TIL Cells Research Articles

Association of aging related genes and immune microenvironment with major depressive disorder

ObjectiveTo study the relationship between aging related genes (ARGs) and Major Depressive Disorder (MDD). MethodsThe datasets GSE98793, GSE52790 and GSE39653 for MDD were obtained from the GEO database, and ARGs were obtained from the Human Aging Genome Resources database. Differential expression genes (DEGs) screening and GO, KEGG enrichment analysis were performed to uncover the underlying mechanisms. To identify key ARGs associated with MDD (key ARG-DEGs), we employed machine learning methods such as LASSO, SVM, and Random Forest, as well as the plug-ins CytoHubba-MCC and MCODE methods. SsGSEA was used to analyze the immune infiltration of MDD and healthy controls. Furthermore, we created risk prediction nomograms model and ROC curves to assess not only the ability of key ARG-DEGs to diagnose MDD, but also predicted miRNAs and transcription factors (TFs) that might interact. Finally, a two-sample Mendelian randomization (MR) study was performed to confirm the association of identified key ARG-DEGs with depression. ResultsDEGs of ARGs between MDD and healthy controls led to the identification of eight ARG-DEGs. GO and KEGG analysis revealed that the pathways associated with these eight ARG-DEGs were primarily concentrated in Foxo pathway, JAK-STAT pathway, Pl3K-AKT pathway, and metabolic diseases. A comprehensive analysis further narrowed down the 8 ARG-DEGs to 4 key ARG-DEGs: MMP9, IL7R, S100B, and EGF. Immune infiltration analysis indicated significant differences in CD8(+) T cells, macrophages, neutrophils, Th2 cells, and TIL cells between MDD and control groups, correlating with these four key ARG-DEGs. Based on these four key ARG-DEGs, a risk prediction model for MDD was developed. The miRNA-TF-mRNA interaction network of the key ARG-DEGs highlights the complexity of the regulatory process, providing valuable insights for future related research. The MR study suggested a potential causal relationship between MMP9 and the risk of depression. ConclusionThe process of aging, immune dysregulation, and MDD are closely interconnected. MMP9, IL7R, S100B, and EGF may be used as novel diagnostic biomarkers and potential therapeutic targets for MDD, especially MMP9.

Abstract 3116: Bioluminescent 3D tumors with immune cell co-culture for high throughput screening (HTS)

Abstract In the past decade, cancer immunotherapy has revolutionized the cancer treatment strategy. Immune-oncology (IO) drugs play an important role in treating various malignancies. Immunotherapies work by re-educating and boosting patients’ immune systems to recognize and eliminate cancer cells. The immune co-culture assay provides invaluable information to understand the tumor microenvironment (TME) and observe the immune response after IO drug treatment. Recent advancements in the Champions TumorGraft3D (CTG3D) platform provides more consistent and accurate information for preclinical drug evaluation, but also efficiently simulate the tumor-Immune microenvironment (TIME) and interactions between cancer organoids and immunocyte. However, current analytic approaches in organoid-immune co-culture studies are still highly reliant on flow cytometry and high-resolution confocal imaging endpoints to provide the study outcome. There is a need for a fast and cost-effective co-culture platform to help set up experimental conditions. Easily traceable tags (e.g. bioluminescent and fluorescent) can quickly help establish reliable the effector (E) cell to tumor (T) cell conditions, thus enabling the user to test multiple immune-cell activator or engagers concentrations in a cost-effective and rapid manner. At Champions Oncology we provide stable Luc/GFP-tagged CTG3D models. We have built a reliable CTG3D-immune co-culture platform with various indications (including colon, prostate, gastric, non-small cell lung cancer, and leukemia) which can be used in co-culture with autologous TIL, allogenic TIL and NK cells, thus providing users a fast and cost-effective option to optimize the E:T ratio and monitor the drug performance. Citation Format: Fu-Ju Chou, Samaneh Kamali, Mara Gilardi, Brandon Walling, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. Bioluminescent 3D tumors with immune cell co-culture for high throughput screening (HTS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3116.

Integrated analysis of histone lysine lactylation (Kla)-specific genes suggests that NR6A1, OSBP2 and UNC119B are novel therapeutic targets for hepatocellular carcinoma

