Background/Aims Recurrence is a major factor limiting the long-term survival of patients with intrahepatic cholangiocarcinoma (ICC). The molecular characteristics and potential therapeutic targets in ICC remain largely undefined. Methods Following our previous whole-exome sequencing study, we performed targeted sequencing, Sanger sequencing, and quantitative PCR to assess all coding exons and copy number variations of LATS2 in 400 primary ICC samples. Kaplan–Meier survival curves were used to assess the impact of LATS2 mutation, copy number loss, and low expression levels on recurrence-free survival and overall survival in ICC patients. In addition, we investigated the functional role and underlying mechanisms of LATS2 variation in ICC tumor progression and resistance to anti-PD-1 therapy. Results Among a total of 400 ICC cases, the overall frequencies of LATS2 somatic mutation and copy number loss were 3% (12/400) and 34% (136/400), respectively. Both types of variation were correlated with decreased LATS2 protein expression, increased tumor recurrence, and poor overall survival. Biofunctional investigations revealed a tumor-suppressor role of LATS2. Inactivation of LATS2 suppressed the Hippo signaling pathway, leading to aberrant activation of YAP, which upregulated PD-L1 expression and CCL2 secretion, suppressed CD8+ T cell infiltration, and enhanced recruitment of M2-like macrophages, thereby promoting immune evasion, tumor progression, and resistance to anti-PD-1 therapy. Conclusions Our study reveals a pivotal clinical association and mechanistic role of LATS2-inactivating variation in ICC, which may serve as a useful biomarker for precision therapy.

BACKGROUND/AIMS:Recurrence is a major factor limiting the long-term survival of patients with intrahepatic cholangiocarcinoma (ICC). The molecular characteristics and potential therapeutic targets in ICC remain largely undefined. METHODS:Following our previous whole-exome sequencing study, we performed targeted sequencing, Sanger sequencing, and quantitative PCR to assess all coding exons and copy number variations of LATS2 in 400 primary ICC samples. Kaplan-Meier survival curves were used to assess the impact of LATS2 mutation, copy number loss, and low expression levels on recurrence-free survival and overall survival in ICC patients. In addition, we investigated the functional role and underlying mechanisms of LATS2 variation in ICC tumor progression and resistance to anti-PD-1 therapy. RESULTS:Among a total of 400 ICC cases, the overall frequencies of LATS2 somatic mutation and copy number loss were 3% (12/400) and 34% (136/400), respectively. Both types of variation were correlated with decreased LATS2 protein expression, increased tumor recurrence, and poor overall survival. Biofunctional investigations revealed a tumor-suppressor role of LATS2. Inactivation of LATS2 suppressed the Hippo signaling pathway, leading to aberrant activation of YAP, which upregulated PD-L1 expression and CCL2 secretion, suppressed CD8+ T cell infiltration, and enhanced recruitment of M2-like macrophages, thereby promoting immune evasion, tumor progression, and resistance to anti-PD-1 therapy. CONCLUSIONS:Our study reveals a pivotal clinical association and mechanistic role of LATS2-inactivating variation in ICC, which may serve as a useful biomarker for precision therapy.

BACKGROUND:The tumor microenvironment has shown abilities to influence the progression and prognosis of intrahepatic cholangiocarcinoma (iCCA). However, little is known about the effect of IgG4+ plasma cells in iCCA. METHODS:We stained IgG4+plasma cells by immunohistochemistry and performed Kaplan-Meier survival analysis to detect the prognostic value. We also stained CD3, CD4, CD8 and Foxp3 positive T cells to explore its associations with IgG4+plasma cells. RESULTS:We found that tumor infiltrated IgG4+plasma cells were associated with poor prognosis of iCCA, rather than IgG4+plasma cells in adjacent liver tissue. The number of IgG4+plasma cells was associated with CD8+ T cells. We divided the iCCA patients into 3 groups by IgG4+plasma cells to CD8+ T cells ratio. The group I (IgG4-/CD8+) had the best prognosis. The group II (IgG4-/CD8- or IgG4+/CD8+) and group III (IgG4+/CD8-) were independent risk factors of recurrence-free survival (HR = 1.69, group II; HR = 2.11, group III) and overall survival (HR = 1.76, group II; HR = 2.38, group III) compared with group I. CONCLUSIONS:We examined the distribution of IgG4+ plasma cells in iCCA and discovered its prognostic utility when combined with CD8+ T cell distribution in iCCA.

