CIITA Research Articles

Differential modulation of mutant CALR and JAK2 V617F-driven oncogenesis by HLA genotype in myeloproliferative neoplasms.

It has been demonstrated previously that human leukocyte antigen class I (HLA-I) and class II (HLA-II) alleles may modulate JAK2 V617F and CALR mutation (CALRmut)-associated oncogenesis in myeloproliferative neoplasms (MPNs). However, the role of immunogenetic factors in MPNs remains underexplored. We aimed to investigate the potential involvement of HLA genes in CALRmut+ MPNs. High-resolution genotyping of HLA-I and -II loci was conducted in 42 CALRmut+ and 158 JAK2 V617F+ MPN patients and 1,083 healthy controls. A global analysis of the diversity of HLA-I genotypes revealed no significant differences between CALRmut+ patients and controls. However, one HLA-I allele (C*06:02) showed an inverse correlation with presence of CALR mutation. A meta-analysis across independent cohorts and healthy individuals from the 1000 Genomes Project confirmed an inverse correlation between the presentation capabilities of the HLA-I loci for JAK2 V617F and CALRmut-derived peptides in both patients and healthy individuals. scRNA-Seq analysis revealed low expression of TAP1 and CIITA genes in CALRmut+ hematopoietic stem and progenitor cells. In conclusion, the HLA-I genotype differentially restricts JAK2 V617F and CALRmut-driven oncogenesis potentially explaining the mutual exclusivity of the two mutations and differences in their presentation latency. These findings have practical implications for the development of neoantigen-based vaccines in MPNs.

Elimination of GGTA1, CMAH, β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions.

Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles. Thus, we generate a 4-gene (GGTA1, CMAH, β4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously. We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment. 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.

MHC Class II Deficiency: Clinical, Immunological, and Genetic Insights in a Large Multicenter Cohort

BackgroundMajor Histocompatibility Complex Class II (MHC-II) deficiency, a combined immunodeficiency, results from loss of Human Leukocyte Antigen class II expression on antigen-presenting cells. Currently, hematopoietic stem cell transplantation (HSCT) stands as the sole curative approach, though factors influencing patient outcomes remain insufficiently explored. ObjectiveOur aim was to elucidate the clinical, immunological, and genetic profiles associated with MHC-II deficiency and identify prognostic indicators that affect survival rates. MethodsIn this multicenter retrospective analysis, we gathered data from 35 patients diagnosed with MHC-II deficiency across 12 centers in Turkey. We recorded infection histories, gene mutations, immune cell subsets, and surface MHC-II expression on blood cells. We conducted survival analyses to evaluate the impact of various factors on patient outcomes. ResultsPredominant symptoms observed were pneumonia (n=29, 82.9%), persistent diarrhea (n=26, 74.3%), and severe infections (n=26, 74.3%). The RFXANK gene mutation (n=9) was the most frequent, followed by mutations in RFX5 (n=8), CIITA (n=4), and RFXAP (n=2) genes. Patients with RFXANK mutations presented with later onset and diagnosis compared to those with RFX5 mutations (p=0.0008 and p=0.0006, respectively), alongside a more significant diagnostic delay (p=0.020). A notable founder effect was observed in 5 patients with a specific RFX5 mutation (c.616G>C). The overall survival rate for patients was 28.6% (n=10), showing a significantly higher proportion in individuals with HSCT (n=8, 80%). Early demise (p=0.006) and higher CD8+ T-cell counts were observed in patients with the RFX5 mutations compared to RFXANK-mutant patients (p=0.006 and p=0.009, respectively). ConclusionThe study delineates the genetic and clinical panorama of MHC-II deficiency, emphasizing the prevalence of specific gene mutations such as RFXANK and RFX5. These insights facilitate early diagnosis and prognosis refinement, significantly contributing to the management of MHC-II deficiency.

Association of CIITA (rs8048002) and CLEC2D (rs2114870) gene variants and type 1 diabetes mellitus.

