Tumor-specific Lymphocytes Research Articles

Abstract 22: Development and characterization of human tumor specific cytotoxic lymphocytes for preclinical drug development

Abstract Heterogeneity within a tumor allows it to better adapt to changing conditions and can have therefore a negative impact on a patient’s prognosis, response to treatment and overall health. Breast cancer is a highly heterogeneous disease caused by distinct genetic mutations in mammary epithelial cells, resulting in significantly different forms of the disease and a need for highly personalized treatment schemes for patients. Hence, it is important to develop and improve (immuno)therapies for breast cancer patients. Adoptive T-cell therapy with autologous CD8+ cytotoxic T cell lymphocytes (CTL) is a promising method that has already shown promising effects in melanoma patients. Unfortunately, there is almost no information about the use in breast cancer patients. We developed an in vitro protocol to generate tumor-specific CTLs by priming on HER2+ breast cancer cell lines JIMT-1 and SKBR-3. Subsequently, these primed CTLs were tested in 2D and 3D immune cell killing assays in a life cell imaging device in combination with an endpoint viability assay. The activity was compared with unspecific CD8+T cells activated by phytohemagglutinin (PHA) or αCD2/αCD3/αCD28, HER2-peptide specific T cells and HER2-CAR T cells serving as a positive control. We investigated the feasibility to freeze CTLs for use in subsequent studies and validated the relevance of HER2 as the main druggable target in the tumor lines using an siRNA approach. The CTLs were most effective on JIMT-1 in a 3D setting, whereas the SKBR-3 CTLs displayed similar activity in 2D and in 3D. The respective CTLs were tumor line specific, as SKBR-3 CTLs did not kill efficiently the JIMT-1 cells and vice versa. The same CTLs displayed the same efficacy pattern after thawing but less pronounced. Thus, within one experimental set-up it is recommended to use either one. The Her2 knockdown in the target cells reduced the killing activity of the CTLs slightly. However, this effect was not statistically significant proving the importance of CTLs as treatment option as well as neoantigens as targets in drug development. In summary, the CTLs proved to capture the respective tumor heterogeneity showing high killing rates in both SK-BR3 and JIMT-1 being more active than HER2-peptide specific T cells. The availability of CTLs in a preclinical setting facilitates screening approaches for combinational studies. Beyond that they can serve as starting material to develop cancer vaccines and identify highly immunogenic neoantigens. Citation Format: Ina Rohleff, Lea Gröne, Kanstantsin Lashuk, Julia B. Schueler. Development and characterization of human tumor specific cytotoxic lymphocytes for preclinical drug development [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 22.

Abstract 4224: The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment

Abstract We developed an in vitro protocol to generate tumor-specific cytotoxic lymphocytes (CTL) by priming T cells from healthy donors on HER2+ breast cancer cell line SKBR-3. Subsequently, these primed CTLs were tested in a 3D immune cell killing assays in a life cell imaging device. An image analysis workflow was applied to understand the T cell movement in spatial relation to the tumor and the kinetic of tumor cell killing. The activity of the CTLs was compared with non-activated CD8+T cells, staurosporin and CD8+ T cells, unspecifically activated by αCD2/αCD3/αCD28 beads. The tumor cells were expressing mKate whereas the T cells were label-free. By analyzing a combination of different masks, we could computationally measure tumor spheroid characteristics and dynamic attributes such as changes in shape, percent of cells expressing red fluorescentce, and relative average distance of T cells from the spheroid in each timelapse assay. This process was applied at each frame to determine the average T cell distance from its corresponding spheroid center and subsequently calculate the average T cell flux over the course of each assay by linking data between image frames. Additional measurements allowed for the quantification of tumor debris accumulation over time both relative to the primary tumor mass and in absolute terms. Our analyses revealed that bead-activated T cells approached the tumor spheroid with the highest speed and infiltrated the spheroid homogeneously. In contrast the CTLs approached the spheroid slower and started tumor cell killing from the rim of the spheroid towards the center. The reduction of spheroid area over time displayed a very similar kinetic when co-cultured with CTL or bead-activated T cells. At the end of the experiment the CTLs induced a more pronounced shrinkage of spheroid size. Interestingly the size of the spheroid and the red fluorescence intensity were not necessarily aligned. The positive control Staurosporin induced a fast reduction of the mKate signal, whereas the spheroid area did not change over time. In summary, the bead-activated T cell approached the spheroid fast, infiltrated the complete area and induced tumor cell killing homogenously across the tumor area. The CTL approached the spheroids slower but started killing earlier from the outside towards the center of the spheroid. The observed differences in infiltration and killing characteristics of the investigated T cell types lead to a better understanding of T cell biology in the context of the tumor biology, which can be translated into applications during dug development. Taken together, with the applied image analysis algorithm we were able to deconvolute the biological processes in the assay in much more detail. The additional read-outs increase the translational value of the assay tremendously in the context of drug development as well as precision medicine. Citation Format: Ryan Kerwin, Ina Rohleff, Martin Blanchard, Kanstantsin Lashuk, Bryan Walker, Julia B. Schueler. The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment [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 4224.

