodal therapies, immuno-suppression and immuno-responsiveness.

The major anticipated deliverable of the IOTN collaborative consortium is the acceleration of clinical translation of basic discoveries to overcome barriers that limit the efficacy of immunotherapy across the spectrum of human cancers.

In women, breast cancer is the most common cancer and the first leading cancer-caused deaths worldwide.

The humanized anti-Globo H mAb was incorporated into the asymmetric IgG-like bispecific antibody platform developed by our institute.

In summary, the asymmetric IgG-like anti-Globo H bispecific antibody possesses great efficacy in killing breast cancer both in vitro and in vivo.

Immune checkpoint inhibitor (anti-PD1) therapy is approved for use in MSI(hi) and EBV+ advanced metastatic gastric cancer (GC) patients;

Using a clinically annotated primary GC cohort [1], we analyzed the immune context of each tumor using multiplex immunohistochemistry (mIHC) and transcriptome analysis (Affymetrix array).

Increased GC patient survival correlated with infiltration of the tumor core by significantly increased T cells, in particular CD4+FOXP3+ T cells (Figure 1F).

Our study showed a new biomarker (CD8highCD4FOXP3high) in primary GC patients for good clinical outcome, this biomarker was prognostic across all GC molecular sub-groups.

This study was approved by the Peter MacCallum Cancer Center Human Research Ethics committee, approval number 12/25

Background: A standard treatment for refractory advanced SCAC has not yet been established;

Methods: This is a single-arm Phase 2 trial in patients ≥18 years old with disease progression during or following 1 or 2 prior systemic treatments for metastatic SCAC, which must have included a platinum-based treatment.

Results: As of July 22 2019, 32 patients have received at least one dose of INCMGA00012.

The study was approved by institutional review boards or independent ethics committees of participating institutions.

CD40 plays multiple roles in anti-tumor immune responses, including within the tumor microenvironment (TME).

CDX-1140 monotherapy DE through 3.0 mg/kg across 10 tumor types (n=32;

To date, CDX-1140 has shown the expected PK, PD, and safety profile up to 3 mg/kg.

The study was approved by the following IRBs: Providence Health and Services (Oregon) (#2017000532), WIRB for Gabrail Cancer Center Research (#1188814), University of Pennsylvania (# 828733), Mount Sinai School of Medicine (#18-00202), WIRB for Honor Health (#1180854), WIRB for Nebraska Cancer Specialist (#1189918), WIRB for North Side Hospital (#1249506), Memorial Sloan Kettering Cancer Center (#18-225A[2])

84 HLA-A2+ advanced melanoma patients received tebentafusp.

The most frequent treatment-related AEs (any grade) per investigator that are also cytokine mediated per Lee criteria were mostly mild to moderate and included fever (57%), fatigue (42%), nausea (40%), headache (23%).

We confirmed the low incidence of severe CRS reported by investigators and regrouped cytokine-mediated AEs under the umbrella of CRS.

Actin cytoskeleton dynamics act as platforms for gene regulation and key signaling transduction pathways involved in the cross-talk among tumor cells and cellular and non-cellular components of the tumor microenvironment (TME).

The actin regulatory protein hMENA undergoes tissue specific splicing, generating two alternatively expressed isoforms hMENA11a and hMENAΔv6 that respectively inhibit or increase cell invasiveness [1], TGFβ [2] and β1 integrin signaling and the secretion of several key extracellular matrix (ECM) proteins [3] and is crucial in TGFβ mediated-EMT [2].

The pattern of hMENA isoform expression, TLS presence and localization and stromal fibronectin were evaluated in 94 primary tumors of node negative NSCLC patients by immunohistochemistry.

We found that TLS localized within the tumor area (TLS-AT) are predictive of a longer disease free survival in N0 NSCLC patients, whereas the presence of peritumoral TLS (TLS-PT) does not associate with the patient survival, but with higher tumor grade.

Our findings indicate that the alternative splicing of hMENA is crucial in the reciprocal signaling between tumor cells and their immune microenvironment, by participating in tertiary lymphoid structure neo-genesis and spatial distribution.

We are testing in vitro 2 candidate vaccine adjuvants (lipid A structures) that are generated by using bacterial enzymatic combinatorial chemistry (BECC).

Mice were vaccinated 3X IM with 2 ug RG1-VLP + 25 ug of BECC438 or BECC470 formulated +/- Alhydrogel (alum) and compared to alum alone or PHAD, a commercial TLR4L.