Transcription of distinct loci of human endogenous retroviruses (HERVs) and in some cases, translation of these transcripts have been consistently observed in many types of cancer. It is still debated whether HERV activation serves as a trigger for carcinogenesis or rather occurs as a consequence of epigenetic alterations and other molecular sequelae that characterize cellular transformation. Here we review the known molecular and epigenetic mechanisms of HERV activation in cancer cells as well as its potential contribution to carcinogenesis. Further, we describe the use of HERV expression in cancer diagnostic and characterize the potential of HERV-derived antigens to serve as novel targets for cancer immunotherapy. We believe this review, which summarizes both what is known as well as unknown in this rapidly developing field, will boost interest in research on the therapeutic potential of targeting HERV elements in tumors and the impact of HERV activation in oncogenesis.

Background We discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T cell-mediated killing. Here, we described the cloning of a novel T cell receptor (TCR) targeting a CT-RCC HERV-E-derived antigen specific to ccRCC and characterized antitumor activity of HERV-E TCR-transduced T cells (HERV-E T cells).Methods We isolated a CD8+ T cell clone from a patient with immune-mediated regression of ccRCC post-allogeneic stem cell transplant that recognized the CT-RCC-1 HERV-E-derived peptide in an HLA-A11-restricted manner. We used 5’Rapid Amplification of cDNA Ends (RACE) to clone the full length HERV-E TCR and generated retrovirus encoding this TCR for transduction of T cells. We characterized HERV-E T cells for phenotype and function in vitro and in a murine xenograft model. Lastly, we implemented a good manufacturing practice-compliant method for scalable production of HERV-E T cells.Results The HLA-A11-restricted HERV-E-reactive TCR exhibited a CD8-dependent phenotype and demonstrated specific recognition of the CT-RCC-1 peptide. CD8+ T cells modified to express HERV-E TCR displayed potent antitumor activity against HLA-A11+ ccRCC cells expressing CT-RCC HERV-E compared with unmodified T cells. Killing by HERV-E T cells was lost when cocultured against HERV-E knockout ccRCC cells. HERV-E T cells induced regression of established ccRCC tumors in a murine model and improved survival of tumor-bearing mice. Large-scale production of HERV-E T cells under good manufacturing practice conditions generated from healthy donors retained specific antigen recognition and cytotoxicity against ccRCC.Conclusions This is the first report showing that human ccRCC cells can be selectively recognized and killed by TCR-engineered T cells targeting a HERV-derived antigen. These preclinical findings provided the foundation for evaluating HERV-E TCR-transduced T cell infusions in patients with metastatic ccRCC in a clinical trial (NCT03354390).

In vivo expansion of genetically modified T cells in cancer patients following adoptive transfer has been linked to both anti-tumor activity and T cell-mediated toxicities. The development of digital PCR has improved the accuracy in quantifying the in vivo status of adoptively infused T cells compared to qPCR or flow cytometry. Here, we developed and evaluated the feasibility and performance of nanoplate-based digital PCR (ndPCR) to quantify adoptively infused T cells engineered with a T cell receptor (TCR) that recognizes a human endogenous retrovirus type E (HERV-E) antigen. Analysis of blood samples collected from patients with metastatic kidney cancer following the infusion of HERV-E TCR-transduced T cells established the limit of detection of ndPCR to be 0.3 transgene copies/mu L of reaction. The lower limit of quantification for ndPCR was one engineered T cell per 10,000 PBMCs, which outperformed both qPCR and flow cytometry by 1 log. High inter-test and test-retest reliability was confirmed by analyzing blood samples collected from multiple patients. In conclusion, we demonstrated the feasibility of ndPCR for detecting and monitoring the fate of TCR-engineered T cells in adoptive cell therapy.

