Adoptive Cellular Immunotherapy Research Articles

Comparing the efficacy of combined versus single immune cell adaptive therapy targeting colorectal cancer.

Colorectal cancer (CRC) is the most frequent cancer with limited therapeutic achievements. Recently, adoptive cellular immunotherapy has been developed as an antitumor therapy. However, its efficacy has not been tested in CRC. This study investigated the ability of an immune cell cocktail of dendritic cells (DCs), T cells, and natural killer (NK) cells to overcome immunological hurdles and improve the therapeutic efficacy of cell therapy for CRC. CRC lysate-pulsed monocyte-derived DCs (Mo-DCs), CRC antigen-specifically expanded T cells (CTL), and in vitro-expanded NK cells were cultured from patient peripheral blood mononuclear cells (PBMC). The ability of the combined immune cells to kill autologous tumor cells was investigated by co-culturing the combined immune cells with patient-derived tumor cells. The Mo-DCs produced expressed T cell co-stimulating molecules like CD80, CD86, human leukocyte antigen (HLA)-DR and HLA-ABC, at high levels and were capable of activating naive T cells. The expanded T cells were predominantly CD8 T cells with high levels of CD8 effector memory cells and low levels of regulatory T cells. The NK cells expressed high levels of activating receptors and were capable of killing other cancer cell lines (K562 and HT29). The immune cell cocktail demonstrated a higher ability to kill autologous tumor cells than single types. An in vivo preclinical study confirmed the safety of the combined immune cell adaptive therapy showing no therapy-related death or general toxicity symptoms. The results suggested that combined immune cell adaptive therapy could overcome the limited efficacy of cell immunotherapy.

The Therapeutic Potential of Cytokine-Induced Killer in Patients with Cancer.

Despite the promising results of immunotherapy, further experiments need to be considered because of several factors ranging from physical barriers to off-tumor adverse effects. It is surprising that adoptive cellular immunotherapy, particularly dendritic cell and cytokine-induced killer (DC-CIK) therapy, is far less emphasized in the treatment of cancer diseases. DC-CIK therapy in cancer patients presents auspicious results with low or no side effects, which should not be overlooked. More interestingly, almost all DC-CIK clinical trials are ongoing in China that highlight the limitations of therapeutic strategies and require large-scale research. To date, it is advisable to consider combination therapy with chemotherapy since it has shown promising outcomes with higher efficacy. In this article, the efficacy of DC-CIK therapy in patients with cancer is summarized by underscoring the lack of experiments on soft cancers on an unprecedented scale. In brief, DC-CIK therapy is a safe and effective therapeutic agent for malignant and nonmalignant diseases that enhances short-term and long-term effects.

Dynamics aspects and bifurcations of a tumor-immune system interaction under stationary immunotherapy

We consider a three-dimensional mathematical model that describes the interaction between the effector cells, tumor cells, and the cytokine (IL-2) of a patient. This is called the Kirschner–Panetta model. Our objective is to explain the tumor oscillations in tumor sizes as well as long-term tumor relapse. We then explore the effects of adoptive cellular immunotherapy on the model and describe under what circumstances the tumor can be eliminated or can remain over time but in a controlled manner. Nonlinear dynamics of immunogenic tumors are given, for example: we prove that the trajectories of the associated system are bounded and defined for all positive time; there are some invariant subsets; there are open subsets of parameters, such that the system in the first octant has at most five equilibrium solutions, one of them is tumor-free and the others are of co-existence. We are able to prove the existence of transcritical and pitchfork bifurcations from the tumor-free equilibrium point. Fixing an equilibrium and introducing a small perturbation, we are able to show the existence of a Hopf periodic orbit, showing a cyclic behavior among the population, with a strong dominance of the parental anomalous growth cell population. The previous information reveals the effects of the parameters. In our study, we observe that our mathematical model exhibits a very rich dynamic behavior and the parameter μ̃ (death rate of the effector cells) and p̃1 (production rate of the effector cell stimulated by the cytokine IL-2) plays an important role. More precisely, in our approach the inequality μ̃2>p̃1 is very important, that is, the death rate of the effector cells is greater than the production rate of the effector cell stimulated by the cytokine IL-2. Finally, medical implications and a set of numerical simulations supporting the mathematical results are also presented.

Gamma/delta T cells as cellular vehicles for anti-tumor immunity.