Abstract 3845: High-intensity focused ultrasound ablation increases tumor specific lymphocytes in prostate cancer

Abstract The treatment of localized prostate cancer has been revolutionized by High-Intensity Focused Ultrasound (HIFU), an FDA-approved procedure. Thermal HIFU achieves the destruction of tumor tissue through the noninvasive delivery of ultrasonic energy, causing rapid heating of the targeted area. Given that prostate cancer typically exhibits a low presence of tumor-infiltrating lymphocytes crucial for antitumor immunity, this study aimed to investigate whether HIFU has the potential to activate antitumor immunity and enhance lymphocyte infiltration within the tumor. RM1 tumors were implanted on the flanks of syngeneic C57BL/6J mice, with one tumor subjected to HIFU treatment. The growth of the contralateral tumor was monitored, and at the end of the study, tumors were collected to evaluate immune cell infiltration. Blood samples were obtained from patients both before and after prostatectomy or HIFU treatment. Peripheral blood mononuclear cells (PBMCs) were then isolated to analyze the immune cell population. In murine experiments, the application of HIFU to one tumor resulted in a reduction in the growth of the contralateral (untreated) tumor, specifically when the contralateral tumor was of the same type, but not when it was a different tumor type. Notably, HIFU led to an augmented infiltration of CD4+ and CD8+ lymphocytes into the contralateral (untreated) tumor. Depletion studies further affirmed that the antitumor immune response triggered by HIFU relies on CD4+ and CD8+ lymphocytes. In human subjects, HIFU was observed to elevate the CD4+ and CD8+ lymphocyte populations in PBMCs. HIFU induces a potent antitumor immune response that effectively inhibits the progression of murine prostate tumors. This immune response is contingent upon CD4+ and CD8+ lymphocytes. HIFU demonstrates significant promise in activating antitumor immune responses in patients diagnosed with prostate cancer. Subsequent research endeavors should delve into models of metastatic prostate cancer, exploring the potential synergy of cytoreductive prostate tumor ablation with systemic immunotherapies to elicit a robust and comprehensive antitumor immune response. Citation Format: Shengchen Su, Yanping Wang, Eric M. Lo, Hyung L. Kim. High-intensity focused ultrasound ablation increases tumor specific lymphocytes in prostate cancer [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 3845.

CTIM-21. T-CELL RECEPTOR REPERTOIRE ANALYSIS IN PBMCS FROM PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT), which involves transferring tumor-specific lymphocytes, has shown promising clinical efficacy across various cancer types. However, accurately tracking these transferred cells has been challenging. To overcome this limitation, we utilized T-cell receptor (TCR) sequencing to investigate the TCR repertoire in peripheral blood mononuclear cells (PBMCs). Specifically, we examined PBMCs stimulated ex vivo using autologous dendritic cells (DCs) loaded with total tumor RNA, which we applied for infusion in pediatric patients. We also collected blood samples prior to and following immunotherapy to compare the TCR repertoire dynamics. METHODS We isolated total RNA from patient PBMCs obtained before the initiation of adoptive cellular therapy (ACT) and continued collection on a weekly basis for one month, followed by monthly collections after immunotherapy treatment. To generate cDNA, we used RACE technology with the addition of a common adapter at the 5' end. The raw sequencing data obtained was then preprocessed using MiXCR software. Subsequent TCR repertoire analysis was performed using the Immunarch R-package and VDJtools software. RESULTS& CONCLUSIONS The analysis of T-cell receptor (TCR) diversity across ex vivo extended T-cell samples showed a range of 593 to 34,000 unique CDR3 clones among 28 patients with recurrent medulloblastoma. The observed range suggests that different patients exhibit distinct levels of TCR diversity. Our results revealed significant instability in the TCR repertoire within the peripheral blood during the initial 28 days after ACT for all patients. Subsequently, the regeneration was mainly driven by de novo thymic production of naïve T-cells. TCR sequencing of PBMCs stimulated with autologous DCs loaded with total tumor RNA allowed us to gain a deeper understanding of the TCR repertoire in the context of immunotherapy. This approach enabled us to track and analyze the tumor-specific T-cells in the repertoire, providing valuable insights into their behavior and response to the treatment.