African American (AA) women are disproportionately affected by obesity and hyperlipidemia, particularly in the setting of adverse social determinants of health (aSDoH) that contribute to health disparities. Obesity, hyperlipidemia, and aSDoH appear to impair NK cells. As potential common underlying mechanisms are largely unknown, we sought to investigate common signaling pathways involved in NK cell dysfunction related to obesity and hyperlipidemia in AA women from underresourced neighborhoods. We determined in freshly isolated NK cells that obesity and measures of aSDoH were associated with a shift in NK cell subsets away from CD56dim/CD16+ cytotoxic NK cells. Using exvivo data, we identified LDL as a marker related to NK cell function in an AA population from underresourced neighborhoods. Additionally, NK cells from AA women with obesity and LDL-treated NK cells displayed a loss in NK cell function. Comparative unbiased RNA-sequencing analysis revealed DUSP1 as a common factor. Subsequently, chemical inhibition of Dusp1 and Dusp1 overexpression in NK cells highlighted its significance in NK cell function and lysosome biogenesis in a mTOR/TFEB-related fashion. Our data demonstrate a pathway by which obesity and hyperlipidemia in the setting of aSDoH may relate to NK cell dysfunction, making DUSP1 an important target for further investigation of health disparities.

435 Background: Human endogenous retrovirus type E (HERV-E) is specifically expressed in ccRCC providing a safe target for T cell-based therapies. We investigated T cells transduced with a TCR targeting HERV-E (HERV-E T cells) for the treatment of mccRCC. Methods: This first-in-human study assessed the safety & efficacy of escalating doses of HERV-E T cells and manufacturing/clinical endpoints correlative analysis. HLA-A*11+ mccRCC patients (pts) were treated with a conditioning regimen, infusion of HERV-E T cells & IL-2. (NCT03354390). Results: Nineteen of185 pts tested were found to express HLA-A*11. 17 HLA-A*11 + pts enrolled on the study: 3 pts on each DL1-3 & 6 pts on DL4. 2 pts did not receive HERV-E T cells given disease progression during manufacturing period. Median age was 57 years. 86% received ≥ 3 prior systemic treatment (range 1-8). The manufacturing failure rate after first apheresis was 12% (n=2); both met target dose after second apheresis and repeat in vitro expansion. All HERV-E T cell products met release criteria for infusion including INF-γ production in response to HERV-E/HLA-A11-expressing tumor cells. Median HERV-E vector copy number (VCN) was 1.9. No dose-limiting toxicities (DLT), off-target toxicities or treatment-related deaths occurred. Pt#17 is on DLT monitoring period. 7 pts completed the planned 14 doses of IL-2 and all received at least 8 doses. Reasons for IL-2 discontinuation: hemodynamic (57%), cardiovascular (28%), pulmonary (28%), renal (14%) criteria & pts decision (14%). The best response was partial response in 7% & stable disease at least 8 weeks in 29% pts. HERV-E mRNA expression was detected in 5 primary & 9 metastatic specimens. HERV-E T cells were measurable in circulation post-dosing, with peak concentrations in the peripheral blood mononuclear compartment on day(D)+7. [DL1: 0.3 %, DL2: 1.2%, DL3: 0.5%, DL4: 12.3%]. Median HERV-E T cell VCN showed no correlation with HERV-E T cells peak concentration on D+4 & D+7. Conclusions: Proof of concept that HERV-T cells can induce tumor regression without evidence of causing off-target toxicities has been established by this trial. Infused HERV-E T cells were detectable transiently in vivo and induce effector cytokine production. Our initial results support the further development of HERV-E-directed therapies that focus on methods to improve in vivo persistence of TCR engineered T-cells and to target HERV-E antigens expressed on more commonly expressed HLA alleles.[Table: see text]