Adoptive cellular immunotherapy as a new paradigm to treat cancers is exemplified by the FDA approval of six chimeric antigen receptor-T cell therapies targeting hematological malignancies in recent years. Conventional αβ T cells applied in these therapies have proven efficacy but are confined almost exclusively to autologous use. When infused into patients with mismatched human leukocyte antigen, αβ T cells recognize tissues of such patients as foreign and elicit devastating graft-versus-host disease. Therefore, one way to overcome this challenge is to use naturally allogeneic immune cell types, such as γδ T cells. γδ T cells occupy the interface between innate and adaptive immunity and possess the capacity to detect a wide variety of ligands on transformed host cells. In this article, we review the fundamental biology of γδ T cells, including their subtypes, expression of ligands, contrasting roles in and association with cancer prognosis or survival, as well as discuss the gaps in knowledge pertaining to this cell type which we currently endeavor to elucidate. In addition, we propose how to harness the unique properties of γδ T cells for cellular immunotherapy based on lessons gleaned from past clinical trials and provide an update on ongoing trials involving these cells. Lastly, we elaborate strategies that have been tested or can be explored to improve the anti-tumor activity and durability of γδ T cells in vivo.

Advances in adoptive cellular immunotherapy in multiple myeloma

Advances in adoptive cellular immunotherapy in multiple myeloma

DIALing-up the preclinical characterization of gene-modified adoptive cellular immunotherapies.

The preclinical characterization of gene modified adoptive cellular immunotherapy candidates for clinical development often requires the use of mouse models. Gene-modified lymphocytes (GML) incorporating chimeric antigen receptors (CAR) and T-cell receptors (TCR) into immune effector cells require in vivo characterization of biological activity, mechanism of action, and preclinical safety. Typically, this characterization involves the assessment of dose-dependent, on-target, on-tumor activity in severely immunocompromised mice. While suitable for the purpose of evaluating T cell-expressed transgene function in a living host, this approach falls short in translating cellular therapy efficacy, safety, and persistence from preclinical models to humans. To comprehensively characterize cell therapy products in mice, we have developed a framework called "DIAL". This framework aims to enable an end-to-end understanding of genetically engineered cellular immunotherapies in vivo, from infusion to tumor clearance and long-term immunosurveillance. The acronym DIAL stands for Distribution, Infiltration, Accumulation, and Longevity, compartmentalizing the systemic attributes of gene-modified cellular therapy and providing a platform for optimization with the ultimate goal of improving therapeutic efficacy. This review will discuss both existent and emerging examples of DIAL characterization in mouse models, as well as opportunities for future development and optimization.

The Therapeutic Efficacy of CD19 Chimeric Antigen Receptor T Cells in Immune Thrombocytopenia Model

Abstract Introduction: ITP is a hemorrhagic autoimmune disease characterized by excessive platelet destruction and impaired platelet production,resulting in a high risk of bleeding. The pathogenesis of ITP involves immune intolerance towards platelet antigen, with platelet autoantibodies produced by plasma cells playing a significant role. Current treatments targeting this pathogenesis mainly rely on CD20 monoclonal antibodies, such as rituximab. However, as CD20 expression is present at the pre-B cell and mature B cell stages but absent on the surface of mature plasma cells. This can explain why a subset of autoantibody-positive patients do not respond to B-cell depletion therapy. CAR-T cell therapy, a form of adoptive cellular immunotherapy, has shown great potential in hematological malignancies and solid tumors. Recent studies have demonstrated its potential effectiveness of CAR-T cell therapy in various autoimmune diseases. CD19, expressed at all stages of B cell differentiation, presents an opportunity for CAR-T cell therapy in ITP. This study aims to evaluate the efficacy of CD19 CAR-T cell immunotherapy in an active murine ITP model. Methods: CD42-knockout mice were immunized with wild type (WT) C57BL/6 mice and splenocytes from these immunized mice were transplanted into irradiated WT mice to construct a murine model of active ITP. The ITP mice were then randomized into two groups, with the experimental group receiving an intraperitoneal injection with 1×10 6 CD19 CAR-T cells. Platelet counts were monitored weekly, and after 3 weeks, the ITP mice were euthanized and sacrificed. Single-cell suspensions were prepared from harvested spleens for analysis. Anti-murine antibodies were analyzed using flow cytometry (Beckmann Coulter, Brea, CA, USA). All data were analyzed by Statistical Package for the Social Sciences (SPSS) software, version 25.0 (IBM Corp., Armonk, NY, USA). P < 0.05 was considered statistically significance. Results: Our results demonstrated that the platelet counts of ITP mice reached their lowest level on day 7 after spleen cell transplantation, gradually recovering within a week. Platelet counts were significantly higher in the CD19 CAR-T cell therapy group compared to the control group on day 7 and persisted until day 21. There were no significant differences in weight between two groups, indicating no severe complication or CRS were identified. In the third week CD19 CAR-T cell therapy, the proportion of plasma cells in the spleen was determined, showing a marked decrease in the percentage of CD138+ plasma cells in the CAR-T group compared to the control group. Additionally, at day 14, the titers of anti-platelet CD42-specific antibodies were significantly lower in the CAR-T cell treated group compared to the control group. Furthermore, the mean fluorescence intensity (MFI) of CD19+ B cells in the spleen cells of the CD19 CAR-T cell immunotherapy group was significantly decreased compared to the control group. Conclusion: This study demonstrates the effectiveness of CD19 CAR-T cell therapy in treating thrombocytopenia by suppressing plasma and B cells in a mouse model of active ITP. These findings provide valuable insights into the potential therapeutic application of CD19 CAR-T cell immunotherapy for immune thrombocytopenia. Disclosure: No conflict of interest.