Histone lysine lactylation (Kla) plays a vital role in the tumorigenesis of hepatocellular carcinoma (HCC). Hence, we focused on Kla-specific genes to select novel therapeutic targets. Differentially expressed Kla-specific genes (DEKlaGs) were identified from TCGA with the cut-off criteria |log2(FlodChange (FC))| > 2, p-value < 0.05, following investigating the prognostic value. The correlation between lactate accumulation and prognostic DEKlaGs expression was further investigated. On the other hand, we explored the roles of Kla activation in the immune microenvironment, immunotherapy, and drug resistance. We conducted gene set enrichment analysis (GSEA) to predict the pathways influenced by Kla. The predictive power of Cox model was further identified in ICGC and GEO databases. A total of 129 DEKlaGs were identified, and 32 molecules might be potential prognostic biomarkers. A Cox model including ARHGEF37, MTFR2, NR6A1, NT5DC2, OSBP2, RNASEH2A, SFN, and UNC119B was constructed, which suggested unfavorable overall survival in high-risk score group, and risk score could serve as an indicator for large tumor size, poor pathological grade and advanced stage. NR6A1, OSBP2 and UNC119B could inhibit NK cell as well as TIL cell infiltration, and impair Type-I and II IFN responses in HCC, thereby contributing to unsatisfactory prognosis and immunotherapy resistance. OSBP2 and UNC119B were identified to be related to chemotherapy resistance. GSEA showed that WNT, MTOR, MAPK and NOTCH signaling pathways were activated, indicating that these pathways might play a crucial role during the Kla process. On the other hand, we showed that NR6A1 and OSBP2 were overexpressed in GEO. OSBP2 and UNC119B contributed to poor survival and advanced stage in ICGC. In summary, histone Kla was related to HCC prognosis and might serve as an independent biomarker. NR6A1, OSBP2 and UNC119B were associated with the prognosis, immunotherapy, and chemotherapy resistance, suggesting that NR6A1, OSBP2 and UNC119B might be novel candidate therapeutic targets for HCC.

The development of adult T cell leukemia/lymphoma in renal transplant recipients: report of two cases with literature review

BackgroundsTherefore, reports on the risk of HTLV-1-related diseases in organ transplantation have increased in recent years, and the management of HTLV in renal transplantation remains a challenge.Patients and methodsWe retrospectively analyzed four HTLV-1-positive recipients or donors among 89 renal transplantation cases from 2006 to 2021.ResultsAmong the four HTLV-1-positive recipients, two patients developed adult T cell leukemia/lymphoma (ATL) derived from recipients at approximately 3 years (1016 days and 1195 days) after renal transplantation. Case 1 developed lymphoma-type ATL (an extranodal primary cutaneous variant), including skin and pulmonary lesions. The patient achieved CR with FK tapering and CHOP therapy following cord blood stem cell transplantation. However, the patient died 101 days after ATL development because of a severe fungal infection. Case 2 developed acute-type ATL with an unusual phenotype of CD4+8+30+. The patient was treated with FK tapering and palliative therapy because of poor PS. Notably, in case 1, histopathological findings showed high numbers of PD-1-positive TIL cells in ATL, suggesting exhausted T cells and a correlation with the early onset of ATL. Furthermore, in Case 2, histopathological findings revealed CD 30 expression in ATL cells, suggesting the importance of CD 30 in ATL development. Importantly, case 2 showed typical driver mutations, including CCR4 truncation mutations of the C-terminal, TBL1XR1 mutation, and TP53 mutation in the splice site. Notably, our present study and our previous study on renal transplantation strongly indicated that two out of two and one out of 59 “recipient” positive cases developed ATL, respectively. Furthermore, our previous nationwide study 4 out of 10 “donor” positive cases developed HAM. These findings showed that ATL may be derived from HTLV-I+ recipient cells and HAM may be derived from HTLV-1+ donor cells, although the precise mechanism remains unknown.ConclusionsThus, early onset and rapid progression of ATL with poor outcomes should be considered in HTLV-1 endemic areas. Furthermore, immunological or genetic mechanisms may be related to the development of ATL after renal transplantation. We believe that the mechanism of onset of ATL after transplantation may be important when considering the immune environment of ATL itself.

Immunohistochemical analysis of the tumor microenvironment in acral lentiginous melanoma (ALM).

