Cellular Immunotherapy Of Cancer Research Articles

Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens.

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.

CAR T cells outperform CAR NK cells in CAR-mediated effector functions in head-to-head comparison

BackgroundCAR NK cells as vehicles for engineered “off-the-shelf” cellular cancer immunotherapy have attracted significant interest. Nonetheless, a comprehensive comparative assessment of the anticancer activity of CAR T cells and CAR NK cells carrying approved benchmark anti-CD19 CAR constructs is missing. Here, we report a direct head-to-head comparison of CD19-directed human T and NK cells.MethodsWe generated CAR T and CAR NK cells derived from healthy donor PBMC by retroviral transduction with the same benchmark second-generation anti-CD19 CAR construct, FMC63.28z. We investigated IFN-γ secretion and direct cytotoxicity in vitro against various CD19+ cancer cell lines as well as in autologous versus allogeneic settings. Furthermore, we have assessed anticancer activity of CAR T and CAR NK cells in vivo using a xenograft lymphoma model in an autologous versus allogeneic setting and a leukemia model.ResultsOur main findings are a drastically reduced capacity for CAR-mediated IFN-γ production and lower CAR-mediated cytotoxicity of CAR NK cells relative to CAR T cells in vitro. Consistent with these in vitro findings, we report superior anticancer activity of autologous CAR T cells compared with allogeneic CAR NK cells in vivo.ConclusionsCAR T cells had significantly higher CAR-mediated effector functions than CAR NK cells in vitro against several cancer cell lines and autologous CAR T cells outperformed allogeneic CAR NK cells both in vitro and in vivo. CAR NK cells will likely benefit from further engineering to enhance anticancer activity to ultimately fulfill the promise of an effective off-the-shelf product.

Abstract 1584: The interplay between natural killer cells and pancreatic stellate cells in pancreatic ductal adenocarcinoma

Abstract Natural Killer (NK) cells have been shown to possess potent anti-tumor activity against multiple cancer types and consequently have been harnessed as a cellular cancer immunotherapy. Furthermore, targeting of pancreatic stellate cells has received increasing interest in the treatment of pancreatic ductal adenocarcinoma (PDAC), with several clinical trials testing the hypothesis that rendering activated pancreatic stellate cells/cancer associated fibroblasts (aPSC/CAF) into a quiescent state (qPSC) can enhance anticancer treatment. Despite this interest, our understanding of the interplay between these two cell types remains limited. We hypothesized that PSC/CAF may exhibit a dynamic interaction with NK cells that may be harnessed for therapeutic strategies and patient stratification. Employing a series of in vitro assays, we demonstrate a significant bi-directional interaction between natural killer cells and pancreatic stellate cells in the context of PDAC. NK cells were found to effectively target and eliminate both aPSC and qPSC, with PSC activation conferring some protection from NK-lysis. Furthermore, we highlight a significant myofibroblastic shift in PSCs regardless of previous activation status when cultured with NK cells; an interaction that was direct contact dependent. NK cells were also found to demonstrate cellular modulation of NKG2A and TIM3 in response to PSC co-culture. Drawing on Luminex ELISA analysis, we suggest that engagement of the IFN-γ signaling pathway may be driving phenotypic modulation. Global proteomic analyses demonstrated differential protein changes induced by NK cells in aPSC compared to qPSC, as well as alterations in the NK cell proteome upon such co-culture. Utilizing multiplex immunohistochemical analysis of PDAC patient samples, we highlight differential spatial distribution of phenotypically distinct NK cells and PSC/CAF subtypes in patient samples between long- (n=16) and short- (n=48) survivors. Moreover, NK-PSC proximity, and not total immune infiltrate, was found to play a prognostic role in PDAC. Our work provides a global overview of the cross-talk between NK and PSC in PDAC, demonstrating a significant bi-directional relationship which offers novel insights into potential therapeutic avenues. Furthermore, we demonstrate, for the first time to our knowledge, the prognostic role of NK-PSC proximity in patient survival and suggest its potential for patient stratification. Citation Format: Rachel Elizabeth Fincham, Parthiban Periasamy, Craig Ryan Joseph, Jia Meng, Jeffrey Chun Tatt Lim, Felicia Wee, Konstantinos Stasinos, Michelle R. Goulart, Jiangfeng Ye, Li Yen Chong, Denise Goh, Joe P. Yeong, Hemant M. Kocher. The interplay between natural killer cells and pancreatic stellate cells in pancreatic ductal adenocarcinoma [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 1584.