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft Versus Leukemia Response with Adoptive Cellular Therapy

Donor lymphocyte infusion (DLI) is an established therapy for relapsed acute myeloid leukemia (AML) after hematopoietic stem cell transplant (HSCT), but response rates are poor (~20%). Interactions between leukemia and immune cells within the leukemia microenvironment may determine responsiveness to adoptive cellular immunotherapies. We hypothesized that systematic characterization of the leukemic marrow microenvironment over treatment course with DLI would define leukemia-immune cell interactions critical to response and hence provide mechanistic understanding of the graft-versus-leukemia (GvL) effect. In particular, we anticipated that use of multi-modal single-cell sequencing across patients and time points before and after therapy accounting for temporal dependencies would facilitate disentangling of the complex microenvironment of leukemic marrow. To infer cell-cell interactions in a temporally resolved fashion from single cell RNA-sequencing (scRNA-seq) data, we developed DIISCO, a Bayesian model using a Gaussian process regression network. In total, we profiled 30 bone marrow aspirates (14 pre- and 16 post-DLI) from 9 patients (5 responder [R], 4 nonresponder [NR]) with post-HSCT relapsed AML treated with DLI by scRNA-seq, single cell TCR sequencing (scTCR-seq) and surface protein characterization (scCITE-seq). We obtained 51,371 high-quality transcriptomes from the marrow samples along with 43,918 cells from the DLI products of these patients (4 R, 3 NR) for a total of 95,289 cells. Cells were clustered with Phenograph resulting in 57 clusters, including 2 CD4+ T cell, 6 CD8+ T cell (C0, C5, C21, C25, C26, C40), 3 NK, 10 B cell, 3 AML leukemia, and several myelo-erythroid clusters. DIISCO revealed a cascading response post-DLI that centered around CD8 C0, CD8 C5, AML, and a B cell cluster in R, not observed in NR. Furthermore, C0 appeared to expand after DLI only in R. We observed a strong negative interaction from CD8 C0 to AML after DLI, suggesting an anti-leukemia immune response, supporting the notion that immune cell populations formed a coordinated interactome associated with effective GvL. Evaluation of inferred interacting pairs using DIISCO and with the tool CellPhoneDB pointed toward an activation circuit in R between CD226 on CD8 C0 and PVR/NECTIN2 on AML. Conversely, inhibitory interactions between the exhaustion marker TIGIT on CD8 C0 and AML were observed in NR. Because DIISCO pinpointed CD8 C0 as a central hub for response, we characterized the identity of this cluster and its post-DLI dynamics. scTCR-seq revealed that this cluster, but not other T cell clusters, underwent marked clonal expansion after DLI ( Figure 1A). scRNA-seq revealed high expression of activation/effector genes ( GZMB, IFNG, CX3CR1, PRF1) compared to other T cells. The clonally expanded cells of C0 also highly expressed ZNF683, a transcription factor associated with T cell cytotoxicity. scCITE-seq demonstrated expression of CD45RA and CD57 but not CD28, consistent with an effector memory re-expressing RA (TEMRA) phenotype. We validated post-DLI expansion of CD8 TEMRA cells (CD3+, CD8+, CD62L-, CD45RA+) in AML R by multi-parameter flow cytometry in an independent cohort of 53 AML patients (29 R, 24 NR) with post-HSCT relapse treated with DLI at our institution (fold change 2.56 pre to post for R, p<0.001; 1.3 pre to post for NR, p=0.474). Moreover, to examine whether CD8+ TEMRA cells were specifically activated after encounter with leukemia, we devised an in vitromodified mixed lymphocyte reaction. For 6 AML R, post-DLI donor-derived CD8+ TEMRA cells that were cultured with pre-DLI recipient leukemia cells consistently demonstrated greater CD137 (p=0.004) and IFN-γ secretion compared to co-culture of the leukemia cells with other T cell subsets (p=0.015) ( Figure 1B). CD8+ TEMRA cells cultured in the absence of leukemia cells did not show elevated expression of either CD137 or IFN-γ, indicating antigen-specific functional activation of CD8+ TEMRAs but not other cell types after encounter with autologous leukemia. Our results reveal clonal expansion and tumor-specific activation of CD8+ TEMRA cells, supporting an antigen specific functional role of these cells in mediating GvL in AML DLI responders. Further evaluation of this CD8 TEMRA population with the ability to recognize leukemia cells may translate to strategies to optimize adoptive cellular therapy or predict responsiveness to DLI.

