Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function.

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells. Moreover, inhibitory signals from malignant cells could be transformed into stimulatory signals by synthetic fusion molecules with extracellular inhibitory receptors fused to an intracellular stimulatory domain. Here, we designed a variety of different TIM-3-CD28 fusion proteins to turn inhibitory signals derived by TIM-3 engagement into T-cell activation through CD28. In the absence of anti-CD19 CAR, two TIM-3-CD28 fusion receptors with large parts of CD28 showed strongest responses in terms of cytokine secretion and proliferation upon stimulation with anti-CD3 antibodies compared to controls. We then combined these two novel TIM-3-CD28 fusion proteins with first- and second-generation anti-CD19 CAR T cells and found that the fusion receptor can increase proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells. These additionally armed CAR T cells showed excellent effector function. In terms of safety considerations, the fusion receptors showed exclusively increased cytokine release, when the CAR target CD19 was present. We conclude that combining checkpoint fusion proteins with anti-CD19 CARs has the potential to increase T-cell proliferation capacity with the intention to overcome inhibitory signals during the response against malignant cells.

Genome-wide off-target analyses of CRISPR/Cas9-mediated T-cell receptor engineering in primary human T cells.

OBJECTIVES: Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T-cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off-the-shelf options to emerging adoptive T-cell transfer (ACT) approaches. Targeted CRISPR/Cas9-mediated double-strand breaks in the DNA enable knockout or knock-in engineering. METHODS: Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells. RESULTS: TCRα chain and TCRß chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering. CONCLUSION: The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects.

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections.

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. T cell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive T cell transfer (ACT) approaches. T cell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific T cells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated ex vivo and preclinically engineered into the TCR locus of allogeneic third-party primary T cells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited T cells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, ex vivo isolated TCRs with clinical proven protection through ACT could be redirected into T cells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.

CLEC12A and CD33 coexpression as a preferential target for pediatric AML combinatorial immunotherapy.

Emerging immunotherapies such as chimeric antigen receptor T cells have advanced the treatment of acute lymphoblastic leukemia. In contrast, long-term control of acute myeloid leukemia (AML) cannot be achieved by single lineage-specific targeting while sparing benign hematopoiesis. In addition, heterogeneity of AML warrants combinatorial targeting, and several suitable immunotargets (HAVCR2/CD33 and HAVCR2/CLEC12A) have been identified in adult AML. However, clinical and biologic characteristics of AML differ between children and the elderly. Here, we analyzed 36 bone marrow (BM) samples of pediatric AML patients and 13 age-matched healthy donors using whole RNA sequencing of sorted CD45dim and CD34+CD38-CD45dim BM populations and flow cytometry for surface expression of putative target antigens. Pediatric AML clusters apart from healthy myeloid BM precursors in principal-component analysis. Known immunotargets of adult AML, such as IL3RA, were not overexpressed in pediatric AML compared with healthy precursors by RNA sequencing. CD33 and CLEC12A were the most upregulated immunotargets on the RNA level and showed the highest surface expression on AML detected by flow cytometry. KMT2A-mutated infant AML clusters separately by RNA sequencing and overexpresses FLT3, and hence, CD33/FLT3 cotargeting is an additional specific option for this subgroup. CLEC12A and CD33/CLEC12Adouble-positive expression was absent in CD34+CD38-CD45RA-CD90+ hematopoietic stem cells (HSCs) and nonhematopoietic tissue, while CD33 and FLT3 are expressed on HSCs. In summary, we show that expression of immunotargets in pediatric AML differs from known expression profiles in adult AML. We identify CLEC12A and CD33 as preferential generic combinatorial immunotargets in pediatric AML and CD33 and FLT3 as immunotargets specific for KMT2A-mutated infant AML.

CRISPR-Cas9-Mediated Glucocorticoid Resistance in Virus-Specific T Cells for Adoptive T Cell Therapy Posttransplantation.

Immunosuppression posttransplantation exposes patients to an increased risk for refractory viral infections as an important cause of morbidity and mortality. Protective T cell immunity can be restored by adoptive T cell transfer, but ongoing immunosuppression limits efficacy of T cell responses. In order to deliver protection against viral pathogens and allow at the same time necessary steroid therapy, we generated glucocorticoid-resistant T cells by CRISPR-Cas9-mediated knockout of the glucocorticoid receptor in primary human virus-specific T cell products. Characterization of the T cell product revealed high efficiency of glucocorticoid receptor knockout and high purity of virus-specific T cells. This tandem T cell engineering preserved protective T cell functionality, such as cytotoxicity, CD107a degranulation, proliferative capacity, and cytokine release patterns. Virus-specific T cells with glucocorticoid receptor knockout were resistant to the suppressive effect of dexamethasone treatment on lymphocyte proliferation and cytokine secretion (tumor necrosis factor alpha [TNF-α], interleukin-4 [IL-4], IL-6, and sFas). Additionally, glucocorticoid receptor knockout cells remained sensitive to cyclosporine A treatment, thereby providing a rescue approach for patients in case of safety issues. This novel approach provides a therapeutic option for the treatment of patients with viral infections after transplantation who are receiving glucocorticoid therapy.