High-Dimensional Analyses Reveal IL15 Enhances Activation of Sipuleucel-T Lymphocyte Subsets and Reverses Immunoresistance.

Sipuleucel-T (sip-T) is the only FDA-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). To elucidate parameters of the response profile to this therapy, we report high-dimensional analyses of sip-T using cytometry by time of flight (CyTOF) and show a lymphoid predominance, with CD3+ T cells constituting the highest proportion (median ∼60%) of sip-T, followed by B cells, and natural killer (NK) and NKT cells. We hypothesized that treatment of sip-T with homeostatic cytokines known to activate/expand effector lymphocytes could augment efficacy against prostate tumors. Of the cytokines tested, IL15 was the most effective at enhancing activation and proliferation of effector lymphocytes, as well as augmenting tumor cytotoxicity in vitro. Co-culture of sip-T with IL15 and control or prostate-relevant antigens showed substantial activation and expansion of CD8+ T cells and NKT cells in an antigen-specific manner. Adoptive transfer of IL15-treated sip-T into NSG mice resulted in more potent prostate tumor growth inhibition compared with control sip-T. Evaluation of tumor-infiltrating lymphocytes revealed a 2- to 14-fold higher influx of sip-T and a significant increase in IFNγ producing CD8+ T cells and NKT cells within the tumor microenvironment in the IL15 group. In conclusion, we put forward evidence that IL15 treatment can enhance the functional antitumor immunity of sip-T, providing rationale for combining IL15 or IL15 agonists with sip-T to treat patients with mCRPC.

Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity

Background aimsNatural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. MethodsUsing wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry–based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. ResultsMean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. ConclusionsTogether, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

T cell immunotherapy for cervical cancer: challenges and opportunities.

Cancer cellular immunotherapy has made inspiring therapeutic effects in clinical practices, which brings new hope for the cure of cervical cancer. CD8+T cells are the effective cytotoxic effector cells against cancer in antitumor immunity, and T cells-based immunotherapy plays a crucial role in cellular immunotherapy. Tumor infiltrated Lymphocytes (TIL), the natural T cells, is approved for cervical cancer immunotherapy, and Engineered T cells therapy also has impressive progress. T cells with natural or engineered tumor antigen binding sites (CAR-T, TCR-T) are expanded in vitro, and re-infused back into the patients to eradicate tumor cells. This review summarizes the preclinical research and clinical applications of T cell-based immunotherapy for cervical cancer, and the challenges for cervical cancer immunotherapy.

5. Progress in Cellular Immunotherapy for Cancer

5. Progress in Cellular Immunotherapy for Cancer

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Selected Articles from This Issue