e21588 Background: Acral lentiginous melanoma (ALM) is a rare subtype of melanoma occurring on the palms, soles and nail beds. Molecularly, ALM is characterized by a higher frequency of chromosomal structural and copy number variants, but lower tumor mutational burden (TMB). Meanwhile, immune checkpoint inhibitors (ICI) are less efficacious in this subtype. In order to better understand these differences in outcomes to ICI, we conducted an immunohistochemical analysis of ALM to explore putative immune biomarkers. Methods: Clinically annotated samples of ALM were identified from patients treated at the USC Keck School of Medicine between 2010-2018. Immunohistochemistry stains included CD8, PD-L1, CD56, CD68, FOXP3, CTLA-4, HLA-ABC and HLA-G, and were characterized based on the intensity of staining. The percentage of tumor cells and infiltrating immune cells (IC) within the tumor microenvironment for the aforementioned antigens were recorded. Data are reported as percent of total cells ( &gt; 1%, &gt; 5%, &gt; 25%, &gt; 50%) expressing marker in the entire sample. Results: A total of 28 ALM specimens from 21 different patients were analyzed, including 19 primary and 9 metastatic tumors. The median age of all patients was 67 years (range 32-92); the majority were men (66%) and Hispanic (66%). Primary ALM tumors were located more often on the plantar foot (62%), compared to the plantar hand (29%). The median tumor thickness was 2.8 mm (range 0.25 – 6.5 mm). Among all patients, 57% had localized disease, while 19% had regional metastases and 24% had distant metastases. CD8+ TIL cells were observed in 95% (18/19) of samples, with 68.4% (13/19) showing &gt; 50% CD8+ staining on IC. PD-L1 expression &gt; 1% on tumor cells was noted in 37% (10/27) of samples, and 19% (5/27) on IC; PD-L1 &gt; 5% was noted in only 18.5% (5/27) on tumor cells and on 11% (3/27) on IC. Both CD56 and CD68 were noted to have at least 1% expression on IC among nearly all (94%) of samples analyzed (7/8 and 9/9, respectively). FoxP3 expression &gt; 1% was noted on IC in 92.3% (24/26) of samples, and &gt; 5% in 50% (13/26). CTLA-4 expression &gt; 1% was observed among 81.5 % (22/27) of samples, while 63% (17/27) had expression &gt; 5%; CTLA-4 expression &gt; 50% was noted among 100% on melanophages (7/7 samples). HLA-ABC expression was detected in 100% of samples analyzed (10/10). The percentage of samples with HLA-G expression &gt; 1% was 60.7% (17/28), while 28.6% (8/28) demonstrated &gt; 5% expression. Further correlations with clinical outcomes will be reported. Conclusions: Our study suggests a unique immune tumor microenvironment in ALM, characterized by low PD-L1 expression, despite infiltration by CD8+, as well as CD56 and CD68 immune cells. Furthermore, the presence of FOXP3+ Tregs and HLA-G expression, both which mediate immune response evasion, may explain the poor outcomes of ICI therapy among these patients and warrant further investigation.

Harnessing the power of tumor-infiltrating lymphocytes: A first-in-human study of GT201 as monotherapy in advanced solid tumors.

2551 Background: Genetically engineered TIL has shown features such as prolonged memory that can improve therapeutic efficacy in tumor mouse models. GT201 is an engineered TIL that expresses a membrane-bound IL-15 (mbIL-15), and preclinical studies have demonstrated enhanced antitumor reactivity. A phase 1 study was initiated to evaluate the preliminary safety of GT201 and to identify Optimal Biological Dose (OBD) suitable for monotherapy expansion. Methods: The dose-escalation study (using TITE-BOIN design) intended to enroll patients (pts) with advanced treatment-refractory solid tumors. Once the OBD is identified, a monotherapy expansion phase will be opened for patients with various solid tumors. Enrolled patients were preconditioned with a nonmyeloablative (NMA) lymphodepletion regimen and treated by autologous infusion of the GMP-grade G201 cell product, followed with supportive low dose IL-2 administration. Results: As of January 17, 2023, the dose-escalation study enrolled 4 pts, all of whom were female with a median age of 52.6 years. The median number of infused TIL cells was 5.3E+09. All pts experienced TRAEs, with the majority being grade 1 or 2. The most common grade 3/4 TRAEs were lymphopenia (100%), leukopenia (25%) and neutropenia (25%), which were related to the NMA regimen. No dose-limiting toxicities (DLTs) or TIL-related grade ≥3 TRAEs were observed. Median time on study was 17 wks (6–30 wks). One pt with highly treatment-refractory cervical cancer experienced a confirmed partial response (PR) after 16 wks that was durable for 30 wks (RECIST 1.1). Two pts (cervical cancer and melanoma) experienced disease stabilization, while another malignant melanoma pt experienced progressive disease. Despite varying doses of TILs (5.0E+09 to 1.40E+10) and IL-2 (up to 3.0E+05 IU/kg), all patients receiving GT201 therapy showed robust T cell expansion post-infusion, with higher doses of IL-2 leading to more rapid TIL expansion. GT201 TILs could be detected in patients after one month post-infusion, indicating promising activation and long-term persistence of the infused TILs. Based on safety, tolerability, PKs, and preliminary efficacy, a TIL dose of 1.0E+10 and an IL-2 dose of 3.0E+05IU/kg was selected as the OBD for the study. Conclusions: The results of the first-in-human study of GT201, a mIL-15 expressed on TILs, demonstrate promising preliminary findings as a monotherapy in pts with advanced cancers. The administration of GT201 was associated with robust T cell expansion, as well as prolonged persistence of infused TILs. Results of mbIL-15 expressed TIL demonstrates that GT201 has good tolerability with encouraging preliminary antitumor activity as a monotherapy. Further investigations, including correlative biomarker analyses and updated safety and clinical outcome data, will be presented at the meeting. Clinical trial information: NCT05430360 .