IMMU-33. PRECISION ANTIGEN-DIRECTED IMMUNOTHERAPY TREATMENT OF BRAIN TUMORS INCREASES TUMOR-INFILTRATING EFFECTOR LYMPHOCYTE RESPONSES

Abstract BACKGROUND Despite considerable advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized tumor antigens expressed within brain tumors that can mediate tumor rejection; the limited mutational burden within these cancers that limits the abundance of targetable neoantigens; and the immunologically “cold” tumor microenvironment that hampers the generation of sustained and productive immunologic responses. Our OBJECTIVE is to develop a precision immunotherapy approach for brain tumor patients utilizing prospectively identified tumor-specific antigens using cancer immunogenomics and selective amplification of patients’ tumor-specific antigens for generating tumor antigen-specific lymphocyte therapy. METHODS We identified neoantigens and tumor-associated antigens that were uniquely expressed in preclinical glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (ORAN). A tumor antigen-specific RNA library was created for each tumor model and tumor-bearing animals were treated with ex vivo expanded tumor-specific lymphocytes and tumor antigen-specific RNA-loaded dendritic cell (tsRNA-DC) vaccines. In a different approach, animals were treated with a combination of tsRNA-DC vaccines and anti-PD-1 checkpoint-blockade therapy. Tumor progression and survival outcomes were determined. The immune cell response was evaluated using flow cytometry, TCR sequencing, and single-cell RNAseq. RESULTS Our results demonstrate the effectiveness of tsRNA-DC vaccines in eliciting robust anti-tumor immune responses in combination with adoptive cellular therapy and checkpoint blockade therapy. Additionally, our findings substantiate a robust increase in tumor-infiltrating lymphocytes characterized by enhanced effector function both intratumorally and systemically after tumor antigen-specific RNA-directed immunotherapies. CONCLUSION This powerful approach to generating immunotherapy recognizing a plurality of tumor antigens uniquely addresses the challenge of dealing with tumor antigenic heterogeneity and confronting the reality of patient-to-patient variation in antigen expression in the development of antigen targeting strategies.

Abstract 6638: Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model

Abstract Background: Although anti-PD1 therapy is standard of care in multiple tumor types, unmet need remains for those non-responsive to immunotherapy. Pulsed Electric Field (PEF) treatment may alter the tumor micro-environment, converting an immune “cold” tumor into an immune “hot” tumor, triggering an adaptive immune response and abscopal effect. We evaluated PEF in combination with anti-PD1 in a highly aggressive orthotopic triple negative breast tumor mouse model. Methods: Murine mammary 4T1 cells (2 × 105) non-responsive to anti-PD1 therapy were orthotopically implanted in mammary fat of 48 female Balb/C mice. Once tumors were established (5 mm diameter), mice were randomized into six groups: Sham+IgG controls, aPD-1-only, PEF (1X), PEF (2X), aPD-1 + PEF (1X), and aPD-1 + PEF (2X). Groups 4 and 6 received two PEF treatments, five days apart. Anti-PD1 and isotype matched control IgG was administered (200 ug, intraperitoneal) weekly, starting 3 days after PEF treatment. Tumor volumes were recorded 3x/week and standard survival analyses were conducted per IACUC protocol. In the cohort of tumors collected 30 days after PEF, flow cytometry (FC) evaluated T-cell immune infiltrates and associated markers. Blood was collected at different time points to evaluate systemic immunological response. Tumor-specific cytotoxic lymphocytes (CTL) were isolated from tumor-infiltrating and circulating lymphocytes by gp70 MHC tetramer formation assay. Results: Single PEF reduced tumor growth compared to IgG and aPD-1-only groups by 65% and 62%, respectively. Double PEF treatment reduced tumor growth by 74% and prolonged survival. FC demonstrated circulating helper T-cells and CTL frequency was increased two weeks post-PEF compared to the control group. The tetramer detected substantial increases in antigen-specific T-cell populations in both single and double PEF treatment groups. PEF groups had significant tumor and spleen infiltration of CD8 and CD4 T-cells, including increased tetramer levels. Anti-PD1-only animals showed no meaningful reductions in tumor growth, prolonged survival, or significant changes in CD8, CD4 T cells or in the tetramer assay. Conclusions: PEF treatments prior to initiation of anti-PD1 in orthotopic 4T1 mouse model triggers a tumor-specific adaptive immune response in an immune “cold” tumor model, irrespective of anti-PD-1 therapy. PEF increases circulating and tumor-infiltrating T-cells and antigen-specific T-cells, with these effects observed after a single or two treatments with PEF. Collectively, our results demonstrate that PEF treatment prior to initiation of anti-PD1 treatment can reduce tumor growth, prolong survival, and activate an adaptive immune response in an immune “cold” tumor. These findings suggest that PEF treatment merits further evaluation in IO-non-responsive cancer patients. Citation Format: Ebtesam Nafie, Mukta Wagh, Chiara Pastori, Mo Trikha, Robert Neal. Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6638.

