Tumor Killing Activity Research Articles

Abstract B033: MUC16 - Potential Target for Antibody-based Immunotherapy in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most devastating and lethal cancer. PDAC patients with the current standard of care produce dismal overall survival benefits with high toxicities. Thus, there is an unmet clinical need to develop targeted therapeutics against PDAC. Approximately 50 to 70% of PDAC patients overexpress MUC16, which is associated with disease progression, metastasis, drug resistance, and poor patient survival. We demonstrated that MUC16 enhances PDAC tumor growth via binding and activating epidermal growth factor (EGF) receptor family members and its downstream target PI3K/AKT signaling pathways. Here, we show the efficacy of humanized anti-MUC16 antibody (huAR9.6) against MUC16-positive PDAC. Methods: We tested the in vitro efficacy of humanized anti-MUC16 antibody (huAR9.6) against MUC16+ PDAC cells, patient-derived xenografts (PDXs), and patient-derived organoids (PDOs). We evaluated the in vivo therapeutic efficacy of the huAR9.6 antibody in MUC16+ PDAC cells implanted in subcutaneous and orthotopic tumor models. Fucosylated and afucosylated huAR9.6 induced direct tumor-killing activity was evaluated by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with MUC16 positive PDAC cells. Results: Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16+ PDAC cells and patient-derived organoids (PDOs). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC, PDOs, and PDX-derived cells. HuAR9.6 treatment induces ADCC and CDC against MUC16+ cells. Removing fucose residue on the N-glycans of the huAR9.6-IgG substantially increased huAR9.6’s ADCC activity. More importantly, huAR9.6 treatment caused PDAC regression in subcutaneous and orthotopic tumor models. Conclusion: The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs (detected in ∼ 50-70% of patients). Citation Format: Cory L Brooks, Prakash Radhakrishnan. MUC16 - Potential Target for Antibody-based Immunotherapy in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B033.

Parthenolide Inhibits Synthesis and Promotes Degradation of Programmed Cell Death Ligand 1 and Enhances T Cell Tumor-Killing Activity.

Parthenolide is a germacrane sesquiterpene lactone separated from the traditional medicinal plant feverfew. Previous studies have shown that parthenolide possesses many pharmacological activities, involving anti-inflammatory and anticancer activities. However, the antitumor mechanism of parthenolide has not been fully elucidated. Thus, we investigate the potential antitumor mechanisms of parthenolactone. We predicted through network pharmacology that parthenolide may target HIF-1α to interfere with the occurrence and development of cancer. We found that parthenolide inhibited PD-L1 protein synthesis through mTOR/p70S6K/4EBP1/eIF4E and RAS/RAF/MEK/MAPK signaling pathways and promoted PD-L1 protein degradation through the lysosomal pathway, thereby inhibiting PD-L1 expression. Immunoprecipitation and Western blotting results demonstrated that parthenolide inhibited PD-L1 expression by suppressing HIF-1α and RAS cooperatively. We further proved that parthenolide inhibited cell proliferation, migration, invasion, and tube formation via down-regulating PD-L1. Moreover, parthenolide increased the effect of T cells to kill tumor cells. In vivo xenograft assays further demonstrated that parthenolide suppressed the growth of tumor xenografts. Collectively, we report for the first time that parthenolide enhanced T cell tumor-killing activity and suppressed cell proliferation, migration, invasion, and tube formation by PD-L1. The current study provides new insight for the development of parthenolide as a novel anticancer drug targeting PD-L1.

Groenlandicine enhances cisplatin sensitivity in cisplatin-resistant osteosarcoma cells through the BAX/Bcl-2/Caspase-9/Caspase-3 pathway

Groenlandicine is a protoberberine alkaloid isolated from Coptidis Rhizoma, a widely used traditional Chinese medicine known for its various biological activities. This study aims to validate groenlandicine’s effect on both cisplatin-sensitive and cisplatin-resistant osteosarcoma (OS) cells, along with exploring its potential molecular mechanism.The ligand-based virtual screening (LBVS) method and molecular docking were employed to screen drugs. CCK-8 and FCM were used to measure the effect of groenlandicine on the OS cells transfected by lentivirus with over-expression or low-expression of TOP1. Cell scratch assay, CCK-8, FCM, and the EdU assay were utilized to evaluate the effect of groenlandicine on cisplatin-resistant cells. WB, immunofluorescence, and PCR were conducted to measure the levels of TOP1, Bcl-2, BAX, Caspase-9, and Caspase-3. Additionally, a subcutaneous tumor model was established in nude mice to verify the efficacy of groenlandicine.Groenlandicine reduced the migration and proliferation while promoting apoptosis in OS cells, effectively damaging them. Meanwhile, groenlandicine exhibited weak cytotoxicity in 293T cells. Combination with cisplatin enhanced tumor-killing activity, markedly activating BAX, cleaved-Caspase-3, and cleaved-Caspase-9, while inhibiting the Bcl2 pathway in cisplatin-resistant OS cells. Moreover, the level of TOP1, elevated in cisplatin-resistant OS cells, was down-regulated by groenlandicine both in vitro and in vivo. Animal experiments confirmed that groenlandicine combined with cisplatin suppressed OS growth with lower nephrotoxicity.Groenlandicine induces apoptosis and enhances the sensitivity of drug-resistant OS cells to cisplatin via the BAX/Bcl-2/Caspase-9/Caspase-3 pathway. Groenlandicine inhibits OS cells growth by down-regulating TOP1 level.Therefore, groenlandicine holds promise as a potential agent for reversing cisplatin resistance in OS treatment.