Highlights| January 17 2023 Selected Articles from This Issue Author & Article Information Online Issn: 1557-3265 Print Issn: 1078-0432 ©2023 American Association for Cancer Research2023American Association for Cancer Research Clin Cancer Res (2023) 29 (2): 297. https://doi.org/10.1158/1078-0432.CCR-29-2-HI Related Content A commentary has been published: Human Metastatic Cholangiocarcinoma Patient-Derived Xenografts and Tumoroids for Preclinical Drug Evaluation A commentary has been published: Triple-Negative PAM50 Non-Basal Breast Cancer Subtype Predicts Benefit from Extended Adjuvant Capecitabine A commentary has been published: Intraperitoneal Monocytes plus IFNs as a Novel Cellular Immunotherapy for Ovarian Cancer: Mechanistic Characterization and Results from a Phase I Clinical Trial View more A commentary has been published: High-Sensitivity Mutation Analysis of Cell-Free DNA for Disease Monitoring in Endometrial Cancer View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record January 17 2023 Citation Selected Articles from This Issue. Clin Cancer Res 15 January 2023; 29 (2): 297. https://doi.org/10.1158/1078-0432.CCR-29-2-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Overall survival for advanced ovarian cancer remains poor, particularly for women with platinum-resistant disease. Green and colleagues report the bench to bedside development of a novel cytokine stimulated autologous monocyte therapy for advanced, platinum-resistant or -refractory ovarian cancer. Activated monocytes killed tumor cells in mice via a TRAIL-dependent pathway. Treatment was well tolerated and produced partial responses by RECIST criteria. These findings characterize the mechanism of a novel cellular immunotherapy for ovarian cancer, which is well tolerated with evidence of clinical activity in a heavily pretreated patient population. This therapy may serve as the backbone for a combination immunotherapy regimen incorporating additional agents targeting T-regulatory cells and/or myeloid-derived suppressor cells (MDSCs). Identifying predictive biomarkers that define the subset of triple-negative breast cancer (TNBC) deriving the most benefit from adjuvant capecitabine is necessary in clinical practice. In this hypothesis-testing study, Asleh and colleagues examined the capacity of candidate RNA biomarkers to... You do not currently have access to this content.

Correction: Intraperitoneal Monocytes plus IFNs as a Novel Cellular Immunotherapy for Ovarian Cancer: Mechanistic Characterization and Results from a Phase I Clinical Trial

In the original version of this article (1) as it was published online on September 28, 2022, author Soumya Korrapati is mistakenly omitted from the author list. The HTML and PDF versions of this article were corrected on December 23, 2022, ahead of print and the author contributions and disclosures were updated as necessary. The authors regret the error.

Cellular Cancer Immunotherapy Development and Manufacturing in the Clinic.

The transfusion of naturally derived or modified cellular therapies, referred to as adoptive cell therapy (ACT), has demonstrated clinical efficacy in the treatment of hematologic malignancies and metastatic melanoma. In addition, cellular vaccination, such as dendritic cell-based cancer vaccines, continues to be actively explored. The manufacturing of these therapies presents a considerable challenge to expanding the use of ACT as a viable treatment modality, particularly at academic production facilities. Furthermore, the expanding commercial interest in ACT presents new opportunities as well as strategic challenges for the future vision of cellular manufacturing in academic centers. Current trends in the production of ACT at tertiary care centers and prospects for improved manufacturing practices that will foster further clinical benefit are reviewed herein.