Radiation induced lymphopenia - a possible critical factor in current oncological treatment.

The effect of ionizing radiation on the immune system during the treatment of malignant tumors has long remained a point of great interest. This issue is currently gaining importance, especially in connection with the advancing development and availability of immunotherapeutic treatment. During cancer treatment, radiotherapy has the ability to influence the immunogenicity of the tumor by increasing the expression of certain tumor-specific antigens. These antigens can be processed by the immune system, stimulating the transformation of naïve lymphocytes into tumor-specific lymphocytes. However, at the same time, the lymphocyte population is extremely sensitive to even low doses of ionizing radiation, and radiotherapy often induces severe lymphopenia. Severe lymphopenia is a negative prognostic factor for numerous cancer dia-gnoses and negatively impacts the effectiveness of immunotherapeutic treatment. In this article, we summarize the possible influence of radiotherapy on the immune system, with a particular emphasis on the impact of radiation on circulating immune cells and the subsequent consequences of this influence on the development of cancer. Lymphopenia is an important factor influencing the results of oncological treatment, with a com-mon occurrence during radiotherapy. Strategies to reduce the risk of lymphopenia consist of accelerating treatment regimens, reducing target volumes, shortening the beam-on time of irradiators, optimizing radiotherapy for new critical organs, using particle radiotherapy, and other procedures that reduce the integral dose of radiation.

Pharmacologic Inhibitors of STAT3 or BCL6 Transcriptional Function Sensitize Lymphoma Cells to the Novel PD-1 Cis-Targeted PD1-IL2v Immunocytokine in a Murine Model

Although immune-based therapies like immune checkpoint inhibitors or CAR-T cells have led to dramatic advances in treating relapsed and refractory lymphomas, most patients still do not derive a durable response. Lymphoma cells, while being immunogenic, can have intrinsic mechanisms of resistance to immune-based killing and may also establish an immuno-suppressive microenvironment in which immune effector cells have diminished activity. This immune resistance may be a direct result of the inappropriate and constitutive activation of transcription factors whose physiologic function is to promote survival and resistance to immune-based killing. Two transcription factors that play such a role in lymphomas are STAT3 and BCL6. We therefore wished to test the hypothesis that drugs that block the transcriptional function of these proteins would enhance the efficacy of an immune-based therapy. We chose to use the A20 murine lymphoma system, which grows as an aggressive tumor in syngeneic Balb/c mice, and is generally resistant to immune checkpoint inhibitors such as PD-1 antagonists. A20 cells are characterized by constitutive STAT3 activation and BCL6 expression. To enhance the translational relevance of this study, we used inhibitors of STAT3 or BCL6 transcriptional function that could rapidly be introduced into clinical trials. We identified drugs that inhibit the transcriptional effects of these proteins based on a computational search of drugs that are known to be safe in humans, and which lead to gene expression changes opposite to that driven by each transcription factor. Using this approach, we identified atovaquone as a STAT3 inhibitor and disulfiram as a BCL6 inhibitor. The activity of each drug was then confirmed by in vitro testing. Furthermore, the resultant changes in gene expression in A20 cells would be predicted to make these cells more immunogenic. To stimulate the endogenous anti-tumor immune response, we utilized a novel murine specific PD-1 cis-targeted PD1-IL2v immunocytokine. muPD1-IL2v is, a fusion protein consisting of a PD-1 antibody and a C-terminally fused variant form of the cytokine IL-2 (IL2v) that does not bind to CD25. CD25 is the alpha chain that confers high affinity to the IL-2 receptor and which is highly expressed on immunosuppressive T regulatory cells (Tregs). This design enables muPD1-IL2v to preferentially activate PD-1 expressing antigen specific T effector cells by removing the competitive advantage of immunosuppressive T regulatory cells. At the same time, the PD-1 targeting allows the preferential delivery of IL-2v to PD-1+ tumor specific lymphocytes while bypassing the binding to Tregs. We then tested the activity of atovaquone or disulfiram in combination with muPD1-IL2v in an immunocompetent model with A20 lymphoma cells. As a single agent, muPD1-IL2v had a modest effect in delaying the growth of these tumors. Similarly, atovaquone or disulfiram each showed only a small change in the rate of growth of A20 tumors. However, when used in combination with muPD1-IL2v, each of these transcriptional inhibitors led to a significant fraction of animals having complete and long term regression of these tumors. Furthermore, tumors resected from animals treated with atovaquone showed evidence of on-target inhibition of STAT3 function, and tumors resected from animals treated with disulfiram showed evidence for on-target effects on BCL6 target gene expression. These findings suggest that inhibition of oncogenic transcription factors like STAT3 and BCL6, in conjunction with novel immune-targeted therapeutics like PD1-IL2v, have the potential for long term disease control in lymphomas. This research collaboration was supported through the imCORE network.