BECC compounds improve HPV16-L2-specific Ab production in mice BECC compounds improve neutralizing Ab production to several HPV strains BECC compounds accelerate anti-HPV humoral responses BECC compounds accelerate appearance of HPV-neutralizing Abs Induction of T cell responses by BECC

Combining the adjuvants Alhydrogel and BECC compounds increases the magnitudes of antibodies specific to HPV16 L1 and the RG1-specific L2 epitope as well as of neutralizing titers to HPV16/18/39/6.

Certain cancers occur at higher rates in people living with HIV (PLWH) as compared with the general population and are a leading cause of mortality among PLWH in developed countries [1].

LOIs including anti-PD(L)1 agents (nivolumab, pembrolizumab, atezolizumab, durvalumab) submitted to CTEP with study approval between January 2014 and May 2018 were included in this analysis.

CTEP advocacy had a meaningful impact on inclusion of PLWH in anti-PD(L)1 clinical trials independent of study phase.

SC administration of immuno-oncology (I-O) therapies is being explored as a less-invasive and more-convenient alternative to IV administration.

Literature published from 2009–2019 was identified through OVID®-indexed databases and supplemented with conference proceedings, recursive searching, and additional clinicaltrials.gov searches.

Our SLR suggests that SC and IV formulations had generally similar efficacy and tolerability profiles, while SC administration led to substantial cost and time savings and was usually preferred by patients and HCPs.

The immune system is a dynamic homeostatic entity which fluctuates in response to antigenic stimulation but provides overall stability, with the capacity to generate an active immune response when required.

B16 F0 melanoma was transplanted into C57BL/6 mice and a peri-tumoural dose of 20,000 IU of IL-2 (or PBS vehicle control) was given on different days after transplantation to observe for any effect on tumour growth determined by the timing of the day of dose delivery.

IL-2 exerted a repeatable effect observed only on Day-7 after transplantation causing statistically significant reduced tumour growth over comparable control (PBS) treated animals.

There appears to be a differential in-vivo effect on control of tumour growth depending on the day of delivery of IL-2 after transplantation and first antigen exposure.

The bi-directional communication mediated by paracrine loop between cancer cells and cancer associate fibroblasts (CAFs) critically affects tumor growth and aggressiveness.

The role of hMENA isoforms in TGF-β-CAF activation was analyzed by loss and gain of function experiments.

We found that hMENA/hMENAΔv6 overexpression identify a subset of CAFs with pro-tumoral functions in NSCLC and PDAC.

In conclusion these novel findings provide the evidence for a crucial role of hMENA and its tissue specific splicing program as regulator of TGF-β-mediated cross-talk between tumor cells and CAFs.

Immunosuppressive tumour microenvironments (TME) reduce the effectiveness of immunotherapies for the treatment of cancer [1].

Using a Balb/c immunogenic, syngeneic model of colorectal cancer, we characterised silayltransferase expression and the cell surface sialic acid profile of tumour associated MSCs exposed to tumour cell secretome, using RNA sequencing and lectin-based flow cytometry respectively.

Our results show overexpression of silayltransferase expression and α2,6-sialic acid cell surface expression on MSCs/CAFs exposed to tumour cell secretome for 72 hours.

These results indicate for the first time that non-haematopoietic stromal cells in the tumour-microenvironment express high levels of sialic acid which contribute to their ability to suppress activated T-effector cells.

Cancer stem-like cells (CSCs) are a small subpopulation of cancer cells that have unique characteristics such as tumor-initiating capacity and cellular stress tolerance.

We have studied the immunopathological properties of CSCs derived from colon, breast, and gynecological cancers to develop CSC-targeted immunotherapy.

#1 CSC-specific antigens: CSCs expressed several germ cell-specific genes, and some of them had crucial roles in the maintenance of tumor initiation capacity.

Based on these unique properties of CSCs, we propose a niche-targeted combination immunotherapy for cancer.

The experiments were conducted with the approval of the Sapporo Medical University Ethics Review Board, approval number 282-134 and 282-1055.

Anticancer versus protumor behavior of tumor microenvironment (TME) largely depends on tumor cell-macrophage interactions which are regulated by nitric oxide (NO) in mouse models as well as patients.

4T1 mammary tumor model in wild type (wt) and Nos2 knockout (Nos2-/-) BALB/c mice was utilized to study the effects of host Nos2 and Cox2 on tumorigenesis and pulmonary metastasis.

Absence of Nos2 in the immune system reduces pulmonary metastasis while pharmacological inhibition of Cox2 delays tumor growth preferentially in Nos2-/- mice.

High levels of NO such as those from inflammatory murine macrophages regulate several effects in neighboring cells due to a cumulative effect of the metabolic changes occurring in Nos2high single cells through localized extracellular NO flux.