Ensuring commercial readiness in the cellular cancer immunotherapy space

David McCall, Editor, Cell & Gene Therapy Insights, talks to three experts from Gamida Cell’s leadership team – Julian Adams PhD, CEO, Michele Korfin PhD, COO & CCO & Ronit Simantov MD, CMO & CSOJulian Adams joined Gamida Cell’s leadership team as CEO in November 2017, bringing more than 35 years of drug discovery and development experience to his role. Prior to his CEO appointment, Julian served as President and Chief Scientific Officer at Clal Biotechnology Industries (CBI), where he oversaw the Boston office, evaluating investment opportunities and supporting portfolio companies, including Gamida Cell. Before joining CBI, he served as president of research and development at Infinity Pharmaceuticals and as senior vice president of drug discovery and development at Millennium Pharmaceuticals, now part of Takeda Oncology. At Millennium, he played a key role in the discovery of Velcade (bortezomib), a therapy widely used for treatment of multiple myeloma. He was also instrumental at developing an oral inhibitor of phosphoinositide 3-kinase (PI3K), also known as duvelisib. In 2018, the FDA approved COPIKTRA (duvelisib, Verastem, Inc.) for adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). Earlier in his career, while at Boehringer Ingelheim, he was credited with discovering Viramune (nevirapine) for HIV. He has also worked in research and development leadership roles at LeukoSite and at ProScript. Julian serves as Chairman at Elicio Therapeutics since 2017. He also serves on the Stand Up to Cancer Scientific Advisory Committee, AACR Council of Scientific Advisors, and MGH Center for Cancer Research Scientific Advisory Board.Julian has won several awards for his drug development efforts throughout his career, holds more than 40 patents from the United States Patent and Trademark Office and has authored more than 100 papers and book chapters in peer-reviewed journals. Julian holds a BSc from McGill University and a Ph.D. from the Massachusetts Institute of Technology. He also holds a ScD, honoris causa, from McGill University.Michele Korfin joined Gamida Cell’s leadership team as Chief Operating and Chief Commercial Officer in July 2020. Prior to Gamida Cell, Michele served as Chief Operating Officer at TYME Technologies. From 2016–2018, she was Vice President of Market Access at Kite Pharma, where she oversaw market access strategy, including payer relations, reimbursement and government affairs for YESCARTA®, the first approved CAR-T therapy in lymphoma. She also worked closely with the manufacturing and supply chain teams at Kite to prepare for FDA approval and commercialization. Before joining Kite, Michele spent more than a decade at Celgene in a variety of key strategic and operational roles, including in commercial leadership and oversight of the global development programs for REVLIMID®, a foundation therapy for multiple myeloma and MDS del 5Q. She also led the Celgene oncology sales force of over 120 representatives who were responsible for $650 million in revenue for ABRAXANE®, which is now a standard of care in pancreatic cancer. Michele has also held positions at Merck & Co. as a manufacturing scientist, Bain & Company as a consultant and Schering-Plough in sales and marketing. Michele holds an MBA from Harvard Business School and a BSc in pharmacy from Rutgers University. She is also on the Board of Trustees of BioNJ, the organization that represents the biotechnology industry for New Jersey.Ronit Simantov joined Gamida Cell’s leadership team as Chief Medical Officer in July 2017, bringing more than 20 years of experience in hematology and oncology research, development, registration and product launch. Prior to joining Gamida Cell, Ronit served as Head of Oncology Global Medical Affairs at Pfizer, where she was responsible for multiple programs including Sutent® (sunitinib), Inlyta ® (axitinib), Ibrance® (palbociclib), Bosulif® (bosutinib) and Xalkori ® (crizotinib). Ronit previously led phase 1-3 studies as Vice President of Clinical Research at OSI Pharmaceuticals. She also served as Chief Medical Officer at CuraGen Corporation (acquired by Celldex), where she led development of small molecules and antibody-drug conjugates. At Bayer HealthCare Pharmaceuticals, Ronit led the phase 3 study of Nexavar® (sorafenib) resulting in the first approval of a tyrosine kinase inhibitor in renal cell carcinoma. Prior to joining industry, Ronit spent seven years on the academic faculty at Weill Medical College of Cornell University, where she directed the fellowship program and conducted angiogenesis and vascular biology research. She has authored over 40 peer-reviewed manuscripts.Ronit holds an MD from New York University School of Medicine and a BSc. from Johns Hopkins University. She completed a residency in internal medicine at New York Hospital Cornell Medical Center, and a fellowship in hematology and oncology at Weill Cornell Medicine.

Combination of 4-1BB and DAP10 promotes proliferation and persistence of NKG2D(bbz) CAR-T cells.

Chimeric antigen receptor (CAR)-T cell therapy has been shown to have considerable therapeutic effects in hematological malignancies, and NKG2D(z) CAR-T cell therapy has been verified to be safe based on clinical trials. However, due to the poor persistence of NKG2D(z) CAR-T cells, their therapeutic effect is not obvious. Here, we constructed NKG2D(bbz) CAR-T cells that can simultaneously activate 4-1BB and DAP10 costimulatory signaling. They were found to be cytotoxic to the target cells in vitro and in vivo. They exhibited low differentiation, low exhaustion, and good proliferation. Importantly, the proportions of central memory T (Tcm) and stem cell-like memory T (Tscm) cell subsets were strikingly increased. After long-term incubation with the target cells, they displayed reduced exhaustion compared to NKG2D(z) CAR-T cells. Further, in the presence of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, they exhibited reduced exhaustion and apoptosis, upregulated Bcl2 expression, and an increased proportion of Tcm cell subsets. Finally, NKG2D(bbz) CAR-T cells had better antitumor effects in vivo. In summary, the results showed that NKG2D(bbz) CAR-T cells may be valuable for cellular immunotherapy of cancer.