Adoptive Cellular Immunotherapy for the Control of Primary Membranous Nephropathy

Adoptive Cellular Immunotherapy for the Control of Primary Membranous Nephropathy

AFM24 in combination with autologous NK cells (SNK01) in patients with advanced/metastatic epidermal growth factor receptor (EGFR) expressing solid tumors: Initial results from the phase 1 dose-escalation study.

26 Background: AFM24 is a novel tetravalent bispecific IgG1-scFv fusion antibody (scFv-IgAb) binding specifically to EGFR on target cells and CD16A on NK cells, mediating specific lysis of EGFR+ tumor cells via NK cell-mediated antibody-dependent cellular cytotoxicity. SNK01 cell therapy is an NK cell-derived adoptive cellular immunotherapy technology that can produce large scale ex vivo expanded and activated autologous NK cells (1,000–10,000-fold expansion of NK cells with >95% cell purity), even in patients with low primary NK cell counts. The antitumor activity of SNK01 may be enhanced when combined with AFM24. Methods: This is a proof-of-concept phase 1, 3+3 dose escalation study to determine the maximum tolerated dose and/or recommended phase 2 dose of AFM24 in combination with SNK01 (NCT05099549). Secondary endpoints are safety/tolerability, preliminary efficacy and pharmacokinetics (PK). Patients with advanced or metastatic, histologically or cytologically confirmed EGFR+ solid tumors (≥1% of positive tumor cells) refractory to standard-of-care therapies were enrolled. AFM24 starting dose was 160 mg followed by 480 mg. AFM24 is administered once weekly, concomitantly with 4.0 x109 SNK01 cells once weekly until disease progression, intolerable toxicity, patient withdrawal. Cycles are 28 days and CTs are at screening, cycle 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of January 27, 2023, 6 patients (5 microsatellite stable colorectal and 1 head and neck cancer) received combination therapy, 3 in each cohort. Median (range) age was 59 (41–64), mean number (range) of prior therapies was 5 (3–7). ECOG performance status was 0 for 3 patients and 1 for the other 3. No dose limiting toxicities or unexpected toxicities were observed. Preliminary results suggest consistent safety events with both drugs in monotherapy. The most frequent treatment-emergent adverse events related to AFM24 was Grade 1/2 infusion related reactions occurring in 5/6 patients. There were no Grade ≥3 events related to AFM24 or SNK01. In the low dose cohort (160 mg), 2 patients had stable disease for 106 days (15 weeks) and 100 days (14 weeks), respectively. At 480 mg, 1 patient with SD had 2% tumor burden reduction and 2 subsequent carcinoembryonic antigen reductions. The patient proceeded to Cycle 3. No difference in PK data was observed in comparison to AFM24 alone. Enrollment continues at 480 mg. Conclusions: In this first study combining a bispecific NK cell engager with autologous NK cell infusions; two dose cohorts have completed, and escalation has proceeded. Early PK data are consistent with AFM24 monotherapy with no new safety signals, suggesting that the combination of AFM24 and SNK01 cells is tolerated and warrants further investigation. Clinical trial information: NCT05099549 .