Human endogenous retroviruses (HERVs) comprise 8% of the human genome and can be abnormally expressed in different tumor cells. Our lab discovered a unique and highly immunogenic HERV-E, CT-RCC HERV-E, that is selectively expressed in most clear cell renal cell carcinoma (ccRCC) cells. Although the function of select HERVs in some tumors has been defined, the role of CT-RCC HERV-E in ccRCC remains unclear. To characterize the impact of HERV-E expression on tumor oncogenesis in ccRCC, we developed a highly efficient strategy to ablate CT-RCC HERV-E through dual-guide CRISPR-Cas9. For isolation of edited cells, we knocked in a truncated CD19 (tCD19) into the same gene edited locus using adeno-associated virus (AAV) as donor templates. In order to ablate the entire 8.8 kb HERV-E region, we designed three single guide RNAs (sgRNAs) flanking the CT-RCC HERV-E genomic locus: one at the upstream site, sgRNA HERV-E Upst(1), and two downstream, sgRNA HERV-E Dwst(3) and sgRNA HERV-E Dwst(4). A single sgRNA targeting β2M was used as a negative control. HERV-E knockout was verified via real-time PCR, and the combination of sgRNAs Upst1-Dwst3 were chosen as the guides for all experiments, due to their higher knockout efficiency. We tested the MOIs of 2, 4, and 10 × 105 for AAV transduction following CRISPR knockout. Expression of CD19 was the highest at the MOI of 10 × 105, which was used for all experiments. Two RCC cell lines, RCC-TIU and RCC-UOK220, were used to validate this methodology. Edited ccRCC cells were enriched by CD19 magnetic microbeads, resulting in CD19 expression of over 96%. Genomic DNAs isolated from the two RCC cell lines confirmed simultaneous knockout of HERV-E and knock-in of tCD19. To isolate a population of cells that had homogenous HERV-E knockout, we single-cell sorted and then expanded CD19+ ccRCC cells in vitro. 252 and 145 clones were harvested from RCC-TIU and RCC-UOK220 respectively. PCR analysis of genomic DNA from 48 selected clones of each of the aforementioned cell lines unveiled both monoallelic and biallelic knockout of HERV-E. Eight biallelic knockout clones were harvested and further expanded, which gave rise to four clones from RCC-TIU and three clones from RCC-UOK220. Additional PCR screening confirmed that RCC-TIU A7, A9, and RCC-UOK220 D3 were biallelic knockout clones. Finally, we used digital PCR to examine the expression of two CT-RCC HERV-E transcripts, Env and RCC-8; No expression of these transcripts was found in the aforementioned clones. In summary, we established a highly efficient method to reliably ablate a human endogenous retrovirus in human tumor cells. The dual-guide CRISPR-Cas9 genome editing method utilized here allowed for the successful isolation of tumor cells that had a biallelic knockout of the CT-RCC HERV-E. These knockout cell lines will be utilized to explore the oncogenic impact of CT-RCC HERV-E expression in ccRCC cells. Citation Format: Long Chen, Elena Cherkasova, Stephanie Pierre, Savannah England, Muna Igboko, Joseph Clara, Stefan Barisic, Angie Parrizzi, Rosa Rios Nadal, David Allan, Mala Chakraborty, Robert Reger, Richard Childs. Ablation of an immunogenic human endogenous retrovirus in renal cell carcinoma cells through dual-guide CRISPR-Cas9 genome editing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5210.

