Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a significant bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to cGMP compliant manufacturing and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.

IMMU-23. NEOANTIGEN-DIRECTED CELLULAR THERAPY IN A PRECLINICAL MOUSE MODEL OF MALIGNANT GLIOMA

Adoptive cellular therapy in the form of CAR T cells or TCR engineered T cells has emerged as a novel approach in the treatment of both solid and hematologic malignancies. Neoantigens generated by tumor somatic mutations represent potentially attractive therapeutic targets in this context owing to their tumor-specific expression and circumvention of immunological tolerance. However, existing cell therapy systems generally target self-proteins or virally overexpressed antigens that fail to recapitulate the features of endogenous tumor neoantigens. Thus, there exists a need for a model in which tumor-specific neoantigens can be targeted via adoptive cellular therapy. Prior work from our lab identified the Imp3D81N mutation (mImp3) within GL261 as a neoantigen recognized by CD8 T cells in both intracranial tumors and draining cervical lymph nodes. To generate a system for targeting this neoantigen, we isolated and cloned mImp3-specific TCRs through a single-cell sort followed by a nested multiplexed PCR reaction. The specificity and functionality of these isolated TCRs was determined through introduction into a T cell hybridoma, identifying a top candidate based upon a high degree of cytokine production and specificity for the mutant epitope. A TCR transgenic mouse was then generated in which more than 90% of all T cells were CD8 T cells bearing this mImp3-specific TCR. T cells isolated from this mouse display specificity for the mImp3 peptide and display in vitro reactivity to GL261 and other cell lines in a mImp3-dependent manner. Therefore, this model represents the first TCR transgenic targeting a brain tumor neoantigen, opening the door for further investigation into cell therapy against this class of antigens.

TAMI-78. STEM CELL IMMUNOTHERAPY MODULATES IMMUNOREGULATORY PATHWAYS IN THE TUMOR MICROENVIRONMENT

INTRODUCTION A major obstacle in efficacious therapeutics against high-grade gliomas has been an inability to overcome powerful regulatory mechanisms within the TME that hamper immune activation. Fortunately, immunotherapy has enabled the enhancement of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition. We found that co-transfer of hematopoietic stem cells (HSCs) with either adoptive immunotherapy or PD-1 blockade significantly improves therapeutic outcomes by manipulating the cellular components that make up the TME in high-grade gliomas. HSC co-transfer with immunotherapy leads to increased in situ downregulation of multiple immune regulatory pathways, activation of tumor-reactive T cells, and significant reduction in the frequency of MDCs and TAMs within the TME. METHODS C57BL/6 mice with orthotopic KR158B-gliomas received either adoptive immunotherapy using activated tumor-reactive T cells or αPD-1, either with or without HSC co-transfer. After treatment of late-stage tumors, brains were sectioned and digital spatial profiling (NanoString GeoMx) of the TME was conducted in situ. Briefly, tumor-bearing brain sections were stained for nuclei, CD3, CD45, and GFP (HSC-derived cells); regions of interest (ROIs) containing 200 nuclei were selected and processed for whole genome sequencing. Areas rich in immune cells within the TME were chosen as ROIs and compared between groups. Results were corroborated with flowcytometry and PCR. RESULTS Mice that received HSCs with either adoptive cellular therapy or αPD-1 had reductions in expression of multiple regulatory markers in the TME including iNOS, TGFβ, and PD-L1. This was accompanied by reductions in the frequencies of MDSCs and TAMs. An increased relative abundance of activated CD8+ T cells within the TME was also observed. Interestingly, we found that HSC-derived cells provided rich amounts of dendritic cells at the TME when co-transferred with immunotherapy. Host-derived myeloid cells were significantly displaced from the TME in mice receiving HSC plus adoptive cellular therapy or αPD-1.

Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Advances in Adoptive Cellular Therapy (ACT)

Adoptive T cell therapy (ACT) is getting acknowledged as the Advanced Therapy Medicinal Products (ATMPs) in many countries and it has evolved as one of the newest regimens to treat cancer. Developed gradually by the basic understanding of cells, involved in innate and adaptive immunity, ACT has emerged as one of the successful immunotherapies in recent times. It broadly includes various cell types such as stem cells, T cells, dendritic cells and Natural Killer cells. By the applications of genetic engineering and advanced cell culture techniques, these cells from patients’ blood, can be manipulated to train them for better efficacy against specific tumor cells. However, only some cells’ subsets have shown promising regression for certain cancer cells types. To understand the reason behind this, technical knowledge about the tumor antigens presentation, tumor microenvironment (TME), hosts’ immune responses and possible issues in the manufacturing of adoptive cellular material for infusion in patients are being explored further. This chapter brings together development of immune cells from basic research to clinical use, newer approaches which have been taken to address the resistance of ACT and future promises of this therapy.