Immunotherapy in Head and Neck Cancer: Where Do We Stand?

Head and neck cancer (HNC) comprises a group of malignancies, amongst which squamous cell carcinoma accounts for more than 90% of the cases. HNC has been related to tobacco use, alcohol consumption, human papillomavirus, Epstein-Barr virus, air pollution, and previous local radiotherapy. HNC has been associated with substantial morbidity and mortality. This review aims to summarize the recent findings regarding immunotherapy in HNC. The recent introduction of immunotherapy, with the use of programmed death 1 (PD-1) inhibitors pembrolizumab and nivolumab, which have been FDA approved for the treatment of metastatic or recurrent head and neck squamous cell carcinoma, has changed the field in metastatic or recurrent disease. There are many ongoing trials regarding the use of novel immunotherapeutic agents, such as durvalumab, atezolizumab, avelumab, tremelimumab, and monalizumab. In this review, we focus on the therapeutic potential of novel immunotherapy treatment modalities, such as combinations of newer immune-checkpoint inhibitors; the use of tumor vaccines such as human papillomavirus-targeted vaccines; the potential use of oncolytic viruses; as well as the latest advances regarding adoptive cellular immunotherapy. As novel treatment options are still emerging, a more personalized approach to metastatic or recurrent HNC therapy should be followed. Moreover, the role of the microbiome in immunotherapy, the limitations of immunotherapy, and the various diagnostic, prognostic, and predictive biomarkers based on genetics and the tumor microenvironment are synopsized.

Cytokines induced memory-like NK cells engineered to express CD19 CAR exhibit enhanced responses against B cell malignancies.

CD19 chimeric antigen receptor (CAR) engineered NK cells have been used for treating patients with relapsed and/or refractory B cell malignancies and show encouraging outcomes and safety profile. However, the poor persistence of NK cells remains a major challenge for CAR NK cell therapy. Memory-like NK cells (MLNK) induced by IL-12, IL-15, and IL-18 have shown enhanced and prolonged responses to tumor re-stimulation, making them an attractive candidate for adoptive cellular immunotherapy. Here, we show efficient and stable gene delivery of CD19 CAR to memory-like NK cells using retroviral vectors with transduction efficiency comparable to those achieved with conventional NK cells. Analysis of surface molecules revealed a distinct phenotypic profile in CAR engineered memory-like NK cells (CAR MLNK), as evidenced by increased expression of CD94 and downregulation of NKp30 as well as KIR2DL1. Compared to conventional CAR NK cells, CAR MLNK cells exhibited significantly increased IFN-γ production and degranulation in response to CD19+ target cells, resulting in enhanced cytotoxic activity against CD19+ leukemia cells and lymphoma cells. Furthermore, memory properties induced by IL-12/-15/-18 improved the in vivo persistence of CAR MLNK cells and significantly suppressed tumor growth in a exnograft mouse model of lymphoma, leading to prolonged survival of CD19+ tumor-bearing mouse. Altogether, our data indicate that CD19 CAR engineered memory-like NK cells exhibited superior persistence and antitumor activity against CD19+ tumors, which might be an attractive approach for treating patient with relapse or refractory B cell malignancies.

Advances in Cancer Nanovaccines: Harnessing Nanotechnology for Broadening Cancer Immune Response.

Many advances have been made recently in the field of cancer immunotherapy, particularly with the development of treatments such as immune checkpoint inhibitors and adoptive cellular immunotherapy. The efficacy of immunotherapy is limited, however, owing to high levels of tumor heterogeneity and the immunosuppressive environments of advanced malignant tumors. Therefore, therapeutic anticancer vaccines have gradually become powerful tools for inducing valid antitumor immune responses and regulating the immune microenvironment. Tumor vaccines loaded in nanocarriers have become an indispensable delivery platform for tumor treatment because of their enhanced stability, targeting capability, and high level of safety. Through a unique design, cancer nanovaccines activate innate immunity and tumor-specific immunity simultaneously. For example, the design of cancer vaccines can incorporate strategies such as enhancing the stability and targeting of tumor antigens, combining effective adjuvants, cytokines, and immune microenvironment regulators, and promoting the maturation and cross-presentation of antigen-presenting cells (APCs). In this review, we discuss the design and preparation of nanovaccines for remodeling tumor antigen immunogenicity and regulating the immunosuppressive microenvironment.

Abstract A13: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets. LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the resombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ, whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A13.