Dual-responsive nanovaccine for cytosolic delivery of antigens to boost cellular immune responses and cancer immunotherapy

Cancer vaccine contributing to the success of the treatment and prevention of tumors has attracted a huge attention as a strategy for tumor immunotherapy in recent years. A major challenge of cancer vaccine is to target cytosols of dendritic cells (DCs) in the lymph nodes (LNs) to enhance efficiency of antigen cross-presentation, which elicits high levels of cytotoxic T-lymphocytes to destruct tumor cells. Here, we address this issue by conjugating ovalbumin (OVA) to PEG-PCL using disulfide bond (-ss-), and the degradable pH-responsive polymer-PEI-PCL as delivery carrier. In addition, the mol ratio of PEG-PCL to PEI-PCL in the mixed micelles was tailored to deliver the OVA to LNs. Subsequently, CpG ODN1826, a TLR-9 agonist, was further introduced into a mixed micelle of 30 nm or less as a unique tumor vaccine. Importantly, the results demonstrated the mixed micelles with 1:1 mol of PCL-PEG and PCL-PEI can effectively migrate to distal LNs where antigen were efficiently captured by DCs, meanwhile, OVA was modified to the surface of mixed micelles via disulfide bonds (-ss-) for promotion efficiency of antigen cross-presentation. More surprisingly, combination of tumor vaccine with anti-PD-1, the therapy of ectopic melanoma (B16-OVA) and lung metastasis melanoma (B16-OVA) is excellent therapeutic effect. Taken together, our works offers a novel strategy for the cytosol delivery of antigens to achieve potent cancer immunotherapy.

Abstract 3557: Toward a personalized off-the-shelf cellular immunotherapy for cancer

Abstract Objectives: We have been developing SV-BR-1-GM, a breast cancer cell line with features of an antigen presenting cell which has been stably transfected with the CSF2 gene encoding GM-CSF. SV-BR-1-GM has been in clinical trials in a regimen including low-dose pre-dose cyclophosphamide to reduce immune suppression, and post-dose local interferon alpha to boost the immune response. The SV-BR-1-GM regimen has been administered alone or in combination with PD-1 inhibitors in patients with advanced breast cancer. We have noted that patients that match the SV-BR-1-GM cell line at least at 1 HLA allele are more likely to derive clinical benefit. Therefore, steps were taken to genetically modify the SV-BR-1 cell line to match more patients. Methodology: We focused on the least polymorphic HLA types in the population: HLA-A (Class I) and HLA-DRB3/4/5 (Class II). The published allele frequencies (Gragert 2013) for HLA-A and HLA-DRB3/4/5 were evaluated for the major demographic groups in the US. The following HLA alleles were selected: A*02:01, A*01:01, A*03:01, A*24:02, A*11:01, A*68:01, A*23:01, A*33:03 for Class I and DRB4*01:01, DRB3*02:02, DRB3*01:01, DRB5*01:01, DRB3*03:01, DRB5*01:02, DRB5*02:02 for HLA-DRB3/4/5. Based on population analysis, this combination of alleles should produce at least a single match in~ 99% of the population, with a ~92% match at Class I HLA-A alleles and a ~98% match at Class II HLA-DRB3/4/5 alleles. SV-BR-1 was modified using CRISPR technology deleting expression of the endogenous HLA-A and HLA-DRB3 alleles. Four lentiviral vectors were constructed to express the HLA alleles, along with the CSF2 gene (which encodes GM-CSF), using a 2A self-cleaving multi-gene expression system. Each lentiviral vector expressed 4 HLA types: 2 HLA-A and 2 HLA-DRB3/4/5 types. Preliminary Data: Following sequential CRISPR treatment, the SV-BR-1 cells were cloned, and one clone selected (clone 17) for further engineering. Lack of expression of HLA-A and HLA-DRB3 was confirmed using flow cytometry, and DNA sequencing. Clone 17 was subsequently transduced separately with the four lentiviral vectors each expressing four HLA genes as well as the CSF2 gene. Following selection and cloning, clones were evaluated by ELISA, flow cytometry and RT-PCR to confirm gene expression. Several clones that secreted GM-CSF and expressed both Class I and Class II HLA alleles have subsequently been transferred to GMP manufacturing. These modified breast cancer cell lines will be used in clinical studies designed to first evaluate the safety of intradermal inoculation with the irradiated cells and later combined with other agents to augment the immune response. Each patient will be treated with the cell line that matches them at least at one HLA allele. Gragert L, et. al. Hum Immunol. 2013;74(10):1313-1320. Citation Format: William V. Williams, Markus D. Lacher, Vivek G. Sunkari, Charles Wiseman, Miguel Lopez-Lago. Toward a personalized off-the-shelf cellular immunotherapy for cancer [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 3557.