NR4A ablation improves mitochondrial fitness for long persistence in human CAR-T cells against solid tumors

BackgroundAntitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells...

Antitumor Effect and Immunomodulatory Mechanism of "Oncolytic Extracellular Vesicles".

Enhancing the antitumor immune response and targeting ability of oncolytic viruses will improve the effect of tumor immunotherapy. Through infecting neural stem cells (NSCs) with a capsid dual-modified oncolytic adenovirus (CRAd), we obtained and characterized the "oncolytic extracellular vesicles" (CRAdEV) with improved targeted infection and tumor killing activity compared with CRAd. Both ex vivo and in vivo studies revealed that CRAdEV activated innate immune cells and importantly enhanced the immunomodulatory effect compared to CRAd. We found that CRAdEV effectively increased the number of DCs and activated CD4+ and CD8+ T cells, significantly increased the number and activation of B cells, and produced higher levels of tumor-specific antibodies, thus eliciting enhanced antitumor activity compared with CRAd in a B16 xenograft immunocompetent mice model. This study provides a novel approach to oncolytic adenovirus modification and demonstrates the potential of "oncolytic extracellular vesicles" in antitumor immunotherapy.

Results of a phase 1/2 study of MHB088C: A novel B7H3 antibody-drug conjugate (ADC) incorporating a potent DNA topoisomerase I inhibitor in recurrent or metastatic solid tumors.

3012 Background: MHB088C is an investigational B7H3-directed ADC, created by coupling a humanized anti-B7H3 monoclonal antibody with a highly potent DNA topoisomerase I inhibitor (5~10 times more potent than DXd). Preclinical studies demonstrated robust binding affinity, superior internalization rates, powerful tumor killing activities (3~10 times more potent than the DS-7300a analog in CDX models) and a favorable safety profile with no unique toxicities observed in the GLP study as compared to other B7H3 ADCs and no occurrences of interstitial lung disease (ILD). We here present the safety and efficacy results from a first-in-human phase 1/2 study of MHB088C. Methods: The study enrolled patients (pts) with recurrent or metastatic solid tumor across two segments: dose-escalation (D-esc) and dose-expansion (D-exp). In the D-esc phase, MHB088C was administrated intravenously at doses of 0.8, 1.6, 3.0mg/kg Q2W; 3.0, 4.0 mg/kg Q3W. Results: As of the data cutoff on Dec 31, 2023, 60 pts were enrolled and received at least one dose of MHB088C (D-esc, n=14; D-exp, n=46). 55 pts remain on the treatment and 12 pts had at least one tumor assessment according to RECIST 1.1. Dose-limiting toxicities (DLTs) were platelet count decreased and febrile neutropenia at 4.0 mg/kg Q3W. The MTD was determined to be 3.0 mg/kg Q3W. The most common TRAEs overall in ≥25% of pts were neutrophil count decreased, lymphocyte count decreased, and white blood cell (WBC) count decreased. The most common Grade ≥3 TRAEs (≥5% of pts) were neutrophil count decreased (33.3%), lymphocyte count decreased (30.0%), WBC count decreased (26.7%), platelet count decreased (23.3%), and anemia (15.0%). No ILD was reported. Of12 response-evaluable pts, 5 partial responses (PRs) were observed (ORR: 41.7%). The disease control rate was 91.7% (11/12). In the subset of 3 SCLC pts, all 3 showed PRs (ORR: 100.0%), with one case demonstrating complete response of the target lesion and another with close to 80% tumor volume reduction. The efficacy of 2 SCLC pts was achieved in 1.6 mg/kg Q2W group without major hematologic side effects. All responses among SCLC pts occurred at the first tumor assessment. Conclusions: MHB088C exhibited a manageable safety profile, with striking efficacy in SCLC pts. The dose optimization and expansion study is continuing to establish the RP2D for MHB088C. Clinical trial information: CTR20231298.