CTIM-31. IMMUNOLOGIC MONITORING AFTER ADOPTIVE CELL THERAPY IN PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA USING IMMUNOGENOMICS APPROACH

Abstract BACKGROUND Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have employed the use of total tumor RNA (TTRNA)-pulsed DCs in the ex vivo expansion of tumor-specific lymphocytes for use in adoptive cellular therapy targeting pediatric malignant brain tumors. METHODS Total RNA was isolated from patient PBMC samples collected prior to adoptive cellular therapy and after 2-weeks and 4-weeks following immunotherapy treatment from 18 patients (Re-MATCH protocol, FDA IND BB-14058). We applied bulk RNA-Seq and high-throughput T cell receptor sequencing to monitor changes in blood draws after ACT. RESULTS We observed an increase of expression level of genes responsible for T cell activation in PBMC after adoptive cell therapy. We found that these genes were involved in T cell signaling pathways, proliferative signals, and cytokine production. The data revealed that certain T-cells were clonally expanded after ACT, with an increasing number of “hyper-expanded” TCR clones (comprising > 1% of all TCR beta or alpha sequences) after adoptive cellular therapy. These TCR clones established a new balance in the peripheral blood that was stable over the month following the completion of cellular immunotherapy. Hyper-expanded TCR clones and increased TCR diversity following ACT was associated with radiographic response to ACT and prolonged progression-free and overall survival. Limited TCR expansion and diversity following ACT was observed in patients with short overall and progression-free survival. CONCLUSIONS These results emphasize the importance of gene expression profiling to achieve higher resolution in monitoring immune responses in patients receiving immunotherapeutic treatment. Also, these findings support further study of the use of TCR sequencing to monitor responses to adoptive cellular therapy and suggest that TCR clonal expansion and increasing TCR diversity following treatment may be associated with positive clinical responses.

Pre-Existing HSV-1 Immunity Enhances Anticancer Efficacy of a Novel Immune-Stimulating Oncolytic Virus.

Oncolytic viruses (OVs) can specifically replicate in the host and cause cancer cell lysis while inducing an antitumor immune response. The aim of this study is to investigate the impact of either pre-existing immunity against herpes simplex virus type-1 (HSV-1) or multicycle treatment with OVs on anticancer efficacy of VG161, an HSV-1 OV in phase 2 clinical trial. VG161 efficacy was tested in CT26 mouse models by comparing the efficacy and immune response in naïve mice or in mice that were immunized with VG161. Moreover, VG161 efficacy in HLA-matched CD34+ humanized intrahepatic cholangiocarcinoma (ICC) patient-derived xenograft (PDX) models was also tested in multicycle treatment and was compared to standard chemotherapy for this type of cancer (gemcitabine). The HSV-1-immunized mice significantly inhibited tumor growth in VG161-treated mice compared to control naïve treated mice. RNA expression profiling and ELISPOT analyses indicated changes in the tumor's immune profile in the immunized and treated group compared to naïve and treated mice, as well as enhanced T cell function depicted by higher numbers of tumor specific lymphocytes, which was enhanced by immunization. In the ICC PDX model, repeated treatment of VG161 with 2 or 3 cycles seemed to increase the anticancer efficacy of VG161. In conclusion, the anticancer efficacy of VG161 can be enhanced by pre-immunization with HSV-1 and multicycle administration when the virus is given intratumorally, indicating that pre-existing antiviral immunity might enhance OV-induced antitumor immunity. Our results suggest potential clinical benefits of HSV-1-based OV therapy in HSV-1-seropositive patients and multicycle administration of VG161 for long-term maintenance treatment.