883TiP A phase I/II open-label study (IOV-GM1-201) of TALEN-mediated PD-1 inactivated autologous tumor-infiltrating lymphocytes (TIL; IOV-4001) in patients with advanced melanoma and NSCLC

Adoptive cell therapy with TIL has demonstrated efficacy in patients (pts) with advanced solid tumors, including melanoma (Sarnaik JCO 2021) and NSCLC (Schoenfeld SITC 2021). IOV-4001 is a TALEN®-mediated PDCD-1 knockout autologous TIL cell therapy product. Preclinical studies suggest that PD-1 inactivation by PDCD-1 gene knockout may enhance TIL cell therapy efficacy, with similar quality attributes and phenotypes to those of non-edited TIL (Natarajan AACR 2022). This first-in-human phase 1/2, open-label, nonrandomized, multicenter study (NCT05361174; open to enrollment) will enroll ∼53 adult pts. During the phase 1 portion, enrollment and dose level decisions will be based on emerging safety and tolerability data in a 28-day dose-limiting toxicity (DLT) observation period. Cohort 1 will include pts with unresectable/metastatic melanoma that has progressed during/within 12 wks of last anti–PD-1/PD-L1 dose (pts must have also received a BRAF ± MEK inhibitor if BRAF V600 mutation-positive). Cohort 2 will include pts with advanced NSCLC who have received ≤3 prior therapies and whose disease progressed either: (1) during/within 12 wks after last anti–PD-1/PD-L1 dose (pts without oncogene-driven mutations) or (2) during/after ≥1 targeted therapy and either platinum doublet chemotherapy or during/within 12 wks after last anti–PD-1/PD-L1 dose (pts with oncogene-driven tumors). Pts must have ECOG PS ≤1, ≥1 resectable lesion(s) (≥1.5 cm), ≥1 remaining RECIST-measurable lesion(s) and recovered from prior surgery/anticancer treatment-related AEs (grade ≤1). IOV-4001 is generated from resected tumor in a centralized GMP process. The regimen includes nonmyeloablative lymphodepletion, IOV-4001 infusion, and a short course of high-dose IL-2. The primary endpoints of phases 1 and 2 are safety (DLTs and AEs) and objective response rate per RECIST v1.1, respectively. Secondary endpoints include complete response rate, duration of response, disease control rate, progression-free survival, overall survival, feasibility, and additional safety. NCT05361174. Medical writing support was provided by Second City Science (Vaniam Group, LLC) and funded by Iovance Biotheraputics, Inc. Iovance Biotherapeutics, Inc.

Abstract 1393: A Novel platform providing automated, consistent and gentle cell isolation from tissues for downstream applications

Abstract Tumor biologists have long studied tumor tissue to understand cancer progression and to ultimately treat the disease. The gold standard would be to analyze tumor and immune cells from the same sample, providing purified cells that are interacting within the tumor. Many tissue homogenization techniques have been developed to release cells for downstream analysis, however, an automated and gentle process of purifying multiple cell types from the same processed tissue has remained elusive. The industry-standard workflows, such as centrifugation, have presented long-standing performance issues in terms of low cell recovery and inefficient removal of molecular debris, which often leads to sequencing results that are difficult to interpret. To ensure the quality of sequencing data from tissues, upstream processes need to improve to get the most from the precious patient tissue. Applied Cells has developed an integrated system based on multiple physics principles to achieve cell wash, concentration, and target cell isolation, with high recovery at an unprecedented flow rate. This platform combines tunable, acoustic cell purification and in-flow, column-free immuno-magnetic separation technologies, enabling automation of the entire cell sample preparation workflow for genomics and transcriptomics analysis. By harnessing the power and precision of acoustic cell manipulation, the MARS System can process a variety of samples including dissociated solid tissue in enzymatic buffer with high cell recovery and superior molecular debris removal compared to standard methods. For instance, when purifying leukocytes from a lung cancer biopsy tissue samples, MARS can remove 99.50% debris and 91% of dead cells in one single step, which is supreme than the conventional centrifuge cell wash while having &amp;gt;90% leukocyte recovery at 1mL/min flow rate. The in-flow microfluidics design drastically reduces residual DNA and RNA fragments that are difficult to remove using centrifuge wash methods. Furthermore, the system enables parallel cell purification and concentration for high throughput sample processing. Tumor or TIL cells can be further enriched by means of positive magnetic selection for genomic or cell biology applications. This novel platform allows improved tumor and TIL recovery in a gentle, consistent manner while having minimal interference with downstream applications, all from a single source starting material. Citation Format: Liping Yu. A Novel platform providing automated, consistent and gentle cell isolation from tissues for downstream applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1393.

Abstract P050: Trial in progress: A phase 2 multicenter study of autologous tumor-infiltrating lymphocyte (TIL, LN-145) cell therapy in patients with metastatic non-small cell lung cancer (mNSCLC)