Type I diabetes mellitus (T1DM) is a significant health challenge, especially for children, owing to its chronic autoimmune nature. Although the exact etiology of T1DM remains elusive, the interplay of genetic predisposition, immune responses, and environmental factors are postulated. Genetic factors control immune reactivity against β-cells. Given the pivotal roles of CIITA and CLEC2D genes in modulating a variety of immune pathologies, we hypothesized that genetic variations in CIITA and CLEC2D genes may impact T1DM disease predisposition. This study was designed to explore the association between gene polymorphisms in CIITA (rs8048002) and CLEC2D (rs2114870) and type 1 diabetes (T1DM), with a focus on analyzing the functional consequence of those gene variants. The study enlisted 178 healthy controls and 148 individuals with type 1 diabetes (T1DM) from Suez Canal University Hospital. Genotyping for CIITA and CLEC2D was done using allelic-discrimination polymerase chain reaction (PCR). Levels of glycated hemoglobin (HbA1c) and lipid profiles were determined through automated analyzer, while fasting blood glucose and insulin serum levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. RegulomeDB was used to examine the regulatory functions of CIITA (rs8048002) and CLEC2D (rs2114870) gene variants. Analysis of the genotype distribution of the CIITA rs8048002 polymorphism revealed a significantly higher prevalence of the rare C allele in T1DM patients compared to the control group (OR = 1.77; P = 0.001). Both the CIITA rs8048002 heterozygote TC genotype (OR = 1.93; P = 0.005) and the rare homozygote CC genotype (OR = 3.62; P = 0.006) were significantly more frequent in children with T1DM when compared to the control group. Conversely, the rare A allele of CLEC2D rs2114870 was found to be significantly less frequent in T1DM children relative to the control group (OR = 0.58; P = 0.002). The heterozygote GA genotype (OR = 0.61; P = 0.033) and the rare homozygote AA genotype (OR = 0.25; P = 0.004) were also significantly less frequent in T1DM patients compared to the control group. Both CIITA (rs8048002) and CLEC2D (rs2114870) gene variants were predicted to have regulatory functions, indicated by a RegulomeDB score of (1f) for each. The rare C allele of CIITA rs8048002 genetic variant was associated with an increased risk of developing T1DM, while the less common A allele of CLEC2D rs2114870 was associated with a reduced risk of T1DM. The online version contains supplementary material available at 10.1007/s40200-024-01402-w.

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

Novel vaccination strategies based on optimal stimulation of CD4+ T helper cells for the treatment of oral squamous cell carcinoma.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant tumor of the oral cavity. Despite recent advances in the field of oral cancer therapy, including the introduction of immunotherapeutic approaches, the 5-year survival rate remains steadily assessed around 50%. Thus, there is an urgent need for new therapeutic strategies. After the characterization of the immune phenotype of three human OSCC cell lines (CAL-27, SCC-25, and SCC-4) and one mouse OSCC cell line (MOC2) showing their similarities to resected patient tumors, we explored for the first time an experimental preclinical model of therapeutic vaccination with mouse OSCC MOC2 cell line stably expressing MHC class II antigens after CIITA gene transfection (MOC2-CIITA). Mice injected with MOC2-CIITA reject or strongly retard tumor growth; more importantly, vaccinated animals that fully reject MOC2-CIITA tumors display anti-tumor immunological memory protective against challenge with parental MOC2 tumor cells. Further experiments of adoptive cell transfer or in vivo cell depletion show that both CD4+ and CD8+ T lymphocytes prove fundamental in tumor rejection. This unprecedented approach for oral cancer opens the way for possible future translation of novel immunotherapeutic strategies to the human setting for the treatment of this tumor.

Differential expression of the inflammatory ciita gene may be accompanied by altered bone properties in intact sex steroid-deficient female rats

ObjectiveThe class II transactivator (CIITA), encoded by the CIITA gene, controls expression of immune response regulators, which affect bone homeostasis. Previously, we investigated a functional CIITA polymorphism in elderly women. Women carrying the allele associated with lower CIITA levels displayed higher bone mineral density (BMD), but also higher bone loss. The present exploratory study in a rat model sought to investigate effects of differential expression of Ciita on bone structural integrity and strength. Two strains DA (normal-to-high expression) and DA.VRA4 (lower expression) underwent ovariectomy (OVX) or sham-surgery at ~ 14-weeks of age (DA OVX n = 8, sham n = 4; DA.VRA4 OVX n = 10, sham n = 2). After 16-weeks, femoral BMD and bone mineral content (BMC) were measured and morphometry and biomechanical testing performed.ResultsIn DA.VRA4 rats, BMD/BMC, cross-sectional area and biomechanical properties were lower. Ciita expression was accompanied by OVX-induced changes to cross-sectional area and femoral shaft strength; DA rats had lower maximum load-to-fracture. Thus, while lower Ciita expression associated with lower bone mass, OVX induced changes to structural and mechanical bone properties were less pronounced.ConclusionThe data tentatively suggests association between Ciita expression and structural and mechanical bone properties, and a possible role in bone changes resulting from estrogen deficiency.