Results of a phase 1/2 study of MHB036C: A potential best-in-class TROP2 antibody-drug conjugate (ADC) incorporating a potent DNA topoisomerase I inhibitor in locally advanced or metastatic solid tumors.

e15007 Background: MHB036C, an investigational ADC, comprises a humanized anti-TROP2 monoclonal antibody engineered to minimize potential toxicities mediated by non-specific uptake, and a highly potent DNA topoisomerase I inhibitor (5~10 times more potent than DXd) conjugated via a stable cleavable linker. In preclinical studies, MHB036C demonstrated superior tumor killing activities (5~10 times more potent than the DS-1062a analog) and a favorable safety profile with no unique toxicities as compared to other TROP2 ADCs and no occurrences of interstitial lung disease (ILD). We here present the safety and efficacy results from a first-in-human phase 1/2 study of MHB036C. Methods: Patients with locally advanced or metastatic solid tumors were enrolled in dose-escalation (D-Esc) and dose-expansion (D-Exp) segments. The D-esc study, utilizing an accelerated titration followed by the traditional 3+3 design, assessed MHB036C at doses ranging from 0.25-2.25 mg/kg administered intravenously (IV) every 3 weeks. Results: At data cutoff (Dec 31, 2023), 26 pts were enrolled and received at least one dose of MHB036C (D-Esc, n=11, D-Exp, n=15). Two pts experienced dose-limiting toxicities (Grade 3 stomatitis) at 2.25 mg/kg. The MTD was determined to be 1.5 mg/kg. The most common TRAEs overall in ≥25% of pts were stomatitis, anemia, decreased appetite, vomiting, white blood cell (WBC) count decreased, fatigue, nausea, and neutrophil count decreased. The most common Grade ≥3 TRAEs (occurred in ≥5% of pts) were stomatitis (34.6%), WBC count decreased (11.5%), neutrophil count decreased (7.7%), and anemia (7.7%). No ILD was reported. Of 21 response-evaluable pts, 7 experienced partial responses. In the subset of 12 NSCLC pts treated with doses ranging from 1.3 to 2.25 mg/kg, the ORR was 33.3% (4/12) and the DCR was 83.3%. In the subset of 5 HER2-negative breast cancer pts encompassing 4 TNBC pts and 1 HR+/HER2- breast cancer patient, the ORR was 60.0% (3/5). Conclusions: MHB036C demonstrated a manageable safety profile without either major hematologic side effects or ILDs and encouraging efficacy in NSCLC and HER2- breast cancer, representing a potential best-in-class TROP2 ADC. The dose optimization and expansion study is ongoing to establish the RP2D for MHB036C. Clinical trial information: CTR20231246.

3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

CDK9 inhibition as an effective therapy for small cell lung cancer

Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

Enhancing antitumor efficacy of oncolytic virus M1 via albendazole-sustained CD8+ T cell activation

The immune response plays a crucial role in the functionality of oncolytic viruses. In this study, Albendazole, an antihelminthic drug known to modulate the immune checkpoint PD-L1, was combined with the oncolytic virus M1 (OVM1) to treat mice with either prostate cancer (RM-1) or glioma (GL261) tumors. This combination therapy enhanced anti-tumor effects in immunocompetent mice, but not in immunodeficient ones, without increasing OVM1 replication. Instead, it led to an increase in the number of CD8+ T cells within the tumor, downregulated the expression of PD1 on CD8+ T cells, and upregulated activation markers such as Ki67, CD44, and CD69 and the secretion of cytotoxic factors including interferon (IFN)-γ, granzyme B, and tumor necrosis factor (TNF)-α. Consistently, it enhanced the invitro tumor-killing activity of lymphocytes from tumor-draining lymph nodes or spleens. The synergistic effect of Albendazole on OVM1 was abolished by depleting CD8+ Tcells, suggesting a CD8+ Tcell-dependent mechanism. In addition, Albendazole and OVM1 therapy increased CTLA4 expression in the spleen, and the addition of CTLA4 antibodies further enhanced the anti-tumor efficacy invivo. In summary, Albendazole can act synergistically with oncolytic viruses via CD8+ T cell activation, and the Albendazole/OVM1 combination can overcome resistance to CTLA4-based immune checkpoint blockade therapy.