Abstract Patients with mNSCLC without actionable driver mutation(s) who have progressed after cytotoxic chemotherapy plus immune checkpoint inhibitors (ICI) ± bevacizumab have limited treatment options and represent an unmet need. The safety and efficacy of TIL cell therapy for patients with mNSCLC who failed to respond or progressed on nivolumab has been evaluated in a Phase 1 clinical trial (Creelan B. AACR 2020), demonstrating an objective response rate (ORR) of 25% including 17% durable CR. This provides a clear rationale for initiation of IOV-LUN-202 study, evaluating TIL cell therapy with LN-145 in patients with mNSCLC without actionable driver mutation(s), who have progressed on or following a single line of approved systemic therapy consisting of combined ICI + chemotherapy ± bevacizumab. Trial Design IOV-LUN-202 (NCT04614103) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase 2 study. Cohorts 1 and 2 (each, n=40) are enrolling patients with mNSCLC based on tumor proportion score (TPS; Cohort 1, TPS &amp;lt;1%; Cohort 2, TPS ≥1%) at metastatic diagnosis prior to ICI use. Cohort 3 (TPS &amp;lt;1%; n=15) uses core biopsies for tumor acquisition in patients unable to undergo a surgical harvest, and Cohort 4 is designated for patients requiring retreatment. LN-145 is a cryopreserved infusion product generated at centralized GMP facilities in a 22 day manufacturing process. All patients receive TIL therapy consisting of nonmyeloablative lymphodepletion with cyclophosphamide (60 mg/kg × 2) + fludarabine (25 mg/m2 × 5), followed by a single infusion of autologous LN-145 (Day 0) and up to 6 doses of IL-2 (600,000 IU/kg). Key eligibility criteria include age ≥ 18 y, 1 prior line of therapy, ≥1 lesion(s) available for TIL generation and a remaining RECIST-measurable lesion, and ECOG PS 0–1. For each cohort the primary endpoint is ORR per RECIST 1.1. Secondary endpoints are safety, complete response rate, duration of response, disease control rate, progression-free survival, overall survival, and efficiency of generating LN-145 from tumor core biopsies (Cohort 3). Citation Format: Erminia Massarelli, Selda Samakoglu, Viktoria Gontcharova, Guang Chen, Madan Jagasia, Friedrich Graf Finckenstein, Ammar Sukari. Trial in progress: A phase 2 multicenter study of autologous tumor-infiltrating lymphocyte (TIL, LN-145) cell therapy in patients with metastatic non-small cell lung cancer (mNSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P050.

P14.04 A Phase 2 Multicenter Study of Iovance Autologous Tumor Infiltrating Lymphocytes (TIL, LN-145) Cell Therapy in Patients With Metastatic NSCLC

Patients with metastatic non-small cell lung cancer (mNSCLC) without actionable driver mutation(s) who have progressed after cytotoxic chemotherapy plus immune checkpoint inhibitors (ICI) have limited treatment options and represent an unmet need. The safety and efficacy of TIL cell therapy for mNSCLC patients who failed to respond or progressed on nivolumab has been evaluated in a Phase 1 clinical trial (Creelan B, AACR 2020), demonstrating an objective response rate (ORR) of 25% including 17% durable CRs.

Immune Microenvironment Change and Involvement of Circular RNAs in TIL Cells of Recurrent Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma is a malignant tumor that is highly prevalent in southern China and the Southeast Asian belt. Recent studies have shown that the T cells play important regulatory roles in tumorigenesis and progression. We test TIL cell of recurrent nasopharyngeal carcinoma and primary nasopharyngeal carcinoma cell. We found that T cell change in recurrent nasopharyngeal carcinoma and primary nasopharyngeal carcinoma cell. Based on GEO database, we selected differently expressed circRNAs in nasopharyngeal carcinoma tissues. qRTPCR show that some circRNAs also highly expressed in TIL cells. In conclusion, immune microenvironment changed in recurrent nasopharyngeal carcinoma. There is involvement of circular RNAs in this progress, with should be researched further.

Abstract CT246: A phase 2 multicenter study of autologous tumor infiltrating lymphocytes (TIL, LN-145) cell therapy in patients with metastatic non-small cell lung cancer (mNSCLC)

Abstract Background Patients with mNSCLC without actionable driver mutation(s) who have progressed after cytotoxic chemotherapy plus immune checkpoint inhibitors (ICI) ± bevacizumab have limited treatment options and represent an unmet need. The safety and efficacy of TIL cell therapy for mNSCLC patients who failed to respond or progressed on nivolumab has been evaluated in a Phase 1 clinical trial (Creelan B., AACR 2020), demonstrating an objective response rate (ORR) of 25% including 17% durable CRs. This provides a clear rationale for initiation of IOV-LUN-202 study, evaluating Iovance Gen 2 TIL cell therapy with LN-145 in patients with mNSCLC without actionable driver mutation(s), who have progressed on or following a single line of approved systemic therapy consisting of combined ICI + chemotherapy ± bevacizumab. Trial Design IOV-LUN-202 (NCT04614103) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase 2 study. Patient cohorts (n=40 ea) based on tumor proportion score (TPS) at metastatic diagnosis prior to ICI use are Cohort 1 (TPS &amp;lt;1%) and Cohort 2 (TPS ≥ 1%). Cohort 3 (TPS &amp;lt;1%; n=15), utilizes core biopsies for tumor acquisition for patients unable to undergo a surgical harvest. LN-145 is generated at centralized GMP facilities and the final cryopreserved infusion product is shipped to the sites. All patients receive TIL therapy consisting of nonmyeloablative lymphodepletion with cyclophosphamide (60 mg/kg X 2) + fludarabine (25 mg/m2 X 5), followed by a single infusion of autologous LN-145 (Day 0) and up to 6 doses of IL-2 (600,000 IU/kg). Key eligibility includes: ≥ 18 y of age; 1 prior line of therapy; ≥ 1 lesion(s) available for TIL generation and a remaining RECIST-measurable lesion; and ECOG PS of 0-1. For each cohort, the primary endpoint is ORR per RECIST 1.1. Secondary endpoints are safety, CR rate, DOR, DCR, PFS, OS and efficiency of generating LN-145 from tumor core biopsies (Cohort 3). Table 1.IOV-LUN-202 Patient CohortsPATIENT POPULATIONCOHORTMetastatic NSCLC without an actionable driver mutation and progressive disease on or following a single line of approved systemic therapy including ICI + Chemo ± BevacizumabSAMPLE SIZE1PD-L1 TPS &amp;lt; 1%N = 402PD-L1 TPS ≥ 1%N = 403PD-L1 TPS &amp;lt; 1%/Core BiopsiesN = 154RetreatmentN = undefined Citation Format: Erminia Massarelli, Zelanna Goldberg, Alex Cacovean, Bhagyashree Yadav, Guang Chen, Madan Jagasia, Friedrich Graf Finckenstein, Maria Fardis, Ammar Sukari. A phase 2 multicenter study of autologous tumor infiltrating lymphocytes (TIL, LN-145) cell therapy in patients with metastatic non-small cell lung cancer (mNSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT246.