Identification of Healthy Donor Phenotypic and Functional Signatures That Predict Allogeneic, Hypoimmune CD19-Directed CAR T Cell Potency

Allogeneic, hypoimmune (HIP) CAR T cells may provide off-the-shelf CAR T cells that allow for expanded access for patients, overcome manufacturing failures, and provide an efficacious alternative to CAR T cells manufactured from heavily pre-treated T cells. However, donor T cells are the primary source of variability during production of allogeneic HIP CAR T cells, and may influence clinical efficacy. Previously, we reported a series of T cell functional assays that differentiate T cell potency as a means to select high-quality CAR T cell donors for clinical trials. Here, we explore donor phenotypic and functional signatures that are associated with desirable pre-clinical attributes for the generation of allogeneic HIP CD19-directed CAR T cells. Human T cells from healthy donors were obtained by leukapheresis and used to generate allogeneic HIP CD19-directed CAR T cells by disrupting B2M, CIITA, and TRAC genes using Cas12b nuclease, and lentiviral transduction to overexpress CD47 and to express the CD19 CAR.Phenotype profiling (PBMC subsets, memory subsets, viability, exhaustion, and activation markers) was assessed on apheresis, CD4 or CD8 enriched subsets and post-CAR manufacturing. Generated CAR T cells were evaluated in vitro by repetitive tumor challenge (3x) performed over a 14-day period using the IncuCyte platform. Bulk cytokine analysis was measured by MesoScale Discovery (MSD). In vivo efficacy was evaluated using a systemic NALM-6 challenge in immune-deficient NSG mice at HIP CD19 CAR T cell doses of 5e5 and 5e6 cells per animal. Gene expression profiling was assessed on pre-production, post-CAR T cell manufacturing, and post-serial tumor challenge CD4+ and CD8+ T cells using the Nanostring nCounter CAR T panel. Functional and phenotypic attributes were used for weighted donor performance scoring. To do this, cell functional assays were defined to produce single, quantitative indices to enable the classification of CAR T cells based on functional performance. We assessed differences in CAR T cell performance and rank scored donor performance. Associations between donor attributes, phenotype/ functional T cell attributes and ranked donor performance were evaluated using Spearman's correlations. Correlative comparison of phenotype and donor attributes in relation to weighted donor performance scores indicated that cellular subtypes at leukapheresis correlate with CAR T performance. Gene expression profiling shows that outperforming donors upregulate expression of mucosal associated invariant T (MAIT) cell canonical TCRb chain TRBV28. Outperforming donors have increased expression of Tc1/Th1 and Tc17/Th17 associated cytokines: IFNg, Il-17A, Il-17F, Il23, Il22, and have reduced expression of Tc2/Th2 associated cytokine Il5. Outperforming donors also showed reduced expression of MHC class II of alpha and beta heterodimers (HLA-DQA1 and HLA-DQB1). The information collected across phenotypic and functional comparisons will be compared with clinical performance.

Development of a Novel, Allogeneic GPRC5D-Directed CAR for Treatment of Multiple Myeloma Patients