Abstract 4586: ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells

Abstract Effector cytolytic CD8 T cells play a major role in the recognition and killing of cancer cells. These cells are short living due to the loss of their self-renewal capacity and rapid exhaustion. Enduring antitumor immune response relies on the capacity of regenerating effector cells upon antigen re-encounter by memory cells. Focusing on molecules that could modulate the differentiation of T cells towards a memory subset with a prolonged anti-tumor activity is strategic for an efficient anti-tumor immunotherapy. Thus, we developed a selective HDAC6 inhibitor currently in Phase I clinical trial, ITF3756, which in vitro drives the differentiation of human CD8 T cells towards a central memory phenotype with reduced expression of exhaustion markers and higher expression of IFNγ and Granzyme b upon tumor cells coculture. This suggests that ITF3756-treated CD8 T cells could have a greater tumor killing activity. Moreover, if this augmented cytotoxic activity is directed not only towards tumor cells expressing MHC I but also towards MHC I negative cells, the anti-tumor efficacy could be improved. We thus selected the MHC I negative K562 leukemic cell line as target in an in vitro model of CD8 T cell killing assay. CD8 T cells were stimulated with anti CD3CD28 beads with or without ITF3756, and then co-cultivated with K562 cells for 4h. ITF3756 treatment improved significantly CD8 mediated K562 killing over this short period of time. However, since the effectiveness of memory cells is based on their capacity of maintaining a long-lasting immune response, we monitored the cytolytic activity over a period of 2 weeks. After 14 days of co-culture, ITF3756 treated CD8 T cells showed a higher cytotoxic capacity compared to control cells. These results indicate that ITF3756-treated CD8 T cells can kill a target tumor cell in a more efficient way independently of MHC I restricted tumor recognition, both after short and long term in vitro co-culture. Since it has been described that CD8 T cells can also kill MHC I-negative tumor cells through the NKG2D-NKG2DL axis, we assessed if ITF3756 could modulate the expression of NKG2D on CD8 T cells. Indeed, ITF3756-treated CD8 T cells had a significantly higher expression of NKG2D compared to untreated cells. In conclusion, we show that ITF3756 promotes the differentiation of cells that possess better effector activity when co-cultured with tumor cells for both short and long periods of time. ITF3756 increased the expression of NKG2D and this could be a driver of the increased cytolytic activity. One mechanism of tumor immune escape is the downregulation of MHC I. Our results suggest that ITF3756 could be used to improve the efficacy of T cell therapy by enhancing the cytolytic activity even in the absence of MHC I. Citation Format: Chiara Ripamonti, Valeria Spadotto, Christian Steinkuhler, Gianluca Fossati. ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells [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 4586.

Abstract 5087: ILB-3101, a novel B7H3-targeting ADC with Eribulin as payload, demonstrates strong tumor killing activities and favorable PK/TOX profile in preclinical evaluation

Abstract B7H3, a B7 family member of immune checkpoint proteins, is overexpressed in a broad spectrum of malignant tumors. It has become an attractive target for cancer therapies in recent years. ILB-3101, a potential best-in-class ADC, was constructed by conjugating a potent mitotic inhibitor Eribulin to a proprietary anti-B7H3 mAb through a proprietary linker. This ADC molecule has been developed and preclinically evaluated as both the 1st line ADC treatment with B7H3 expressed patients in Eribulin sensitive cancer types and the later line treatment for patients whose cancer are B7H3 positive but TopoI inhibitor resistant. The humanized antibody of ILB-3101 is IgG1 type and shows high affinity to both human and monkey B7H3 proteins. It also shows better internalization than benchmark antibodies such as that of DS-7300 analog. The DAR value of ILB-3101 is 3.5. ILB-3101 has potent and dose-dependent cytotoxicity to B7H3-positive tumor cells and shows better activities than DS-7300 analog as positive control. In vivo studies based on xenograft tumor models reveal that ILB-3101 has potent anti-tumor activities in various tumor types including TNBC, sarcoma, ovarian cancer and AML etc., which are superior or comparable to DS-7300 analog. Furthermore, ILB-3101 shows strong anti-tumor activity on Dxd based ADC resistant tumors in xenograft tumor models. The PK study of ILB-3101 in cynomolgus monkeys shows dose-dependent exposure and desirable PK profile. ILB-3101 was well tolerated in cynomolgus monkeys with HNSTD at 6 mg/kg. No serious adverse events were observed, and the main safety findings were limited and reversible hematological and immunological effects, which are consistent with the safety profile of Eribulin as an approved chemotherapy. The in vitro and in vivo preclinical results above demonstrate that ILB-3101 has good efficacy and safety profile and it is a promising and potentially best-in-class B7H3 targeting ADC drug candidate. ILB-3101 is worthy of further translational research and clinical trials. Its IND application has been submitted in China, followed by US FDA filing soon. Citation Format: Yushuan Shang, Junzhuan Qiu, Yanfei Xin, Min Zhao, Qiang Liu, Zhengsheng Wang, Xiaojing Gong, Yan Xu, Yan Li, Mingde Xia, Rulei Chen. ILB-3101, a novel B7H3-targeting ADC with Eribulin as payload, demonstrates strong tumor killing activities and favorable PK/TOX profile in preclinical evaluation [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 5087.