4542 Background: Previously, our group described a novel human endogenous retrovirus type E (CT-RCC HERV-E) that is selectively expressed in most clear cell renal cell carcinomas (ccRCC). Here, we report the characterization and preclinical testing of a T cell receptor (HERV-E TCR) that targets an HLA-A11-restricted 10-mer peptide antigen (CT-RCC-1) that is derived from CT-RCC HERV-E. Methods: From a patient with ccRCC who had prolonged immune-mediated tumor regression following an allogeneic hematopoietic stem cell transplant, we identified a CD8 + T cell clone with HLA-A11-restricted specificity for the CT-RCC-1 peptide. The TCR of this clone was encoded into a retroviral vector containing a truncated CD34 cassette as a selection marker. Gene-engineered T cells expressing the HERV-E TCR (HERV-E T cells) were characterized for tumor recognition, lysis, and cross-reactivity. In vivo anti-tumor activity was assessed in a xeno-murine model using a human subcutaneous ccRCC tumor graft. A good manufacturing practice (GMP)-compliant method to produce HERV-E T cells was implemented for a subsequent clinical trial in humans with metastatic ccRCC. Results: When transduced into human T cells, the HERV-E TCR showed CD8-dependent specific recognition and lysis of HERV-E-expressing ccRCC cells that were HLA-A11+. The extent of tumor cytotoxicity correlated with CT-RCC HERV-E mRNA expression levels and HLA-A11 surface density (r=0.82). Cross-reactivity assessment established that there were no naturally occurring peptides with substantial sequence identity to CT-RCC-1 that could serve as alternative targets for HERV-E T cells. Further, immunopeptidomics and in silico analyses provided strong evidence that presentation of the CT-RCC-1 epitope depends exclusively on the transcription of the CT-RCC HERV-E genomic region. In a murine model, human HERV-E T cells mediated regression of established human ccRCC tumor grafts, significantly prolonging animal survival compared to controls that either received non-transduced T cells or no T cells (median survival 50 days vs. 20 and 20 days, respectively; p<0.001). Finally, the GMP-compliant production of HERV-E T cells yielded pure populations (i.e., >90% TCR-transduced) of HERV-E T cells that were highly cytotoxic to ccRCC tumor cells. The expansion numbers of GMP-produced cells were sufficient for testing the safety of the adoptively infused T cells in a phase I clinical trial. Conclusions: Here, we provide the first data showing T cells armed with a HERV-E TCR acquire specific anti-tumor activity in vitro and in vivo against ccRCC cells. These preclinical data provide the foundation for an ongoing first-in-human phase I clinical trial evaluating the safety of HERV-E T cell infusions in patients with advanced ccRCC.

Background: The diffuse large B-cell lymphoma (DLBCL) is a heterogeneous lymphoma with a dismal outcome, due to approximately 40% patients will be relapsed or refractory to the standard therapy of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Therefore, we need urgently to explore the approach to classify the risk of DLBCL patients accurately and accurately targeting therapy. The ribosome is a vital cellular organelle that is mainly responsible for translation mRNA into protein, moreover, more and more reports revealed that ribosome was associated with cellular proliferation and tumorigenesis. Therefore, our study aimed to construct a prognostic model of DLBCL patients using ribosome-related genes (RibGs). Method: We screened differentially expressed RibGs between healthy donors' B cells and DLBCL patients' malignant B cells in GSE56315 dataset. Next, we performed analyses of univariate Cox regression, the least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analyses to establish the prognostic model consisting of 15 RibGs in GSE10846 training set. Then, we validated the model by a range of analyses including Cox regression, Kaplan-Meier survival, ROC curve, and nomogram in training and validation cohorts. Results: The RibGs model showed a reliably predictive capability. We found the upregulated pathways in high-risk group most associated with innate immune reaction such as interferon response, complement and inflammatory responses. In addition, a nomogram including age, gender, IPI score and risk score was constructed to help explain the prognostic model. We also discovered the high-risk patients were more sensitive to some certain drugs. Finally, knocking out the NLE1 could inhibit the proliferation of DLBCL cell lines. Conclusion: As far as we know, it is the first time to predict the prognosis of DLBCL using the RibGs and give a new sight for DLBCL treatment. Importantly, the RibGs model could be acted as a supplementary to the IPI in classifying the risk of DLBCL patients.