An update on Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cancer of the skin characterized by a neuroendocrine phenotype and an aggressive clinical behavior. It frequently originates in sun-exposed body areas, and its incidence has steadily increased in the last three decades. Merkel cell polyomavirus (MCPyV) and ultraviolet (UV) radiation exposure are the main causative agents of MCC, and distinct molecular features have been documented in virus-positive and virus-negative malignancies. Surgery remains the cornerstone of treatment for localized tumors, but even when integrated with adjuvant radiotherapy is able to definitively cure only a fraction of MCC patients. While characterized by a high objective response rate, chemotherapy is associated with a short-lasting benefit of approximately 3months. On the other hand, immune checkpoint inhibitors including avelumab and pembrolizumab have demonstrated durable antitumor activity in patients with stage IV MCC, and investigations on their use in the neoadjuvant or adjuvant setting are currently underway. Addressing the needs of those patients who do not persistently benefit from immunotherapy is currently one of the most compelling unmet needs in the field, and multiple clinical trials of new tyrosine kinase inhibitors (TKIs), peptide receptor radionuclide therapy (PRRT), therapeutic vaccines, immunocytokines as well as innovative forms of adoptive cellular immunotherapies are under clinical scrutiny at present.

ASXL1 disruption preserves CD8 T cell exhaustion-progenitor epigenetic programming during chronic stimulation

Abstract Differentiation of CD8 T cells into a terminally exhausted state is a major hurdle limiting efficacy of both immune checkpoint blockade and adoptive cellular immunotherapy approaches. Prior studies describing disruption of the epigenetic regulators DNMT3A and TET2 in CD8 T cells have revealed a critical role of DNA methylation in repressing the proliferative capacity and effector potential of CD8 T cells during exhaustion. Disruption of these genes has also been noted to preserve the proliferative capacity of hematopoietic stem cells (HSCs), resulting in clonal hematopoiesis (CH) and establishing the broader concept that these epigenetic regulators control cellular developmental potential. In addition to DNMT3A and TET2, the histone modifier ASXL1 is a critical regulator controlling CH. Here we report that disruption of ASXL1 in CD8 T cells preserved a stem-like phenotype denoted by increased TCF1 expression, IFN-γ production during chronic infection. At the chronic stage of infection, ASXL1 KO CD8 T cells retained a heightened ability to undergo a proliferative burst during PD-L1 blockade. Genome-wide epigenetic profiling demonstrated that disruption of ASXL1 resulted in inhibition of DNA methylation accompanied by changes in chromatin accessibility. These data document crosstalk between histone modifications and DNA methylation in regulating the transition of stem-like progenitor T cells into terminally exhausted T cells. Collectively, these data demonstrate that ASXL1 is a novel epigenetic regulator controlling development of exhausted CD8 T cells and represents a novel immunotherapeutic target for improving responses to T cell-based cancer immunotherapy.

Abstract 3185: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets.LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the recombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ,whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [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 3185.

Abstract 911: Butyrophilin 3A2 expression plays a critical role in phosphoantigen-mediated γδ T cell cytotoxicity of neuroblastoma cells