Abstract 1913: KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with diverse biological functions, including apoptosis, cell survival, inflammation, and immunity. While recombinant human TNFα has demonstrated therapeutic efficacy in isolated limb perfusion for soft tissue sarcoma and melanoma, its systemic administration is limited by severe toxicities, including hypotension. Tumor-specific targeting and enhancement of TNFα's tumor cell killing activity hold promise for improving its therapeutic index. To address these challenges, we developed KD5005, a novel HER-2-targeted immunocytokine fusion protein comprising an anti-HER-2 IgG1 monoclonal antibody (trastuzumab) and human TNFα. Tissue distribution studies revealed that KD5005 specifically accumulated in HER-2-expressing tumors, demonstrating its tumor-targeting capability. In vitro, KD5005 exhibited reduced TNFα-mediated apoptosis in L929 cells compared to recombinant TNFα, indicating its lower non-specific toxicity. Notably, KD5005 induced apoptosis of HER-2-positive human gastric cancer NCI-N87 cells at significantly lower concentrations than trastuzumab, TNFα, or their combination, demonstrating its enhanced tumor cell killing activity. The potent apoptotic activity of KD5005 was HER-2-dependent, as it did not show increased apoptotic activity in WT mouse forestomach carcinoma (MFC) cells and had similar apoptotic activity compared to the combination of trastuzumab and TNFα. This HER-2 dependency further supports the tumor-specific targeting of KD5005.In vivo, KD5005 demonstrated superior anti-tumor efficacy compared to the combination of trastuzumab and TNFα in syngeneic tumor models using CT26 tumor cells transfected with the human HER-2 gene. KD5005 induced robust tumor regression and durable cure in 7 of 8 mice, while the combination of trastuzumab and TNFα elicited weaker therapeutic responses, resulting in tumor rejection in only 3 of 8 mice. Pharmacokinetic and toxicity studies of KD5005 were conducted in cynomolgus monkeys. KD5005 exhibited a significantly prolonged in vivo half-life compared to TNFα, indicating its improved pharmacokinetic profile. KD5005 was administered intravenously every two weeks to the same animal in a dose-escalation study at 0.3 mg/kg, 1.0 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg. The toxicity study results showed that the maximum tolerated dose for KD5005 in cynomolgus monkeys is 10 mg/kg, indicating its favorable toxicity profile. In summary, we have developed KD5005, a HER-2-targeted immunocytokine fusion protein with enhanced tumor-killing activity, improved pharmacokinetic properties, and a favorable toxicity profile. These preclinical findings support the further clinical development of KD5005 as a promising therapeutic agent for HER-2-positive cancers. Citation Format: YI CHEN, PENG LI, GUOBO FANG, HENG WU, CHI ZHANG, ZELING CAI. KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine [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 1913.

Abstract 2373: Functional and specific T-cell engagers against a peptide-MHC tumor target

Abstract Accessing peptides from intracellular tumor antigens displayed on MHC class I (pMHC) can expand the T-cell engager (TCE) target pool for solid tumor applications. A challenge limiting TCE development for pMHC targets is the identification of rare, high-affinity, and high-specificity pMHC-binders. In this work, we describe TCEs in a 1 × 1 format with potent tumor-cell killing activity and high specificity for MAGE-A4, a pMHC tumor antigen expressed in multiple cancer types. We identified functional MAGE-A4 x CD3 TCEs with high specificity and affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA-A:02*01). We strategically selected MAGE-A4-binders with high binding specificity for pMAGE-A4230-239, but not MHC-I or many closely related MHC-restricted peptides. We then paired them with diverse and developable CD3-binders with a range of binding affinities, subunit specificities, and binding kinetic profiles. High-throughput in vitro functional characterization of hundreds of these TCEs enabled identification of molecules with optimal T-cell dependent cellular cytotoxicity in MAGE-A4-expressing tumor cell lines, with comparable tumor-killing activity and cytokine release to a clinical-stage TCE in a 2 × 1 format. Previous studies have highlighted unique challenges associated with pMHC targets, including their potential for cross-reactive binding to pMHCs on healthy tissues, and the absence of pharmacologically relevant species for preclinical toxicity testing. To address these challenges, we have designed and implemented an in vitro and in silico workflow to identify antibody binding to potential off-target peptides. Using this approach, we selected TCEs with several pMHC binding orientations and high specificity and affinity. These TCEs killed tumor cell lines endogenously expressing MAGE-A4 pMHCs and not isogenic cells with MAGE-A4 knocked out. Results demonstrate that our high-resolution, multiparametric antibody discovery capabilities can identify pMHC-binding antibodies with desired functional and specificity attributes. This approach unlocks the discovery and development of optimal TCEs against complex pMHC antigens. Citation Format: Davide Tortora, Peter Bergqvist, Allison Goodman, Ryan Blackler, Nathalie Blamey, Stefania Carrara, Lauren Chong, Gabrielle Conaghan, Cindy-Lee Crichlow, Valentine de Puyraimond, Harveer Dhupar, Patrick Farber, Jessica Fernandes Scortecci, Kate Gibson, Rodrigo Goya, Ahn Lee, Franco Li, Tova Pinsky, Craig Robb, Patrick Rowe, Antonios Samiotakis, Eduardo Solano Salgado, Ping Xiang, Irene Yu, Kelly Bullock, Tara Fernandez, Stephanie K. Masterman, Kush Dalal, Tim Jacobs, Bryan C. Barnhart. Functional and specific T-cell engagers against a peptide-MHC tumor target [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 2373.