Background Adoptive transfer of natural killer (NK) cells with augmented antibody-dependent cellular cytotoxicity (ADCC) capabilities and resistance to CD38 targeting has the potential to enhance the clinical anti-myeloma activity of daratumumab (DARA). Therefore, we sought to develop an efficient CRISPR/Cas9-based gene editing platform to disrupt CD38 expression (CD38 knockout (KO)) in ex vivo expanded NK cells and simultaneously arm CD38(KO) NK cells with a high-affinity CD16 (CD16-158V) receptor. Methods CD38(KO) human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38(KO) NK cells and targeted gene insertion at the CD38 locus to mediate gene knockin (KI). The capacity of these gene-edited NK cells to persist and mediate ADCC in the presence of DARA was tested in vitro and in a MM.1S xenograft mouse model. Results Highly efficient CD38 gene disruption was achieved in ex vivo expanded NK cells without affecting their proliferative or functional capacity. CD38 KO conferred resistance to DARA-induced NK cell fratricide, enabling persistence and augmented ADCC against myeloma cell lines in the presence of DARA in vitro and in a MM.1S xenograft mouse model. CD38(KO) NK cells could be further modified by transfection with mRNA encoding a CD16-158V receptor, resulting in augmented DARA-mediated ADCC. Finally, we observed that a homology-directed repair template targeted to the CD38 locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38(KO)/CD16(KI) NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo. Conclusions Adoptive immunotherapy using ex vivo expanded CD38(KO)/CD16(KI) NK cells has the potential to boost the clinical efficacy of DARA. By incorporating complementary genetic engineering strategies into a CD38 KO manufacturing platform, we generated NK cells with substantially augmented CD38-directed antitumor activity, establishing a strong rationale for exploring this immunotherapy strategy in the clinic.

OBJECTIVE The aim of this study was to describe the clinical and procedural risk factors associated with the unplanned neurosurgical intensive care unit (NICU) readmission of patients after elective supratentorial brain tumor resection and serves as an exploratory analysis toward the development of a risk stratification tool that may be prospectively applied to this patient population. METHODS This was a retrospective observational cohort study. The electronic medical records of patients admitted to an institutional NICU between September 2018 and November 2021 after elective supratentorial brain tumor resection were reviewed. Demographic and perioperative clinical factors were recorded. A prognostic model was derived from the data of 4892 patients recruited between September 2018 and May 2021 (development cohort). A nomogram was created to display these predictor variables and their corresponding points and risks of readmission. External validation was evaluated using a series of 1118 patients recruited between June 2021 and November 2021 (validation cohort). Finally, a decision curve analysis was performed to determine the clinical usefulness of the prognostic model. RESULTS Of the 4892 patients in the development cohort, 220 (4.5%) had an unplanned NICU readmission. Older age, lesion type, Karnofsky Performance Status (KPS) < 70 at admission, longer duration of surgery, retention of endotracheal intubation on NICU entry, and longer NICU length of stay (LOS) after surgery were independently associated with an unplanned NICU readmission. A total of 1118 patients recruited between June 2021 and November 2021 were included for external validation, and the model's discrimination remained acceptable (C-statistic = 0.744, 95% CI 0.675-0.814). The decision curve analysis for the prognostic model in the development and validation cohorts showed that at a threshold probability between 0.05 and 0.8, the prognostic model showed a positive net benefit. CONCLUSIONS A predictive model that included age, lesion type, KPS < 70 at admission, duration of surgery, retention of endotracheal intubation on NICU entry, and NICU LOS after surgery had an acceptable ability to identify elective supratentorial brain tumor resection patients at high risk for an unplanned NICU readmission. These risk factors and this prediction model may facilitate better resource allocation in the NICU and improve patient outcomes.