Abstract 3577: First-in-human phase I study of intraperitoneally administered interferon-activated autologous monocytes in platinum-resistant ovarian cancer

Abstract Background: Ovarian cancer is the most lethal gynecologic cancer and is intrinsically resistant to immunotherapies. Previous work has demonstrated superior response rates with intraperitoneally (IP) directed therapies, providing the rationale for IP immunotherapy. Methods: A previously published model system in which OVCAR ovarian cancer spheroids are killed by IFN-activated human monocytes was further characterized using confocal microscopy, global gene expression analysis, flow cytometry, and knockout/knockdown of key genes in the TRAIL-DR4/DR5 pathway. These results were translated into a phase I dose escalation trial in which 3-6 patients with platinum-resistant or refractory ovarian cancer were enrolled into 4 cohorts and treated IP q28 days with Peginterferon alfa-2b (25-250 mcg) and Interferon gamma-1b (5-50 mcg), with or without autologous monocytes (75-750 x 106 cells), in order to determine the recommended phase II dose (RP2D). Results: Confocal microscopy of IFN-activated monocytes demonstrated migration into OVCAR spheroids and destruction of the spheroids and global gene expression analysis identified TRAIL as a ligand on monocytes upregulated by IFN treatment. Disruption of TRAIL receptors DR4/DR5 or deletion of CASPASE8 on OVCAR cells largely abrogated killing. IFN-activated human monocytes were able to kill IP-injected OVCAR tumor cells in a mouse xenograft model, and IFN-activated monocytes from ovarian cancer patients were able to kill OVCAR spheroids as effectively as those from healthy donors. 18 patients were subsequently enrolled on a phase I trial (median age, 61 years; median 5 prior therapies). 1 of 3 patients at the second dose level experienced a dose-limiting toxicity (grade 3 anemia) and no subsequent DLTs were observed. The RP2D was defined as 250mcg IFNa/50mcg IFNg/750 x 106 cells. The only treatment-related grade 3 or higher adverse events occurring in more than one patient were lymphocyte decrease (33.3%) and abdominal pain (11.1%). The best response observed was partial response (PR) in 2/9 RECIST-evaluable patients, and 1 additional patient had a CA-125 response with >50% reduction. 4/9 patients had stable disease. Four patients were able to receive 5+ cycles (range 1-10 cycles). Biomarker analyses demonstrated that these long-term responders had a lower level of T-regulatory cells at baseline, but exhibited marked increase in myeloid derived suppressor cells (MDSC) at the time of tumor progression. Conclusion: These findings characterize the mechanism of a novel cellular immunotherapy for ovarian cancer which is well tolerated with evidence of clinical activity in a heavily pretreated patient population. This therapy may serve as the backbone for a combination immunotherapy regimen incorporating additional agents targeting T-regulatory cells and/or MDSCs. Citation Format: Christopher B. Cole, Daniel S. Green, Franklin Ning, Kathryn Zoon, Lindsey B. Rosen, Steven M. Holland, Christina M. Annunziata. First-in-human phase I study of intraperitoneally administered interferon-activated autologous monocytes in platinum-resistant ovarian cancer [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 3577.