PD-L1 Expression in Colorectal Carcinoma: Immunohistochemical Study

Background : Colorectal carcinoma is the most common type of GIT malignancy which is a multifactorial process with etiology encompassing genetic factors, environmental exposures and inflammatory conditions of the digestive tract.&#x0D; Method: 40 histologically proven cases of colorectal carcinoma were subjected for IHC expression of PD-L1 using Biocare kit. PD-L1 was taken positive as yellow to brown particles in membrane, cytoplasm or both. It was graded depending upon staining intensity and percentage of the cells and scoring was done by multiplying both and ≥ 3 was taken as positive score.&#x0D; Result : Most of the cases were in 6th and 7th decade of life (62.5%) with M:F ratio being 1.4:1. Most common site involved was rectum. Histologically 29 cases were conventional adenocarcinoma with rest being mucinous and mucin secreting type and most of them being moderately differentiated (70%). On immunohistochemistry, PD-L1 expression was seen in 20 cases with moderate and strong intensity. But PD-L1 score was positive in 14 cases (35%). TIL was positive in 57.5% of the cases. Statistically there was significant correlation between histological type of tumor with PD-L1 (p value=0.03) and TIL (p value=0.017). Although PD-L1 positivity was insignificant with histological differentiation (p value=0.65) but TIL was significant (p value=0.03).&#x0D; Conclusion : PD-L1 expression is relevant to the prognosis as well as for further CRC therapies whereas the TIL cell positivity has become an independent predictor of patients survival.

Enhanced Phenotype Definition for Precision Isolation of Precursor Exhausted Tumor-Infiltrating CD8 T Cells.

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39– Tim3– Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39– Tim3– Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.

Sierra Clinical Trial Dosimetry Results Support Low Dose Anti-CD45 Iodine (131I) Apamistamab [Iomab-B] for Targeted Lymphodepletion Prior to Adoptive Cell Therapy