Chimeric Antigen Receptors (CAR)-T cells are genetically modified to express an extracellular binding domain, transmembrane domain and intracellular signaling domain. The extracellular binding domain is usually derived from antibodies; however, it can be replaced with other ligand-receptor binding systems, such as with an Fc receptor. CD16A has been tested in second-generation 4-1BB-CD3ζ CAR T cells and NK cell lines (NK-92) and enhances tumor killing; however, it is not clear whether these cells provide superior tumor killing compared to cells modified to express CD16A alone. In this study, we designed three CAR constructs containing the extracellular binding domain of high affinity mutant CD16A (CD16A-V158) and intracellular signal through 4-1 BB-CD3ζ that differed in leader sequences and transmembrane domains. CAR1 contained the CD16A leader and CD16A transmembrane domain; CAR2 had the CD16A leader and CD8α transmembrane domain; CAR3 contained the CD8α leader and CD8α transmembrane domain. When transduced into Jurkat cells via lentivirus, CD16A V158 and all three CARs showed stable high surface expression of CD16A (> 70%). In contrast, NK-92 cells were only successfully transduced to express CD16 by CD16A V158 (29.8%), CAR2 (42.3%), and CAR3 (42.5%) with no expression of CD16A by CAR1. Transduced cells were sorted by FACs based on CD16A expression. All sorted cells showed >90% stable expression of the constructs for up to 42 days post sorting. We observed a much higher CD16 MFI in cells transduced with CAR2 and CAR3 compared with CD16A V158. Western blotting with anti-CD3ζ identified the full assembly of CARs. We then tested the function of the transduced cells by a Rituximab binding assay. Although there was no difference in binding to Rituximab at 0.01 and 0.1 mg/mL; CAR2 and CAR3 showed better binding at the 1 and 10 mg/mL. In a 4-hour coculture toxicity assay with 721.221 LCLs, CD16A V158 and CAR3 increased the target lysis by two-fold while CAR2 only showed a 50% increase. When cocultured with 721.221 HLA-E+ LCLs, a 721.221 subclone more resistant to killing by NK-92 cells, we observed similar tumor killing with CD16A V158 and CAR3 (killing doubled) in contrast to CAR2(killing increased by only 50%). We next tested against the B-cell line Raji and found that there was only a mild increase of target lysis with transduced NK-92 cells; however, the CD16A V158 and CAR3 still showed higher toxicity (around a 40% increase). In summary, CAR2 and CAR3 expressed a higher CD16 MFI in NK cells and demonstrated better binding to Rituximab at1 and 10 mg/mL compared to CD16A V158; however, CAR3 and CD16A V158 had similar killing of target cells that was higher than was observed with CAR2. Our data indicate that NK cells transduced to have a CAR structure combining CD16A with 4-1BB-CD3ζ have augmented ADCC, although this augmentation is not superior to NK cells that are simply transduced to express CD16A alone. Citation Format: Long Chen, Vicky Li, David Allan, Robert Reger, Elena Cherkasova, Stephanie Pierre, Stefan Barisic, David Granadier, Emily Levy, Giacomo Waller, Susan Doh, Mala Chakraborty, Kate Stringaris, Richard Childs. The Costimulatory Signal Domains 4-1BB and CD3ζ Do Not Improve the Function of CD16A Chimeric Antigen Receptor Transduced NK Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1434.