Abstract 4080: IBB0979, A novel B7H3/IL-10 immunocytokine of monovalent anti-B7H3 antibody fused with IL-10 homodimer

Abstract Background: This study demonstrates the preclinical evaluation of a novel B7H3/IL-10 immunocytokine IBB0979. IBB0979 was designed to solve the problems of immune cell exhaustion and drug resistance in current immunotherapy. As the tumors progress, the domination of progenitor exhausted T cells (which are the major target cells of current PD-1/L1 based immunotherapy) will be replaced by terminally exhausted T cells (which are the major target cells of IL-10) causing resistance of current PD-1/L1 based therapies. Thus, IL-10 is a potent activator of antigen-specific CD8+ T cells in the tumor microenvironment and can restore the tumor-killing activity of tumor-infiltrating lymphocytes by restoring the oxidative phosphorylation metabolism of terminally exhausted T cells. B7H3 is highly expressed on various tumors but expressed at lower levels in normal tissues and blood vessels, which makes it an ideal target for developing cancer treatment. Also, B7H3 antibody extended the half-life and performed tumor-targeted delivery of IL-10, which improves the safety and efficacy of IL-10. Methods: We designed several structures of B7H3 antibody and IL-10 bifunctional fusion protein. Binding activity to B7H3 and IL-10 receptor was evaluated by both ELISA and FACS. C57BL/6J mice bearing MC38-hB7H3 cell line were used to evaluate the anti-tumor efficacy of IBB0979. Preclinical PK and toxicity study was also performed in cynomolgus monkeys to describe the safety profile of IBB0979. Results: Preclinical study showed that IBB0979 has high affinity to both targets and exhibited potent TGI in C57BL/6J mice bearing MC38-hB7H3 cell line, with TGI of 100% at 0.3mg/kg, 1mg/kg and 3mg/kg. An in vivo study in cynomolgus monkeys showed that after intravenously administered with 1 mg/kg, 2 mg/kg and 6 mg/kg of IBB0979 once a week for 29 days (5 times in total, given on days 1, 8, 15, 22 and 29), MTD was not reached up to 6 mg/kg which is significantly higher than the 10-20 μg/kg tolerated by IL-10 cytokines alone. Conclusion: IBB0979 is the world's first B7H3/IL-10 immunocytokine receiving IND approvals from both the FDA and the NMPA, showing excellent preclinical efficacy and safety profile. The Phase I clinical trial is currently on-going, with the first patient dosed in July 2023 and achieved SD at first efficacy evaluation after 2 cycles of administration. Since B7H3 is overexpressed in a wide range of cancers including lung, prostate, glioma, thyroid, head and neck, rectal, breast, skin, renal cell, and ovarian cancers, IBB0979 has the potential to become a next-generation therapy for resolving T cell exhaustion in cancer patients. Citation Format: Xiaoling Jiang, Chongbing Wu, Zi Chen, Liusong Yin. IBB0979, A novel B7H3/IL-10 immunocytokine of monovalent anti-B7H3 antibody fused with IL-10 homodimer [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 4080.

Interferon Gamma Induces Higher Neutrophil Extracellular Traps Leading to Tumor-Killing Activity in Microsatellite Stable Colorectal Cancer.

Many patients with colorectal cancer do not respond to immune checkpoint blockade (ICB) therapy, highlighting the urgent need to understand tumor resistance mechanisms. Recently, the link between the IFNγ signaling pathway integrity and ICB resistance in the colorectal cancer tumor microenvironment has been revealed. The immunosuppressive microenvironment poses a significant challenge to antitumor immunity in colorectal cancer development. Tumor-associated neutrophils found in tumor tissues exhibit an immunosuppressive phenotype and are associated with colorectal cancer patient prognosis. Neutrophil extracellular traps (NET), DNA meshes containing cytotoxic enzymes released into the extracellular space, may be promising therapeutic targets in cancer. This study showed increased NETs in tumor tissues and peripheral neutrophils of high levels of microsatellite instability (MSI-H) patients with colorectal cancer compared with microsatellite stable (MSS) patients with colorectal cancer. IFNγ response genes were enriched in MSI-H patients with colorectal cancer compared with patients with MSS colorectal cancer. Co-culturing neutrophils with MSI-H colorectal cancer cell lines induced more NET formation and higher cellular apoptosis than MSS colorectal cancer cell lines. IFNγ treatment induced more NET formation and apoptosis in MSS colorectal cancer cell lines. Using subcutaneous or orthotopic CT-26 (MSS) tumor-bearing mice models, IFNγ reduced tumor size and enhanced PD-1 antibody-induced tumor-killing activity, accompanied by upregulated NETs and cellular apoptosis. These findings suggest that IFNγ could be a therapeutic strategy for MSS colorectal cancer.