Tumor cell-based vaccine contributes to local tumor irradiation by eliciting a tumor model-dependent systemic immune response.

Multimodal treatment approaches, such as radio-immunotherapy, necessitate regimen optimization and the investigation of the interactions of different modalities. The aim of this study was two-fold. Firstly, to select the most effective combination of irradiation and the previously developed tumor cell-based vaccine and then to provide insight into the immune response to the selected combinatorial treatment. The study was performed in immunologically different murine tumor models: B16F10 melanoma and CT26 colorectal carcinoma. The most effective combinatorial treatment was selected by comparing three different IR regimens and three different vaccination regimens. We determined the local immune response by investigating immune cell infiltration at the vaccination site and in tumors. Lastly, we determined the systemic immune response by investigating the amount of tumor-specific effector lymphocytes in draining lymph nodes. The selected most effective combinatorial treatment was 5× 5 Gy in combination with concomitant single-dose vaccination (B16F10) or with concomitant multi-dose vaccination (CT26). The combinatorial treatment successfully elicited a local immune response at the vaccination site and in tumors in both tumor models. It also resulted in the highest amount of tumor-specific effector lymphocytes in draining lymph nodes in the B16F10, but not in the CT26 tumor-bearing mice. However, the amount of tumor-specific effector lymphocytes was intrinsically higher in the CT26 than in the B16F10 tumor model. Upon the selection of the most effective combinatorial treatment, we demonstrated that the vaccine elicits an immune response and contributes to the antitumor efficacy of tumor irradiation. However, this interaction is multi-faceted and appears to be dependent on the tumor immunogenicity.

Abstract 573: Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models

Abstract Adoptive cell transfer (ACT) involving autologous tumor-specific lymphocytes (TIL) has shown promising results in the treatment of advanced cancer. Poor in vitro cell expansion, inefficient T-cell migration and T cell exhaustion in the tumor microenvironment greatly limits the broader application of this approach. The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is highly expressed in T cells, where it limits cell activation following TCR engagement, therefore functioning as an important intracellular checkpoint that limits T cell mediated anti-tumor responses. We have developed NX-0255 a highly potent small molecule inhibitor of CBL-B that increases TCR-mediated activation of T cells in the presence or absence of CD28 co-stimulation. Here we used the OT-1 ACT/E.G7-OVA lymphoma and the Pmel-1 ACT/B16 melanoma tumor models to investigate whether adding NX-0255, during the in vitro expansion of tumor-specific T cells would have favorable effects on their ability to reject tumors following adoptive transfer in vivo. OT-1 CD8+ T cells were expanded in vitro using anti-CD3 stimulation and either NX-0255 alone, IL-2 alone, or NX-0255 combined with IL-2 and subsequently transferred in vivo to mice bearing established E.G7-OVA. ACT of OT-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 demonstrated superior anti-tumor activity against E.G7-OVA lymphoma tumors, resulting in significant improvement in survival when compared to mice receiving OT-1 CD8+ T cells cultured with NX-0255 alone or IL-2 alone. Importantly, OT-1 CD8+ T cells cultured in the presence of NX-0255 or the combination of NX-0255 plus IL-2 were found at an increased frequency in the blood and in the tumor and persisted longer in circulation when compared to cells cultured with IL-2 alone. A lower percentage of the tumor-infiltrating OT-1 cells were triple-positive for exhaustion markers PD1+TIM3+LAG3+ expression when cultured with NX-0255 alone or the combination of NX-0255 and IL-2 compared to culture with IL-2 alone. Similarly, ACT of Pmel-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 provides a robust and durable anti-tumor response compared to mice receiving Pmel-1 CD8+ T cells cultured with IL-2 alone in the aggressive B16-OVA model. Flow cytometry analysis showed that Pmel-1 CD8+ T cells cultured in the presence of NX-0255 plus IL-2 were found at an increased frequency in the blood and showed an increased central-memory phenotype (CD44+CD62L+) after adoptive transfer when compared to cells cultured with IL-2 alone. Collectively, these data suggest that adding the CBL-B inhibitor, NX-0255, during the in vitro expansion of tumor-specific T cells increases the frequency and absolute numbers of less exhausted CD8+ memory T-cells, increasing their in vivo persistence and ability to infiltrate the tumor. Citation Format: Marilena Gallotta, Jose Gomez Romo, Alexandra Borodovsky, Austin Tenn-McClellan, Jennifer Stokes, Jennifa Gosling, Gwenn M. Hansen, Arthur Sands, Ryan Rountree, Cristiana Guiducci. Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models [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 573.