Natural killer (NK) cells have the unique capacity to lyse tumor cells without the need for antigen specific receptors. Modulation of NK cell cytokine, chemokine, and activating and inhibitory receptor expression using gene regimens represents an attractive strategy to further bolster NK cell anti-tumor activity for the purpose of NK cell immunotherapy. Current efforts in clinical-grade genetic modification of patient-derived cells, used in immunotherapy protocols, rely on stable transduction mediated by viral vectors. However, primary NK cells are notoriously difficult to transduce using viral vectors. Here we describe a lentiviral vector-based approach that results in an efficient, robust, and highly reproducible method of stable gene transfer into primary human peripheral blood-derived NK cells, which can subsequently be expanded ex vivo for adoptive infusion into humans. In vitro experiments established highly efficient transduction of ex vivo expanded NK cells could be achieved by activation of primary NK cells in vitro for 2-4 days in cytokine containing media followed by transduction of activated NK cells using LV vectors 2-3 days before integration into our existing ex vivo expansion protocol using irradiated EBV-LCL feeder cells. Unless NK cells were first activated in cytokine-containing media, LV transduction was unsuccessful. Cytokine activation in media containing IL-2 for 2-4 days was found to be sufficient to achieve high transduction efficiency, while additional supplementation of the media with IL-15 or IL-21 had negligible effects. NK cell transduction efficiency and viability with the use of RetroNectin® was uniformly superior to polycation-based transduction protocols. In experiments where NK cells were transduced with identical lentiviral constructs with 8 different promoter sequences driving expression of EGFP, PGK, EFS, and SV40 promoters consistently demonstrated the highest transduction efficiencies reproducibly in the range of 25-60%. Remarkably, when transduced NK cells were stimulated ex vivo using a clinical grade irradiated EBV-LCL feeder cell line, 100-1000 fold expansions of transduced NK cells could be achieved with sustained transgene expression over two weeks. Transduced and expanded primary NK cells remained highly cytotoxic against K562 tumor targets without developing functional deficiencies in degranulation, IFNγ, or TNFα production. These data establish a simple method to produce large numbers (>1011) of genetically modified clinical grade NK cells suitable for infusion. The protocol described herein overcomes previous difficulties described with common transduction techniques opening new avenues to investigate the impact of adoptive infusion of primary NK cells stably transduced to express a variety of different transgenes of interest.

CCR7 is a G protein-coupled chemokine receptor. In this study, we used immunohistochemistry with tissue microarrays to measure CCR7 expression in tumor specimens from 122 patients with gastric cancer. We show that CCR7 expression is associated with lymph node metastasis (P = 0.022) and overall survival (OS; P = 0.025), and is an independent factor associated with poorer overall survival (P = 0.032). The CCR7 mechanism was predicted based on bioinformatic analysis and verified in gastric cancer cell lines and primary tumor samples. The data show that CCR7 contributes to TGF-β1-induced epithelial-mesenchymal transition (EMT) and that the effects of TGF-β1 are inhibited by a CCR7 neutralizing antibody or a NF-κB inhibitor. Increased TGF-β1 expression was accompanied by nuclear localization of NF-κB-p65 and higher levels of the mesenchymal marker vimentin in human gastric cancer samples. We conclude that the CCR7 axis mediates TGF-β1-induced EMT via crosstalk with NF-κB signaling, facilitating lymph node metastasis and poorer overall survival in patients with gastric cancer. These findings suggest CCR7 is a novel prognostic indicator and a potential target for gastric cancer therapy.

Immunosenescence contributes to pathogenesis of Alzheimer's disease (AD) in the elderly. In this study, we explored the effects of young wild type (WT) splenocytes (ySCs) on Alzheimer's disease by transplanting ySCs into APPswe/PSENldE9 transgenic mice. Young WT splenocytes not only prevented AD, but also improved the spatial learning and memory of APPswe/PSENldE9 transgenic mice. Young WT splenocytes enhanced Aβ clearance, decreased astrogliosis and increased systemic growth differentiation factor 11 (GDF11) levels. Splenocytes derived from old AD mouse promoted AD. There was an increased number of regulatory T cells (Tregs) among old AD splenocytes. We suggest that alterations of GDF11 and Tregs are involved in AD progression and that rejuvenation of the immune system is a potential therapeutic strategy in AD.

The chemokine system consists of four different subclasses with over 50 chemokines and 19 receptors. Their functions in the immune system have been well elucidated and research during the last decades unveils their new roles in hepatocellular carcinoma (HCC). The chemokines and their receptors in the microenvironment influence the development of HCC by several aspects including: inflammation, effects on immune cells, angiogenesis, and direct effects on HCC cells. Regarding these aspects, pre-clinical research by targeting the chemokine system has yielded promising data, and these findings bring us new clues in the chemokine-based therapies for HCC.