Introduction Nearly all adoptive cell therapies currently being evaluated in the clinic, including CAR-T, TIL, and TCR-based cell therapies, require lymphodepletion to remove cellular cytokine sinks and create a favorable cytokine environment for the incoming transferred cells to proliferate. Targeted conditioning with an antibody radio-conjugate directed to CD45 represents a promising and potentially more effective alternative to the commonly used fludarabine/cyclophosphamide chemotherapy lymphodepletion regimen. Methods SIERRA is an ongoing Phase 3 multicenter trial evaluating anti-CD45 Iodine (131I) Apamistamab [Iomab-B] as targeted conditioning prior to HCT in active, relapsed or refractory acute myeloid leukemia. Prior to administration of the therapeutic dosage, dosimetry is performed using a tracer amount of Iomab-B (range from 7-20 mCi, median 10 mCi) in an out-patient setting to calculate the appropriate patient-specific therapeutic infusion. Blood sample analysis from 57 evaluable Iomab-B treated patients collected pre-dosimetric infusion (Pre-DI), post-dosimetric infusion (Post-DI), day 1 post-dosimetric infusion (D1 post-DI), and pre-therapeutic infusion (Pre-TI, range 6-14 days post-dosimetry) was assessed to determine if residual Iomab-B had any significant effect on blood counts in support of its use as a transient targeted lymphodepletion agent. Results From these data, a significant but transient decrease in lymphocytes and white blood cells was observed compared to pre-DI values. An 85% decrease of lymphocytes was observed at the post-DI time point, a 67% decrease at day 1 post-DI, and a 43% decrease at the time of therapeutic infusion. Peripheral blasts were also transiently decreased at the post-DI time point (35%), indicating that low dose Iomab-B may exert an anti-tumor effect in these patients. Interestingly, the levels of platelets, hematocrit, and neutrophils were unchanged at the Pre-TI time point compared to Pre-DI, reflecting the comparatively lower surface antigen levels of CD45 on these cell types. In addition, data from a subset of treated patients (n=25) was used to calculate the radiation absorbed dose to bone marrow to determine an appropriate amount of Iomab-B that would not impart more than 2 Gy, a threshold that is considered to be non-myeloablative. This analysis determined that 75 mCi Iomab-B could be administered as a non-myeloablative amount and has been proposed as the starting dose for a clinical trial using Iomab-B for targeted lymphodepletion prior to CAR-T. Additional calculations were performed to model the clearance of Iomab-B to determine at what time post-infusion a CAR-T could be administered without the amount of residual radiation to bone marrow exceeding a safe level (0.25 Gy). Based on clinical data from the SIERRA trial, the average effective half-time of Iomab-B was 45.1 hours and the time frame for CAR-T administration following 75 mCi of Iomab-B was 136 hours (5.7 days). Given that administration of radiopharmaceuticals often requires special safety precautions, the proposed range of doses for Iomab-B is considered an outpatient infusion without the need for isolation. Conclusions Despite the importance of lymphodepletion prior to adoptive cell therapies, there has been very little optimization of this step. Clinical data collected using a low dose of Iomab-B for dosimetry has demonstrated that this method of lymphodepletion is specifically targeted to CD45+ immune cells, may have an anti-tumor effect, and can be administered in an outpatient setting. These clinical and logistical attributes are attractive characteristics for lymphodepletion and supportive of using Iomab-B as a novel lymphodepletion regimen prior to adoptive cell therapies such as CAR-T. Table Disclosures Nath: Astellas: Consultancy; Daiichi Sankyo: Consultancy; Actinium: Consultancy. Geoghegan:Actinium Pharmaceuticals: Employment. Spross:Actinium Pharmaceuticals: Employment, Equity Ownership. Lichtenstein:Actinium Pharmaceuticals: Employment, Equity Ownership. Konerth:Versant Medical Physics and Radiation Safety: Consultancy. Fisher:Versant Medical Physics and Radiation Safety: Employment. Liang:Actinium Pharmaceuticals: Employment. Ludwig:Actinium Pharmaceuticals: Employment, Equity Ownership. Reddy:Actinium Pharmaceuticals: Employment. Berger:Actinium Pharmaceuticals, Inc: Employment, Equity Ownership. Gyurkocza:Actinium Pharmaceuticals: Research Funding.

Abstract 1471: Short hairpin RNA-expressing oncolytic adenovirus-mediated inhibition of PDL1 significantly suppresses human cancer cell growth

Abstract In cancer immunotherapy, it is known that aberrant expression of immune checkpoint proteins in tumor cells and their resulting immunosuppression in surrounding tumor microenviroment and even in whole immune system represent the crucial limiting factors toward successful cancer treatment. Multiple studies evidenced that oncolytic viruses carrying biological molecule including cytokines, shRNAs and miRNAs relieve or attenuate immunosuppression, and may be a promising way toward curing tumor. In this study we generated an oncolytic adenovirus with expression of shRNA targeting human PDL1. The adenovirus exhibited proliferation activity more than two orders of magnitude higher in human cancer cells than in human normal primary cells. Western blot showed significantly decreased expressing levels of PDL1 protein in human cancer cells upon infection of the PDL1 shRNA expressing-oncolytic virus. FACS assay also showed decreased expression levels of PDL1 on the cell membrane after the infection of same viruses. In vivo experiments in two different genetic backgrounds mouse model &amp;lt;NOD-SCID mice and BALBC nude mice&amp;gt; demonstrated that the PDL1 shRNA expressing adenoviruses lysed the majority of the tumor cells derived from HCT116 colorectal cancer cells that were subcutaneously inoculated. Moreover, the adenoviruses downregulated PDL1 expression levels within the HCT116 colon cell-derived tumor cells, remarkably attenuated immunosuppression surrounding the HCT116 tumor. Consisted with these observations, TIL cell counts in the tumors derived from mice with shRNA expressing oncolytic adenovirus treatment were significantly higher than that from mice in other control groups. The growth of HCT116 colon cell-derived tumor in mice with oncolytic adenovirus treatment was greatly inhibited for a longer period compared with that in mice in other control groups. The oncolytic adenovirus described in this study features highly selective amplification in human tumor cells versus normal cells and inhibition of PDL1 expression in tumor cells, therefore it may be important toward developing novel method for cancer therapy. Citation Format: Jipo Sheng, Xue Wang, Xiaoming Dong, Jin Fu, Sanmao Kang, Fang Hu. Short hairpin RNA-expressing oncolytic adenovirus-mediated inhibition of PDL1 significantly suppresses human cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1471.