Despite the clinical success of immune checkpoint blockade therapy in providing durable responses in advanced cancers such as melanoma, its use as a monotherapy in patients with pancreatic cancer has shown little efficacy [1].

We utilized TH-302 and a vascular endothelial growth factor receptor-2 (VEGFR-2) blocking antibody (DC101) to assess the relationship between blockade of angiogenesis and the hypoxia reduction activity of TH-302.

We found that orthotopic transplanted pancreatic tumors contain hypoxic tumor niches that lack T cell infiltration.

We found that the combination of DC101 and TH-302 promotes infiltration of CD8+ T cells.

Decitabine, a DNA demethylating agent, is known to induce a viral mimicry interferon response and facilitate T cell killing in a major histocompatibility complexes (MHC)-dependent manner.

We treated human lung cancer cells with decitabine at clinically-relevant low doses, and isolated cell surface proteins using biotin-affinity purification.

Our data demonstrated sensitization effects of decitabine to γδ T cell therapy in lung cancer cells through ICAM1 upregulation.

The study was approved by National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC), approval number 20180077.

The recent success of immunotherapeutic approaches has demonstrated that the immune system plays a critical role in fighting cancer.

We are currently characterizing 5 syngeneic tumor models in C57BL/6 mice (B16, LL2, PAN02, EO771, MC38) and their microenvironment by flow cytometry and RNA sequencing.

The flow cytometry analysis revealed striking differences across the different models, especially in relation to the ratio of CD45+ cells within the tumor.

Thus far, we can conclude that the characterization of syngeneic tumor models and their microenvironment reflects the immunologic diversity observed in the clinic and provides a resource to select relevant models to study and test the efficacy of immunotherapies in vivo.

The NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) model was created by simultaneous CRISPR/Cas9 editing of the Prkdc and Il2rg loci in the NOD/Nju mouse.

Charles River NCG mice (n=14) were inoculated with cord blood derived human CD34+ stem cells.

NCG mice remain humanized up to 34 weeks post-engraftment.

The Charles River NCG mouse has been developed as a new humanized model with stable graft function.

Actin cytoskeleton dynamics act as platforms for gene regulation and key signaling transduction pathways involved in the cross-talk among tumor cells and cellular and non-cellular components of the tumor microenvironment (TME).

The actin regulatory protein hMENA undergoes tissue specific splicing, generating two alternatively expressed isoforms hMENA11a and hMENAΔv6 that respectively inhibit or increase cell invasiveness [1], TGFβ [2] and β1 integrin signaling and the secretion of several key extracellular matrix (ECM) proteins [3] and is crucial in TGFβ mediated-EMT [2].

The pattern of hMENA isoform expression, TLS presence and localization and stromal fibronectin were evaluated in 94 primary tumors of node negative NSCLC patients by immunohistochemistry.

We found that TLS localized within the tumor area (TLS-AT) are predictive of a longer disease free survival in N0 NSCLC patients, whereas the presence of peritumoral TLS (TLS-PT) does not associate with the patient survival, but with higher tumor grade.

Our findings indicate that the alternative splicing of hMENA is crucial in the reciprocal signaling between tumor cells and their immune microenvironment, by participating in tertiary lymphoid structure neo-genesis and spatial distribution [7, 8].

While immune checkpoint inhibitors targeting the programmed-death receptor-1 in recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) (all regions) showed an encouraging 11% increase in 2-year overall survival (OS) (from 6-17%) [1], those data at the same time stress the need for the identification of additional, targetable, suppressive pathways in this disease.

Formalin-fixed paraffin embedded OSCC surgical resection specimen collected between 2005-2014 at our institute (n=56) were stained with anti-human GARP (clone: Plato-1) or an IgG2b isotype control antibody.

We found clear expression of GARP on OSCC tumor cells in over fifty percent of the patient samples.

All patient materials were handled and used following the medical ethical guidelines as described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies or written informed consent was obtained in accordance with the Declaration of Helsinki.

Evaluation of the mutational landscape of cancers has revealed significant correlation between mutation burden and response to immunotherapy in cancers such as melanoma and non-small cell lung carcinoma[1,2].

Whole exome sequencing and RNA sequencing was performed on matched normal and tumor tissue from ten ccRCC patients (nuclear grades I-IV) undergoing surgical resection.

The visualization of immune-repertoire of RCC revealed a higher fraction of lymphoid infiltrate (~70-75%) than myeloid in a relatively stable ratio across both patients, with tumor containing a slightly higher fraction of lymphoid cells.