ScRNA-seq of colorectal cancer shows regional immune atlas with the function of CD20+ B cells

Colorectal cancer (CRC) from different regions exhibits different histological, genetic characteristics, and molecular subtypes, even in response to conventional chemotherapies and immunotherapies. To characterize the immune landscape in different regions of CRC and search for potential therapeutic targets, we analyzed 39,484 single-cell transcription data from 19 samples of CRC and paired normal tissues from four regions to identify the immune characteristics of CRC among anatomic locations, especially in B cells. We discovered that immune cell infiltration in tumors significantly varied among different regions of CRC. B cells from right- and left-sided CRC had different development trajectories, but both had extensive interactions with myeloid cells and T cells. Survival analysis suggested that CD20+ B cells correlated with good prognosis in CRC patients, especially on the right side. Furthermore, the depletion of CD20+ B cells demonstrated that anti-CD20 promoted tumor growth progression and reversed the tumor-killing activity of anti-PD-1 treatment in vivo and in vitro. Our results highlight the characterization of the immune landscape of CRC in different regions. CD20+ B-cell infiltration has been associated with CRC patient prognosis and may promote the tumor-killing role of PD-1 antibodies.

Cytokine modification by the RNA regulatory protein TFL

We found a novel gene, named "transformed follicular lymphoma" (TFL), in a patient who developed diffuse large B-cell lymphoma from follicular lymphoma. TFL modulates several cytokines and chemokines by binding their 3'UTR region of mRNA with its unique RNase motif. TFL is part of a family called zinc finger CCCH-type containing 12A-D. Due to its unique RNase motif, the TFL family is also called regulatory RNase (Regnase 1-4). TFL is expressed in lymphoid tissue and is upregulated by an inflammatory response, contributing to autoimmune diseases such as multiple sclerosis via IL-17 in CNS and worsening inflammation (cachexia) due to lymphoma progression through CXCL-13. Loss of TFL expression is reported to be a valuable biomarker for various cancers, including lung adenocarcinoma, endometrial cancer, and possibly lymphoma. In addition to its mRNA modulation function, Regnase1 is known to have deubiquitinase activity for TRAF2, 3, and 6, attenuating JNK and NFκB activity, and TFL captures and transports naked nonvesicular extracellular mRNA of IL-1β to the nucleus, enhancing the tumor-killing activity in NK cells. Based on its potential to modulate inflammation, TFL could be a future treatment target for autoimmune diseases and cancer.

Manufacturing, Characterization and Anti-Tumor Activity of Allogeneic CD19-CAR Cytokine-Induced Killer (CIK) Cells Engineered with Non-Viral Sleeping Beauty Transposon System and Armored with IL-18

Rationale & Objective: Adoptive cell therapy by chimeric antigen receptor- engineered cytokine-induced killer (CAR-CIK) cells has demonstrated high therapeutic potential with minimal alloreactivity and little to no toxicity in B-cell acute lymphoblastic leukemia patients, but further development is required to improve anti-tumor activity and persistence in vivo. The expression of the proinflammatory cytokine interleukin-18 (IL-18) has been shown to greatly enhance CAR-T cells potency and persistence. The purpose of the work described here was to develop a workflow for the manufacture of functional CAR-CIK cells, using the non-viral Sleeping Beauty transposon system, that co-express a second generation CD19-targeted CAR and the proinflammatory cytokine IL-18, hereafter referred to as CARCIK-1918 cells. The CAR molecule expressed by these cells includes an attenuated CD28 intracellular domain for enhancement of anti-tumor activity and in vivo persistence. Characterization of the manufactured armored CARCIK-1918 cells and evaluation of their anti-tumor activity in vitro and in vivo were performed. Methods: PBMCs isolated from healthy donors were cultured with IFN-γ, stimulated with anti-CD3 and IL-2, electroporated for non-viral genetic modification with Sleepy Beauty transposase RNA (SB100X) and pT4-1928'z-IL18 bicistronic plasmid DNA, and cultured for 15 days (17-day process). In process and final product characterization included monitoring of cell culture composition, cell expansion and viability, and evaluation of the expression of CAR, CIK proteins CD56 and NKG2D, and memory phenotype markers using flow cytometry. The final product CARCIK-1918 cells were further characterized by in vitro stimulation with CD19 + REH tumor cells to assess tumor killing activity and IL-18 secretion. CARCIK-1918 in vivo efficacy and persistence were also investigated in an NSG/Daudi mouse model. Mice were grafted with CD19 + Daudi tumor cells and, after 2 days, received an intravenous injection of 5x10 6 or 10x10 6 CARCIK-1918 cells. Daudi-grafted untreated mice were used as controls. Survival was monitored, and tumor burden and CAR-CIK cell persistence evaluated by using flow cytometry to assess human CD19 + and human CD3 + cells respectively, in peripheral blood, bone marrow, spleen, and kidney. Results: We manufactured >15 lots of CARCIK-1918 cells. After electroporation on day 2, CAR expression was already detected at day 7 and increased during the expansion culture. After the 17-day process, the range of harvested cells was 4.4E+09 to 1.6E+10 viable cells, with a cell viability >94%, and a fold expansion that varied among lots (range 20.1 - 161.4). The final CARCIK-1918 cells were predominantly CD3 +, expressed both the CAR (range 13 - 81%) and CIK-specific markers CD56 (range 27 - 88%) and NKG2D (range 59 - 86%) at variable levels among batches, and showed mostly a central memory/effector memory phenotype. CARCIK-1918 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19 + REH target cell line. Cells secreted IL-18 only in the presence of CD19 + target cells (range 62.17 - 560.58 pg/mL). In addition, CARCIK-1918 cells had a substantial anti-leukemic activity in vivo. CARCIK-1918 injection resulted in a significant dose-dependent reduction of tumor growth and improved survival, as compared with untreated NSG/Daudi mice. CARCIK-1918 cells persisted in vivo in mouse peripheral blood up to 90 days. Post-mortem analysis demonstrated that CARCIK-1918 cells from both dose levels persisted over time and limited the level of leukemic cell dissemination in the animals, with 10x10 6 regimen allowing an almost total control of the disease progression in all tissue examined. Conclusion: In conclusion, this study demonstrates that manufacturing of armored CAR-CIK cells using Sleepy Beauty non-viral transposon system and 2 nd generation CD19 CAR with an attenuated CD28 costimulatory domain and armored with IL-18 is feasible and allows rapid and efficient expansion of highly potent CARCIK-1918 cells. These cells demonstrated potent and specific anti-tumor activity in vitro and a robust anti-leukemic activity in vivo. These findings provide the first reported successful production of IL-18 armored CAR-CIK cells offering a promising strategy for the treatment of B-cell malignancies.