Despite the success of BCMA-targeted therapy in advanced multiple myeloma, the disease is still considered incurable. Relapse following BCMA-directed cellular and antibody-based therapies is commonly associated with decreased surface expression of BCMA on malignant plasma cells. The G protein-coupled receptor, class C group 5 member D (GPRC5D) represents an additional target whose expression is limited to bone marrow and hair follicles, with selectively increased expression on malignant plasma cells. Thus far, the use of GPRC5D-targeted CAR T cells has provided a strong clinical benefit in multiple myeloma with limited on-target, off-tumor effects. To this end, we have validated novel, fully human GPRC5D-specific CARs for use with our hypoimmune (HIP), allogeneic CAR T cell platform. This will allow rapid delivery of allogeneic GPRC5D CAR T cells from healthy donors that simultaneously circumvent “non-self” recognition through TCR, B2M and CIITA gene disruption and promote persistence through innate immune evasion by leveraging CD47 overexpression. For early selection of candidate GPRC5D binders, T cells from healthy donors were transduced with VSV-G pseudotyped lentivirus containing second-generation, fully-human GPRC5D CARs. A clinically validated GPRC5D CAR was produced in similar fashion and used as benchmark control for initial characterization experiments. Transduction efficiency of GPRC5D CARs was analyzed via flow cytometry. GPRC5D CAR efficacy was analyzed in vitro via 24-hour luciferase or long-term Incucyte cytotoxicity assays against RPMI-8226 and MM.1S multiple myeloma target cells. Systemic MM.1S in vivo tumor models were performed in NSG mice to further assess efficacy of lead CAR candidates. Here we show our candidate GPRC5D CARs demonstrate in vitro cytotoxicity and effector cytokine production that is comparable to clinically validated benchmark controls. In 5-day Incucyte cytotoxicity assays, GPRC5D CAR T cells expand up to 10-fold and reduce tumor cell counts at starting E:T ratios of 1:8. Furthermore, in MM.1S in vivo tumor challenge models, these GPRC5D CARs significantly reduce flux and prolonged survival relative to tumor only controls with tumor clearance that was in line with clinical benchmark controls. Using ex vivo VSV-G production, our GPRC5D CAR T cells control multiple myeloma tumor cells both in vitro and in vivo, demonstrating efficacy that is on par with GPRC5D clinical benchmark CAR T cells. Studies to further define the efficacy of HIP-modified GPRC5D CAR T cells are ongoing. Moving forward, further interrogating GPRC5D CAR activity against multiple myeloma tumor cells with varying levels of target antigen expression will be critical to understanding the benefit of deploying GPRC5D CAR T cells within the multiple myeloma clinical landscape.

Hypoimmune, Allogeneic CD22-Directed CAR T Cells That Evade Innate and Adaptive Immune Rejection for the Treatment of Large B Cell Lymphoma Patients That Are Relapsed/Refractory to CD19-Directed CAR T Cell Therapy

Despite the success of CD19CAR therapy against large B-cell lymphoma, up to 60% of CD19-directed CAR T cell treated patients relapse after, or are refractory to, treatment. Of the relapsed/refractory population a significant subset of patients, around 30%, have CD19-negative tumor and a short median survival prognosis - necessitating the implementation of rapid intervention strategies (Atilla, Transl Oncol 2022; Spiegel, Blood 2021). Sana is developing “off-the shelf” allogeneic CD22-directed CAR T cells that may enable rapid intervention and induce clinically meaningful activity for these CD19 CAR T cell relapsed/refractory patients. Here we describe methods to create hypoimmune (HIP), allogeneic CD22-directed CAR T cells using a clinically validated CD22CAR that: 1) support adaptive immune cell evasion via B2M and CIITA gene disruption, 2) promote persistence and innate immune cell evasion by overexpression of CD47, and 3) mitigate GvHD via TRAC gene disruption, (Hu, Nat Commun 2023). HIP, allogeneic CD22-directed CAR T cells (CD22 HIP CAR T cells) were derived from healthy donor CD8 + and CD4 + T cells that were gene-edited to disrupt the B2M, CIITA, and TRAC (TCR) genes and transduced to overexpress CD47 and express a second-generation, clinically validated CD22-directed CAR. Gene-edited Mock T cells and unedited, allogeneic CD47-CD22CAR T cells were generated as donor-matched controls. The CD22 HIP CAR T cells were evaluated for immune evasive activity using a previously described in vitro xCelligence cytotoxicity assay (Hu, Nat Commun 2023). The T cells were further evaluated in vitro for dose-dependent short-term and long-term cytolytic activity analysis via luciferase or IncuCyte assay, respectively. Correlate cytokine responses were performed by meso-scale discovery analysis and T cell expansion by IncuCyte classification. Systemic NALM and RAJI parental or CD19 antigen knockout tumor models (IV injection) were executed in a NSG or NSG-SGM3 mouse background. xCelligence analysis confirmed that CD22 HIP CAR T cells were protected from NK and macrophage recognition; whereas Mock control T cells that lack HLA expression succumbed to NK cell and macrophage killing within 5-hours of the 50-hour assay due to the absence of CD47 protection. Furthermore, CD22 HIP CAR T cells elicited cytotoxicity and robust cytokine responses (e.g. IFNγ, IL-2) in a dose-dependent manner comparable to allogeneic CD47-CD22CAR T cells when cultured with CD22 +/CD19 + or CD22 +/CD19 - NALM, RAJI, or K562 targets. The CD22 HIP CAR T cell and allogeneic CD47-CD22CAR T cell control produced low level IFNγ (<400pg/mL) and Granzyme B (<100pg/mL) when cultured alone or with CD22 - tumor targets. These basal levels of IFNγ and Granzyme B did not have long-term cytolytic effects on CD22 - tumor cells, nor was any significant level of IL-2 produced by CD22 HIP CAR T cells cultured alone, or in the presence of CD22-negative tumor targets. Indeed, CD22 HIP CAR T cell proliferation was dependent upon CD22 + tumor co-culture. NALM and RAJI CD19 knockout in vivo tumor models demonstrated that CD22 HIP CAR T cells significantly reduced flux (Day 21 AUC vs Mock: NALM and RAJI (p=0.0001)) and prolonged survival (Median vs Mock: p<0.004). Furthermore, CD22 HIP CAR T cells elicited comparable, dose-dependent antitumor activity relative to allogeneic CD47-CD22CAR T cell controls in a systemic NALM tumor model. Our data provide evidence that CD22 HIP CAR T cells elicit robust antitumor activity in an antigen and dose-dependent manner. Furthermore, these studies demonstrate that CD22 HIP CAR T cells are agnostic to CD19 expression and selectively elicit pharmacologic activity against, or in response to, CD22 + tumor. Results also demonstrate that the HIP approach to generate allogeneic CAR T cells that evade innate and adaptive immune rejection can be expanded as a platform to generate CAR T cells against target antigens alternative to CD19. Collectively, the data suggest that CD22 HIP CAR T cells display a combination of antigen-specific pharmacologic activity and immune evasion that support their progression into human clinical studies for the treatment of CD19 CAR T cell-refractory patients.