Harnessing the Anti-Tumor Mediators in Mast Cells as a New Strategy for Adoptive Cell Transfer for Cancer.

The emergence of cancer immunotherapies utilizing adoptive cell transfer (ACT) continues to be one of the most promising strategies for cancer treatment. Mast cells (MCs) which occur throughout vascularized tissues, are most commonly associated with Type I hypersensitivity, bind immunoglobin E (IgE) with high affinity, produce anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte macrophage colony-stimulating factor (GM-CSF), and generally populate the tumor microenvironments. Yet, the role of MCs in cancer pathologies remains controversial with evidence for both anti-tumor and pro-tumor effects. Here, we review the studies examining the role of MCs in multiple forms of cancer, provide an alternative, MC-based hypothesis underlying the mechanism of therapeutic tumor IgE efficacy in clinical trials, and propose a novel strategy for using tumor-targeted, IgE-sensitized MCs as a platform for developing new cellular cancer immunotherapies. This autologous MC cancer immunotherapy could have several advantages over current cell-based cancer immunotherapies and provide new mechanistic strategies for cancer therapeutics alone or in combination with current approaches.

Non-invasive fluorescence imaging for tracking immune cells in preclinical models of immunotherapy.

The field of cellular immunotherapy for cancer has experienced exponential growth over the last decade. Several chimeric antigen receptor T cell products have already received FDA approval, which has stimulated growth and enthusiasm for other cellular therapies. Preclinical models are critical steps in the development of these products, and understanding their in vivo trafficking and persistence are critical components of their efficacy and toxicity analogous to volume of distribution and tissue penetration in small molecule therapeutics. Thus, well-established preclinical methodologies for following cells after adoptive transfer are important to understanding immune cell trafficking to, and persistence in, tumors or organs of interest. Here, we describe a quick and reliable method for labeling and in vivo tracking of immune cells adoptively transferred into small animal models by using in vivo fluorescent imaging.

Engineering NK-92 Cell by Upregulating CXCR2 and IL-2 Via CRISPR-Cas9 Improves Its Antitumor Effects as Cellular Immunotherapy for Human Colon Cancer.

Natural killer (NK) cells have shown good application prospects in adoptive cellular immunotherapy against cancer. However, due to its insufficient infiltration and low activity, the therapeutic effect of infused NK cells has been limited in solid tumors, such as colorectal cancer. It has been proved that tumor-produced chemokines regulate the migration of NK cells expressing corresponding chemokine receptors, and cytokines could enhance the antitumor activity of NK cells. In this study, we innovatively upregulated the expression of chemokine receptor CXC chemokine receptor 2 (CXCR2) and cytokine interleukin (IL)-2 on NK-92 cells using CRISPR-Cas9 gene-editing technology. We demonstrated that overexpressing CXCR2 and IL-2 promotes NK-92 cells to increasingly transfer into tumor sites and achieve stronger cell-killing and proliferation activity. Moreover, the inhibitory effects of gene-edited NK-92 cells on the growth of human colon cancer in vivo were also improved. The tumor burden of tumor-bearing mice was reduced, and their survival time was significantly prolonged. Gene-editing modification NK cells are expected to become a novel and promising tumor treatment strategy.