Abstract LB535: A novel IL12-based immunocytokine targeting fibroblast activation protein (FAP) for the treatment of cancer

Abstract Immunocytokines, which consist of the fusion of cytokines to tumor targeting antibodies, represent a novel class of biopharmaceuticals that have great potential for cancer treatment. The use of these products improves the efficacy of the delivered drug by reducing related side effects and significantly enhances the therapeutic index of the payload. Over the past years, nuclear medicine studies have validated the Fibroblast Activation Protein (FAP) as a pantumoral antigen, with more than 28 different cancer types successfully imaged in patients. Since FAP is mostly undetectable in healthy organs, it has been considered as an attractive target for both imaging and therapeutic applications. For this reason, here we describe the generation of a novel fully human monoclonal antibody targeting FAP. The antibody was isolated from a synthetic antibody phage display library and its tumor recognition properties were validated by immunofluorescence performed on a commercial tissue microarray as well as on freshly frozen colon cancer biopsies from human patients. An ex vivo biodistribution analysis in mice confirmed the ability of anti-FAP in IgG1 format to selectively localize to solid tumors while sparing healthy organs. Furthermore, Pharmacokinetics (PK) studies in Cynomolgus Monkey showed no sink effects at early time points and a slow clearance from bloodstream as expected for IgG1 antibodies. Encouraged by these results, an antibody cytokine fusion (mIL12-FAP) based on the novel anti-FAP antibody was generated and characterized in vitro and in vivo, showing activation of the immune system both in immunocompetent and immunodeficient mouse models bearing FAP-positive tumors. mIL12-FAP exhibited strong anticancer activity in mice bearing CT26-FAP colon carcinomas which could be boosted by the combination with immune checkpoint inhibitors, leading to durable cancer eradication. The targeted delivery of mIL12 to the tumor microenvironment increased the infiltrate of tumor-specific lymphocytes and Natural Killer (NK) cells as compared to the control group without apparent evidence of toxicity. Citation Format: Lisa Nadal, Frederik Peissert, Abdullah Elsayed, Chiara Libbra, Dario Neri, Roberto De Luca. A novel IL12-based immunocytokine targeting fibroblast activation protein (FAP) for the treatment of 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 LB535.

2-methoxyestradiol inhibits melanoma cell growth by activating adaptive immunity

Background: The overall survival of melanoma patients remains poor despite advancements in surgical treatment and targeted therapies. Therefore, there is a need to develop new therapeutic strategies for melanoma. 2-methoxyestradiol (2-ME) is a major metabolite of estrogen that has been shown to have anti-tumor effects against many malignancies. However, the effects and mechanisms of action of 2-ME against melanoma remain unclear. Materials and methods: Melanoma cells (B16) were treated with 2-ME in vitro. Cell proliferation was detected by CCK8 and clone formation, transwell was carried out to measure the migration of B16 cells with or without 2-ME. Flow cytometry was performed to measure the apoptosis and cell cycle. C57BL/6 mice were used for tumor-bearing of B16 cells, tumor volumes were measured once a day, and sacrificed after it was over 2000 mm3, then immunofluorescence was implemented to examine the marker of CD3, CD8 and PD-L1. Results: In our study, we found that 2-ME significantly affected the proliferation, migration, apoptosis, and cell cycle of melanoma in vitro. Our results also showed that 2-ME had strong anti-tumor effects against melanoma in vivo and increased the infiltration of tumor-specific cytotoxic lymphocytes CD8+ T cells in the tumor microenvironment. Besides, PD-L1 expression in tumor cells was significantly higher in the 2-ME-treated group than in the control group, indicating that 2-ME could exhibit stronger anti-tumor effects against melanoma if combined with PD-1 blockade therapy. Conclusion: 2-ME suppresses melanoma in vivo and in vitro and is a promising synergistic enhancer of PD-1 blockade immunotherapy.

STING and TLR7/8 agonists-based nanovaccines for synergistic antitumor immune activation.