The Role of Immunotherapy to Overcome Resistance in Viral-Associated Head and Neck Cancer

A subset of head and neck cancers arising in the oropharynx and the nasopharynx are associated with human papillomavirus or Epstein–Barr virus. Unfortunately, limited treatment options exist once patients develop recurrent or metastatic disease in these cancers. Interest has risen in utilizing novel strategies including combination immune checkpoint inhibitors, vaccines, and adoptive cellular therapy, to improve treatment response and outcomes. Several ongoing studies are investigating the potential to overcome resistance to standard of care chemoradiation therapy with monotherapy or combination immunotherapy strategies in these viral-associated head and neck cancers.

Adoptive cellular therapy in solid tumor malignancies: review of the literature and challenges ahead

While immune checkpoint inhibitors (ICIs) have ushered in major changes in standards of care for many solid tumor malignancies, primary and acquired resistance is common. Insufficient antitumor T cells, inadequate function of these cells, and impaired formation of memory T cells all contribute to resistance mechanisms to ICI. Adoptive cellular therapy (ACT) is a form of immunotherapy that is rapidly growing in clinical investigation and has the potential to overcome these limitations by its ability to augment the number, specificity, and reactivity of T cells against tumor tissue. ACT has revolutionized the treatment of hematologic malignancies, though the use of ACT in solid tumor malignancies is still in its early stages. There are currently three major modalities of ACT: tumor-infiltrating lymphocytes (TILs), genetically engineered T-cell receptors (TCRs), and chimeric antigen receptor (CAR) T cells. TIL therapy involves expansion of a heterogeneous population of endogenous T cells found in a harvested tumor, while TCRs and CAR T cells involve expansion of a genetically engineered T-cell directed toward specific antigen targets. In this review, we explore the potential of ACT as a treatment modality against solid tumors, discuss their advantages and limitations against solid tumor malignancies, discuss the promising therapies under active investigation, and examine future directions for this rapidly growing field.

Perspectives of tumor-infiltrating lymphocyte treatment in solid tumors

Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cellular therapy by harvesting infiltrated lymphocytes from tumors, culturing and amplifying them in vitro and then infusing back to treat patients. Its diverse TCR clonality, superior tumor-homing ability, and low off-target toxicity endow TIL therapy unique advantages in treating solid tumors compared with other adoptive cellular therapies. Nevertheless, the successful application of TIL therapy currently is still limited to several types of tumors. Herein in this review, we summarize the fundamental work in the field of TIL therapy and the current landscape and advances of TIL clinical trials worldwide. Moreover, the limitations of the current TIL regimen have been discussed and the opportunities and challenges in the development of next-generation TIL are highlighted. Finally, the future directions of TIL therapy towards a broader clinical application have been proposed.

Cancer-Associated Glycosphingolipids as Tumor Markers and Targets for Cancer Immunotherapy

Aberrant expression of glycosphingolipids is a hallmark of cancer cells and is associated with their malignant properties. Disialylated gangliosides GD2 and GD3 are considered as markers of neuroectoderm origin in tumors, whereas fucosyl-GM1 is expressed in very few normal tissues but overexpressed in a variety of cancers, especially in small cell lung carcinoma. These gangliosides are absent in most normal adult tissues, making them targets of interest in immuno-oncology. Passive and active immunotherapy strategies have been developed, and have shown promising results in clinical trials. In this review, we summarized the current knowledge on GD2, GD3, and fucosyl-GM1 expression in health and cancer, their biosynthesis pathways in the Golgi apparatus, and their biological roles. We described how their overexpression can affect intracellular signaling pathways, increasing the malignant phenotypes of cancer cells, including their metastatic potential and invasiveness. Finally, the different strategies used to target these tumor-associated gangliosides for immunotherapy were discussed, including the use and development of monoclonal antibodies, vaccines, immune system modulators, and immune effector-cell therapy, with a special focus on adoptive cellular therapy with T cells engineered to express chimeric antigen receptors.