This study demonstrates the abundance of immune cell subtypes in the ccRCC TME, identifying a tumor-specific myeloid population.

Cancer immunotherapy, in particular immune checkpoint blockade, has shown great promise in the treatment of advanced cancer.

Transcriptomic data from TCGA and METABRIC were mined for LDHC gene expression in breast cancer.

Interrogation of TCGA and METABRIC RNA-seq data revealed that LDHC was markedly upregulated in TNBC compared to non-TNBC (Figure 1A), with a significant poorer overall and disease-specific survival in TNBC patients with higher LDHC expression (Figure 1B).

Our findings demonstrate a positive correlation between LDHC expression and worse survival in specifically triple negative breast cancer.

This work was supported by a grant from the Qatar Biomedical Research Institute (grant number VR94), awarded to Dr Julie Decock.

Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients.

In this study, we assessed the density and distribution of TIL-Bs in FFPE samples from 2 independent cohorts of OPSCC patients.

Here, we observed that CD20+ TIL-Bs and CD8+ T cells formed non-organized aggregates with clearly interacting cells and the densities of both intraepithelial CD20+ B cells and B/CD8+ T cell interactions have prognostic significance for the overall survival of OPSCC patients.

Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can preselect patients with excellent prognosis who would profit from less invasive treatment.

This study was financially supported by the Grant Agency of Charles University, project number 344216.

All patient studies were conducted in accordance with Declaration of Helsinki and approved by an institutional review board, namely the Ethics Committee of the University Hospital Hradec Kralove and the Ethics Committee of the University Hospital Motol.

Dysregulation of long noncoding RNA (lncRNA) H19 has been implicated in hepatocellular carcinoma (HCC), but the concrete regulatory mechanism is lack of research.

We mined gene expression profiles of 457 HCC samples from TCGA and TJMUCH cohorts (GSE116174 and GSE116182) and further validated in 64 FFPE HCC tissues.

LncRNA H19 overexpression in situ was significantly correlated with poor prognosis of HCC patients, which induced EMT, promoted stemness and accelerated invasion of HCC cells in vitro.

TAMs-induced lncRNA H19 promotes HCC aggressiveness via triggering and activating the miR-193b/MAPK1 axis, mediates the crosstalk between HCC and immunological microenvironment, and causes poor clinical outcomes.

This work was supported by National Natural Science Foundation of China (grant numbers: 81872143, 81702280, 81472473, 81272360) and Key Project of Tianjin Health and Family Planning Commission (16KG126).

TGF-β1 and BMP7 are important members of TGF-β superfamily, which have similar downstream transduction pathways and interfere mutually.

The immunohistochemistry staining method was used to exam the expressions of TGFBR2, TGFBR1, BMP7, BMPR2 and ACVR1 proteins in 64 HCC samples.

In 64 HCC samples, the expression of TGFBR2 was significantly negatively correlated with ACVR1 expression.

In summary, the miR-17 cluster promoted M2-TAMs related cell aggressiveness via inducing the imbalance of TGF-β1/BMP7 pathways in HCC.

This work was supported by National Natural Science Foundation of China (grant numbers: 81872143, 81702280, 81472473, 81272360) and Key Project of Tianjin Health and Family Planning Commission (16KG126).

Vitiligo, an immune-related adverse effect (irAE) which targets normal melanocytes in the skin, is associated with significantly improved overall survival in metastatic melanoma patients treated with immunotherapy [1].

Seven melanoma survivors who received immunotherapy with the irAE of vitiligo were recruited to the study.

TRM-like CD8 T cells were found in all melanoma patients’ vitiligo skins.

These data suggest that TRM-like CD8 T cells with TCR clonotypes matched between tumor and skin maintains a tumor protecting population in long-term melanoma survivors’ vitiligo skin.

This study was approved by Dartmouth College/Dartmouth Hitchcock Medical Center's Committee for the Protection of Human Subjects, approval number 29821

Despite the unprecedented success of immune checkpoint blockade (ICB) therapy, many patients still respond poorly, owing to multiple resistance mechanisms.

To address these knowledge gaps, we employed single-cell sequencing (scRNA-seq) to assess transcriptional and metabolic changes in myeloid cells that associated with resistance to PD-1 blockade in preclinical cancer models.

Our study revealed that the resistant tumor was dominated by immunosuppressive myeloid cells enriched for a fatty acid metabolism gene expression signature, which can be used as biomarker to distinguish responders from non-responders.

Overall, we have identified Pim1 as a metabolic modulator in MDSCs that may cause ICB resistance and can be therapeutically inhibited to sensitize non-responsive tumors to ICB therapy.

Master's Ceremony Fall 2018