Immunostimulatory therapies based on pattern recognition receptors (PRRs) have emerged as an effective approach in the fight against cancer, with the ability to recruit tumor-specific lymphocytes in a low-immunogenicity tumor environment. The agonist cyclic dinucleotides (CDNs) of the stimulator of interferon gene (STING) are a group of very promising anticancer molecules that increase tumor immunogenicity by activating innate immunity. However, the tumor immune efficacy of CDNs is limited by several factors, including relatively narrow cytokine production, inefficient delivery to STING, and rapid clearance. In addition, a single adjuvant molecule is unable to elicit a broad cytokine response and thus cannot further amplify the anticancer effect. To address this problem, two or more agonist molecules are often used together to synergistically enhance immune efficacy. In this work, we found that a combination of the STING agonist CDGSF and the Toll-like receptor 7/8 (TLR7/8) agonist 522 produced a broader cytokine response. Subsequently, we developed multicomponent nanovaccines (MCNVs) consisting of a PC7A polymer as a nanocarrier encapsulating the antigen OVA and adjuvant molecules. These MCNVs activate bone marrow-derived dendritic cells (BMDCs) to produce multiple proinflammatory factors that promote antigen cross-presentation to stimulate specific antitumor T-cell responses. In in vivo experiments, we observed that MCNVs triggered a strong T-cell response in tumor-infiltrating lymphocytes, resulting in significant tumor regression and, notably, a 100% survival rate in mice through 25 days without other partnering therapies. These data suggest that our nanovaccines have great potential to advance cancer immunotherapy with increased durability and potency.Electronic Supplementary MaterialSupplementary material (synthesis of CDGSF, 522, PC7A and OVA; preparation of MCNVs; representative gating strategies for flow cytometry) is available in the online version of this article at 10.1007/s12274-022-4282-x.

Primary Tumor Suppression and Systemic Immune Activation of Macrophages through the Sting Pathway in Metastatic Skin Tumor.

PurposeAgonists of the stimulator of interferon genes (STING) play a key role in activating the STING pathway by promoting the production of cytokines. In this study, we investigated the antitumor effects and activation of the systemic immune response of treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist, in EML4-ALK lung cancer and CT26 colon cancer.Materials and MethodsThe abscopal effects of DMXAA in the treatment of metastatic skin nodules were assessed. EML4-ALK lung cancer and CT26 colon cancer models were used to evaluate these effects after DMXAA treatment. To evaluate the expression of macrophages and T cells, we sacrificed the tumor-bearing mice after DMXAA treatment and obtained the formalin-fixed paraffin-embedded (FFPE) tissue and tumor cells. Immunohistochemistry and flow cytometry were performed to analyze the expression of each FFPE and tumor cell.ResultsWe observed that highly infiltrating immune cells downstream of the STING pathway had increased levels of chemokines after DMXAA treatment. In addition, the levels of CD80 and CD86 in antigen-presenting cells were significantly increased after STING activation. Furthermore, innate immune activation altered the systemic T cell-mediated immune responses, induced proliferation of macrophages, inhibited tumor growth, and increased numbers of cytotoxic memory T cells. Tumor-specific lymphocytes also increased in number after treatment with DMXAA.ConclusionThe abscopal effect of DMXAA treatment on the skin strongly reduced the spread of EML4-ALK lung cancer and CT26 colon cancer through the STING pathway and induced the presentation of antigens.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Synergism between CAR-T Cells and a Personalized Tumor Vaccine in Hematological Malignances

Synergism between CAR-T Cells and a Personalized Tumor Vaccine in Hematological Malignances

The Immunology of Hepatocellular Carcinoma.

Liver cancer is the third leading cause of cancer death worldwide. Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. Liver resection or transplantation offer the only potentially curative options for HCC; however, many patients are not candidates for surgical resection, either due to presentation at advanced stages or poor liver function and portal hypertension. Liver transplantation is also limited to patients with certain characteristics, such as those that meet the Milan criteria (one tumor ≤ 5 cm, or up to three tumors no larger than 3 cm, along with the absence of gross vascular invasion or extrahepatic spread). Locoregional therapies, such as ablation (radiofrequency, ethanol, cryoablation, microwave), trans-arterial therapies like chemoembolization (TACE) or radioembolization (TARE), and external beam radiation therapy, have been used mainly as palliative measures with poor prognosis. Therefore, emerging novel systemic treatments, such as immunotherapy, have increasingly become popular. HCC is immunogenic, containing infiltrating tumor-specific T-cell lymphocytes and other immune cells. Immunotherapy may provide a more effective and discriminatory targeting of tumor cells through induction of a tumor-specific immune response in cancer cells and can improve post-surgical recurrence-free survival in HCC. We herein review evidence supporting different immunomodulating cell-based technology relative to cancer therapy in vaccines and targeted therapies, such as immune checkpoint inhibitors, in the management of hepatocellular carcinoma among patients with advanced disease.

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.