Abstract The clinical use of antibodies targeting the diaslganglioside GD2 improved survival and clearly identified a role for immunotherapy in the aggressive pediatric solid tumor neuroblastoma (NB). Even so, the 5-year survival rate for high risk patients remains 50%, warranting novel immunotherapeutic approaches. Given their MHC-independent cytotoxic activity towards transformed cells, γδ T cells are attractive tools for adoptive cellular immunotherapy. γδ T cell cytotoxicity is mediated through the expression of various activating receptors that contribute to multiple mechanisms of anti-tumor activity. Specifically, their expression of the γδ-TCR works in concert with tumor expression of members of the butyrophilin-3 (BTN3A) subfamily for cytotoxicity in response to phosphoantigens (pAg). In other cancers, stimulation of pAg production through zoledronate (ZOL) supplementation promotes γδ T cell recognition/killing. We have identified that γδ T cells are differentially cytotoxic towards NB cell lines, suggesting variations in expression of key players responsible for γδ T cell susceptibility. This work serves to understand the significance of BTN3A members in NB, and their impact on the susceptibility of NB to γδ-TCR mediated killing. It is hypothesized that the pAg-mediated response can be augmented by increasing intracellular pAg via use of ZOL or by manipulating BTN3A members. To test this, NB cells were pre-treated with either vehicle or ZOL for 24 hours and then co-incubated with expanded γδ T cells for 4 hours at various effector:target (E:T) ratios. Apoptotic death was then determined for NB cells using Annexin-V/7-AAD staining. Across 8 tested NB cell lines, Kelly, NB-1643, NGP, SK-N-AS, SH-SY5Y, NLF, and SMS-SAN cells were resistant to γδ T cells at a 10:1 E:T ratio when untreated with ZOL. IMR-5 demonstrated 12-18% lysis at a 5:1 E:T. When NB cells were pre-treated with ZOL, γδ T cell cytotoxicity was enhanced towards all models except SK-N-AS. We identified three NB cell lines with differential susceptibility to γδ T cell killing, SK-N-AS<NLF<IMR5. Using a high-risk NB gene expression dataset, expression of important BTN members was queried, revealing IMR5 to have BT3.2low expression, NLF to have BT3.2mod expression, and SK-N-AS to have BT3.2high expression. BT3.2 expression levels was confirmed via western blotting. It has been postulated that BT3.2 may be a decoy receptor that interacts with γδ T cells without intracellular signaling, given it lacks the intracellular pAg sensor. Due to its high expression in SK-N-AS, a lentiviral system was used to knockdown BT3.2. SK-N-A-S shBT3.2 models had 48-55% lysis at a 5:1 E:T when untreated and 65-72% lysis when pre-treated with ZOL. In summary, the anti-NB γδ T cell response can be augmented with ZOL pre-treatment. Additionally, the expression of BT3.2 may be responsible for differential sensitivity of NB cell lines to γδ T cells. Citation Format: Andrew Ho, Hunter C. Jonus, Kelly C. Goldsmith. Butyrophilin 3A2 expression plays a critical role in phosphoantigen-mediated γδ T cell cytotoxicity of neuroblastoma cells [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 911.

2022 Chinese expert consensus and guidelines on clinical management of toxicity in anti-CD19 chimeric antigen receptor T-cell therapy for B-cell non-Hodgkin lymphoma.

Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells has emerged as a novel modality for treating relapsed and/or refractory B-cell non-Hodgkin lymphoma (B-NHL). With increasing approval of CAR T-cell products and advances in CAR T cell therapy, CAR T cells are expected to be used in a growing number of cases. However, CAR T-cell-associated toxicities can be severe or even fatal, thus compromising the survival benefit from this therapy. Standardizing and studying the clinical management of these toxicities are imperative. In contrast to other hematological malignancies, such as acute lymphoblastic leukemia and multiple myeloma, anti-CD19 CAR T-cell-associated toxicities in B-NHL have several distinctive features, most notably local cytokine-release syndrome (CRS). However, previously published guidelines have provided few specific recommendations for the grading and management of toxicities associated with CAR T-cell treatment for B-NHL. Consequently, we developed this consensus for the prevention, recognition, and management of these toxicities, on the basis of published literature regarding the management of anti-CD19 CAR T-cell-associated toxicities and the clinical experience of multiple Chinese institutions. This consensus refines a grading system and classification of CRS in B-NHL and corresponding measures for CRS management, and delineates comprehensive principles and exploratory recommendations for managing anti-CD19 CAR T-cell-associated toxicities in addition to CRS.

CTLs, NK cells and NK-derived EVs against breast cancer

Patients with advanced stage breast cancer need novel therapies. New potential treatments have been developed, such as adoptive cellular therapies and alternative cell-free immunotherapies. The goal of this study was to assess the cytotoxicity of three of the patient-derived immune components, CTLs, NK cells and NK-derived EVs, and evaluate the potential for the development of novel therapy against breast cancer. CTLs were activated against MUC-1 antigen. The in vitro cytotoxic activity of three components was assessed with flow cytometry and in vivo study revealed the efficacy of adoptive cell therapy. Overall, CTLs exhibited the highest cytotoxicity against spheroids of MCF7 breast adenocarcinoma, reaching in all cases higher than double the percentage of NK cells’ cytotoxicity. NK-derived EVs exhibited the lowest effect against MCF7 spheroids comparing to the two cell populations. MUC-1 specific CTLs were evaluated with adoptive cell therapy mice study and appeared to be well tolerable and moderately efficacious. More studies need to be performed with CTLs to evaluate safety and efficacy in order to assess their clinical potential, while NK cells and NK-derived EVs are promising candidates that require more experiments to enhance their cytotoxicity.