Novel CD19-Specific γ/δ TCR-T Cells in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background: T cell receptor(TCR)-engineered T cell therapy, by replacing the antigen recognition domain of TCR with an antibody-derived Fab fragment, is another active field of cellular immunotherapy for cancer. We previously developed a human anti-CD19 antibody (ET190L1), and found that ET190L1-TCR-T cells maintained comparable anti-tumor potency with less cytokine release to CD28-costimulated ET190L1-CAR and CD137-based CTL019 T cells (Cell Discov. 2018 Nov 20;4:62.). ET019003-T cells are novel anti-CD19 γδ TCR-T cells generated based on ET190L1-TCR-T cells by adding an independent chimeric signaling receptor(CSR) to further promote T cell activation and reduce cytokine release (Figure 1A). We report outcomes for adult patients with relapsed or refractory diffuse large B-cell lymphoma (RR DLBCL) treated with ET019003-T cells.Methods: Our first-in-human, single-center, phase 1 study (NCT04014894) was designed to evaluate the safety and efficacy of ET019003-T cells in patients with CD19 + malignancies, of whom 8 with RR DLBCL are reported here. Eligible patients must have (1) histologically confirmed DLBCL; (2) CD19 + on malignant cells by IHC; (3) refractory disease as defined in the SCHOLAR-1 study, or recurrent disease within 6 months or at least 2 times after CR; (4) prior therapy including an anti-CD20 monoclonal antibody and an anthracycline. Patients with CNS lymphoma were eligible. Bridging therapy wasn't allowed after apheresis. Cyclophosphamide 250 mg/m 2 on day -5 and fludarabine 25 mg/m 2 on day -5 to -3 were used as the conditioning regimen. Planned dose levels were 2, 4x10 6 TCR+T cells/kg, and repeated infusions were allowed. Primary objectives were incidence of adverse events (AEs) and overall response rate(ORR). CRS and neurotoxicity were graded using the ASTCT criteria, and other AEs using CTCAE v5.0. Response was assessed per Lugano Criteria (Cheson 2014).Results: 8 pts (median age 50, range 33-71) received infusion of ET019003-T cells (6 at 2x10 6/kg, and 2 at 4x10 6/kg) and were included in the study analysis. Patient enrollment was ceased in June, 2020. Pt1 had primary CNS lymphoma, and 62.5% had stage 4 disease against Ann Arbor staging. MYC/BCL2/BCL6 triple expression was detected in 50% of pts, and double expression in 25%. Pts had received a median of 4.5 (range 2-8) prior lines of treatment, and 37.5% received prior PD-1 inhibitors, and 62.5% had primary refractory disease.3 pts (37.5%) experienced grade 1 CRS that resolved spontaneously; 1(pt2) developed grade 3 neurotoxicity (dose-limiting toxicity), manifested by confusion, barylalia, tremor and agitation, which occurred after CRS and responded to corticosteroids. The most common AE was neutropenia (100%), and 62.5% were related to conditioning regimen; other hematologic AEs included thrombocytopenia (37.5%) and anemia (12.5%). Pt8 had pulmonary infection on day 15, and pt1 experienced viral encephalitis at 18 months, and both were manageable. As of July 20, 2021, median follow-up after infusion was 15 months (range 2-24.7). 6 pts(75%) achieved a clinical response, and 5(62.5%) reached CR, of whom 80% kept ongoing CR (all at 18+ months). 3 pts received a second infusion, pt5 for consolidation therapy after CR, and pt2 and pt7 for salvage therapy after disease progression, but response wasn't observed (Figure 1B). Pt1 with primary CNS lymphoma got continuing CR, without CRS or neurotoxicity (Figure 1C). The rate of overall survival, progression-free survival, and duration of response at 12 months was 87.5%, 62.5% and 66.7%, respectively.ET019003 cells showed striking peak expansion during 10-20 days post infusion as measured as ET019003+ cells per milliliter of PB by flow cytometry and copies per microgram of genomic DNA by qPCR, but poor expansion was observed in the second infusions (Figure 1D). ET019003 cells were detectable in cerebrospinal fluid of pt1, and continued to be detectable at 12+ months in PB of pt3 and pt5. Serum cytokine levels increased mildly post-infusion, except elevated IL-6 (>10 folds of upper limit of normal) in 3 pts (pt1 with a high baseline level, coinciding with onset of CRS and neurotoxicity of pt2, and concurrent with pulmonary infection of pt8).Conclusion: These data suggest ET019003-T cells had a good safety profile and could induce durable remission in patients with RR DLBCL, even with primary CNS lymphoma. γ/δ TCR-T cells may present a potential therapeutic option for these patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.