Targeting the Cgas-Sting Pathway Inhibits the Progression and Enhances the Chemotherapy Efficacy of Peripheral T-Cell Lymphoma

Objective Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. Owing to its complex clinicopathological characteristics and diagnostic difficulties, the underlying molecular mechanism leading to the high invasiveness and rapid progression remains significantly unidentified. The efficacy of current first-line treatment is dismal, novel agents and therapeutic strategies are urgently needed to improve the prognosis of PTCL patients, especially for relapsed/refractory (RR) ones. Recent studies have indicated a close association between the cGAS-STING pathway and tumor promotion, which may provide new therapeutic targets. Herein, we investigated the role of the cGAS-STING pathway and its underlying mechanism in PTCL progression. Methods Firstly, we conducted single-cell RNA sequencing (scRNA-seq) to examine cellular identity and investigate the expression heterogeneity of cGAS-STING pathway. The cGAS-STING pathway score (CSPS) was calculated based on the expression of seven marker genes of this pathway. Immunofluorescence was performed to detect cGAS and STING expression in PTCL tissue microarray containing 35 PTCL samples and 8 normal lymph node biopsies. To assess the tumor-killing activity of cGAS-STING pathway inhibitors, here we tested five drugs including cGAS inhibitors G150 and G140, STING inhibitor H151, TANK binding kinase-1 (TBK1) inhibitor TBK1/Ikkε-IN-5 and signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic. Protein mass spectrometry was used to evaluate the protein changes in PTCL cell lines after G150 treatment. Next, we established a xenograft model to test the anti-tumor effect of G150 and TG003 (an inhibitor of CLK1) single-agent or combination therapy in vivo. Furthermore, we applied single-cell dynamic RNA sequencing to investigate the mechanism on the synergistic effect of TG003 and G150 in inhibition of cell proliferation. CCK8 and flow cytometric analysis were performed to detected the cellular viability and the apoptotic rate with the drug combination compared with single-drug treatment. Results scRNA-seq and showed the cGAS-STING pathway was overexpressed in PTCL with high heterogeneity and could be used as a prognostic marke (Fig. A-D). cGAS expression was positively correlated with DNA repair. cGAS inhibition effectively suppressed tumor growth via cGAS-STING-RELB-STAT3 signaling pathway and impairing DNA damage repair in PTCL. G150 treatment led to a concentration-dependent DNA damage (Fig. E-G). Moreover, single-cell dynamic RNA sequencing revealed that TG003 functions through inducing the reduction of proliferation-related nascent RNAs on the basis of G150 treatment (Fig. H,I). CLK1 played a critical role in the survival of residual tumor cells after treatment with cGAS inhibitors, and CLK1 suppression could enhance the sensitivity to cGAS inhibitors (Fig. J). In first-line therapy, chemotherapy-induced DNA damage activated the cGAS-STING pathway, which may ultimately lead to relapse or progression. cGAS inhibitors could synergize with chemotherapeutic agents in tumor killing (Fig. K-N). Fig. O summarized the oncogenic role of cGAS-STING pathway and the interaction between Tfh cells and B cells in AITL. Conclusion Herein, we demonstrated for the first time that the activated cGAS-STING pathway plays an oncogenic role in PTCL progression, in part by enhancing DNA repair and proliferation, which may also trigger chemotherapy resistance and ultimate relapse. CLK1 may serve as an indicator for the sensitivity to cGAS inhibitors. This may partly unveil the deficiency of first-line therapy and lay foundation for clinical application of cGAS-STING pathway-associated inhibitors.