LncRNA SNHG1 regulates the differentiation of Treg cells and affects the immune escape of breast cancer via regulating miR-448/IDO

ObjectiveTo investigate the mechanism of lncRNA SNHG1 in the immune escape of breast cancer (BC). MethodsSNHG1, miR-448 and IL-10 levels were evaluated by qRT-PCR. The protein levels of IDO and Foxp3 were measured by Western blot. SNHG1 and miR-448 interaction was tested by RIP assay and RNA pull-down assay. MiR-448 and IDO interaction was observed by luciferase reporter assay. ResultsCompared with CD4+T cells, miR-448 expression in CD4+ TIL cells was decreased, while the expression of SNHG1, IDO, IL-10 and Foxp3 were increased. Moreover, SNHG1 directly contacted with miR-448, which could negatively regulate IDO. In cells treated with siRNA-SNHG and miR-448 inhibitor, interference SNHG1 up-regulated miR-448 expression and down-regulated IDO expression, while miR-448 inhibitor reversed this effect. In addition, miR-448 inhibitor reversed the inhibitory effect of siRNA-SNHG1 on Treg cell differentiation, and siRNA-SNHG1 could reduce tumor volume and down-regulated the expressions of SNHG1, IL-10, IDO and Foxp3. ConclusionInterference SNHG1 could inhibit the differentiation of Treg cells by promoting miR-448 expression and reducing IDO level, thereby impeding the immune escape of BC.

Blockade of BAFF Receptor BR3 on T Cells Enhances Their Activation and Cytotoxicity.

The BAFF receptor BR3 plays key roles in B-cell activation, maturation, and survival whereas the function of BR3 on T lymphocytes is less well characterized. Previous reports have demonstrated that BR3 costimulates human T-cell activation in vitro in the presence of high nonphysiological levels of plate-bound BAFF. Here, relying on the soluble and membrane-bound BAFF expressed by T cells themselves, we investigated the function of BR3 on activated primary CD4 and CD8 T lymphocytes using a BR3-specific neutralization antibody and shRNA gene down-modulation. Interestingly, the anti-BR3 blocking antibody resulted in significant augmentation of CD25 and IFN-γ expression by both subsets, as did shRNA-mediated down-modulation of BR3. In addition, granzyme B expression was substantially elevated in anti-BR3-treated and BR3-silenced T cells. Anti-BR3 blockade increased the expression of CD25 on cytolytic CRTAM T cells. Importantly, anti-BR3 significantly enhanced redirected killing of P-815 cells by both CD4 and CD8 cytotoxic T cells [cytotoxic T lymphocytes (CTLs)]. Furthermore, anti-BR3-augmented CD4 T-cell-mediated killing of class II melanoma cell line A375 and cervical cancer cell line HeLa in vitro, increasing the level of granzyme B activity as measured by PARP-1 cleavage and active caspase 3. Together, our data indicate that BR3 neutralization increases the activation and cytolytic function of CD4 and CD8 cytotoxic T lymphocytes. Our findings provide a novel strategy for ex vivo T-cell activation applicable to T-cell immunotherapy platforms such as TIL or CAR-T cell therapeutics.

TNF\u03b1 blockade overcomes resistance to anti-PD-1 in experimental melanoma

Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma. However, many patients do not display therapeutic response or eventually relapse. Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies. Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown. Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma. Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers. Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression. TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens. This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.

β-Catenin in desmoid-type fibromatosis: deep insights into the role of T41A and S45F mutations on protein structure and gene expression.

Desmoid‐type fibromatosis (DF) is a rare mesenchymal lesion with high risk of local recurrence. Specific β‐catenin mutations (S45F) appeared to be related to this higher risk compared to T41A‐mutated or wild‐type (WT). We explored the influence of both mutations and WT on structure stability and affinity of β‐catenin for α‐catenin and the pattern of gene expression that may influence DF behavior. Using 33 surgically resected primary DFs harboring T41A (n = 14), S45F (n = 10), or WT (n = 9), we performed a comparative molecular analysis by protein/protein interaction modeling, gene expression by DASL microarrays, human inflammation gene panel, and assessment of immune system‐based biomarkers by immunohistochemistry. Mutated proteins were more stable than WT and formed a weaker complex with α‐catenin. Consensus unsupervised gene clustering revealed the presence of two DF group‐mutated (T41A + S45F) and WT (P = 0.0047). The gene sets ‘Inflammatory‐Defense‐Humoral Immune Response’ and ‘Antigen Binding’ were significantly enriched in T41A. The deregulation of 16 inflammation‐related genes was confirmed. Low numbers of T cells and tumor‐associated macrophages (TAM) infiltrating the tumors and low/absent PD‐1/PD‐L1 expression were also identified. We demonstrated that mutated DFs (T41A or S45F) and WT are two distinct molecular subgroups with regard to β‐catenin stability, α‐catenin affinity, and gene expression profiling. A different inflammation signature characterized the two mutated groups, suggesting mediation either by T41A or by S45F. Finally, all mutated cases showed a low number of TIL and TAM cells and a low or absent expression of PD‐1 and PD‐L1 consistent with β‐catenin activation insensitive to checkpoint blockade.