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR.

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRß chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Leukemia-induced dysfunctional TIM-3+CD4+ bone marrow T cells increase risk of relapse in pediatric B-precursor ALL patients.

Interaction of malignancies with tissue-specific immune cells has gained interest for prognosis and intervention of emerging immunotherapies. We analyzed bone marrow T cells (bmT) as tumor-infiltrating lymphocytes in pediatric precursor-B cell acute lymphoblastic leukemia (ALL). Based on data from 100 patients, we show that ALL is associated with late-stage CD4+ phenotype and loss of early CD8+ T cells. The inhibitory exhaustion marker TIM-3 on CD4+ bmT increased relapse risk (RFS = 94.6/70.3%) confirmed by multivariate analysis. The hazard ratio of TIM-3 expression nearly reached the hazard ratio of MRD (7.1 vs. 8.0) indicating that patients with a high frequency of TIM-3+CD4+ bone marrow T cells at initial diagnosis have a 7.1-fold increased risk to develop ALL relapse. Comparison of wild type primary T cells to CRISPR/Cas9-mediated TIM-3 knockout and TIM-3 overexpression confirmed the negative effect of TIM-3 on T cell responses against ALL. TIM-3+CD4+ bmT are increased in ALL overexpressing CD200, that leads to dysfunctional antileukemic T cell responses. In conclusion, TIM-3-mediated interaction between bmT and leukemia cells is shown as a strong risk factor for relapse in pediatric B-lineage ALL. CD200/TIM-3-signaling, rather than PD-1/PD-L1, is uncovered as a mechanism of T cell dysfunction in ALL with major implication for future immunotherapies.

Low mutational load in pediatric medulloblastoma still translates into neoantigens as targets for specific T-cell immunotherapy.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity. METHODS: We identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients' human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro. RESULTS: Despite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants. CONCLUSION: Detection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.

Strategies of adoptive T -cell transfer to treat refractory viral infections post allogeneic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) can expose patients to a transient but marked immunosuppression, during which viral infections are an important cause of morbidity and mortality. Adoptive transfer of virus-specific T cells is an attractive approach to restore protective T -cell immunity in patients with refractory viral infections after allogeneic HSCT. OBJECTIVES: This narrative review summarizes clinical evidence and developments of almost 30 years of adoptive T -cell transfer. The review is based on evidence extracted from PubMed searches and the clinical and experimental work of the authors. CONTENT: Viral infections after HSCT are frequently caused by the endogenous reactivation of persistent pathogens such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (AdV). Current antiviral medication is not satisfactory and does not treat the underlying pathophysiology which is the lack of specific T -cell immunity. Adoptive transfer of virus-specific T cells could be a potentially curative, pathogen-specific, and non-toxic treatment providing long-term immunity against the virus. The isolation of virus-specific T cells from a healthy donor and infusion into a recipient is known as adoptive T -cell transfer and has been performed in many patients using different treatment protocols. Based on basic research, new isolation protocols aim at a safe and fast availability of cellular products for adoptive T -cell transfer. We summarize preclinical and clinical data on each of the main pathogens and on the technical approaches currently available to target either single antigens or even multiple pathogens. CONCLUSION: Cellular therapy is considered as one of the major recent breakthroughs in medicine. Translation of this individualized treatment into first-line clinical routine is still limited. Main hurdles are availability of the technique, limited compatibility of classical phase III designs with cellular therapy, and regulatory restrictions. Multinational efforts are required to clarify the status of cellular treatment in first-line clinical routine with the overall objective to strengthen evidence-based treatment guidelines for the treatment of refractory viral infections post HSCT.

Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia.

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100 ml peripheral blood from pediatric pre-B ALL patients was processed including CD4+/CD8+-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4+ = 50%, CD8+ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

MicroRNA methodology: advances in miRNA technologies.

There is an emerging trend in microRNA research and thus substantial progress in microRNA technologies. In this chapter we provide insights into the main microRNA specific methodologies and critical steps of microRNA expression profiling, target gene identification, and functional confirmation of microRNA effects up to in vivo application of antagomirs.