CRISPR-Cas9-mediated disruption of B2M and CIITA genes eliminates HLA class I and II expression in human induced pluripotent stem cells (MUSIi001-A-2)

Cell-based therapy offers great promise for treating degenerative diseases. Although autologous cell-based therapy is ideal, it may be impractical due to the high manufacturing cost and long production time. Allogeneic cell-based therapy offers a more cost-effective alternative; however, the risk of graft rejection is a major concern. Here, we generated HLA class-I and -II null iPSC line by knocking out CIITA gene in the B2M-knockout MUSIi001-A-1 cell line using CRISPR/Cas9 system. The MUSIi001-A-2 line provides a valuable model for studying immunological responses against allogeneic T cells and serves as a prototype for developing specific cell types for future cell-based therapy.

Genome editing HLA alleles for a pilot immunocompatible hESC line in a Chinese hESC bank for cell therapies

Robust allogeneic immune reactions after transplantation impede the translational pace of human embryonic stem cells (hESCs)-based therapies. Selective genetic editing of human leucocyte antigen (HLA) molecules has been proposed to generate hESCs with immunocompatibility, which, however, has not been specifically designed for the Chinese population yet. Herein, we explored the possibility of customizing immunocompatible hESCs based on Chinese HLA typing characteristics. We generated an immunocompatible hESC line by disrupting HLA-B, HLA-C, and CIITA genes while retaining HLA-A*11:01 (HLA-A*11:01-retained, HLA-A11R ), which covers ~21% of the Chinese population. The immunocompatibility of HLA-A11R hESCs was verified by in vitro co-culture and confirmed in humanized mice with established human immunity. Moreover, we precisely knocked an inducible caspase-9 suicide cassette into HLA-A11R hESCs (iC9-HLA-A11R ) to promote safety. Compared with wide-type hESCs, HLA-A11R hESC-derived endothelial cells elicited much weaker immune responses to human HLA-A11+ T cells, while maintaining HLA-I molecule-mediated inhibitory signals to natural killer (NK) cells. Additionally, iC9-HLA-A11R hESCs could be induced to undergo apoptosis efficiently by AP1903. Both cell lines displayed genomic integrity and low risks of off-target effects. In conclusion,we customized a pilot immunocompatible hESC cell line based on Chinese HLA typing characteristics with safety insurance. This approach provides a basis for establishment of a universal HLA-AR bank of hESCs covering broad populations worldwide and may speed up the clinical application of hESC-based therapies.

Genetic haplotypes associated with immune response to Leishmania infantum infection in dogs.

Leishmaniasis is a zoonotic parasitic disease, and the main reservoir of the parasite is the dog, although recent years have seen an increase in other mammalian species. In the Mediterranean region, where it is an endemic disease, it is caused by the species Leishmania infantum. The Ibizan hound, an autochthonous breed of this region, appears to have a genetic resistance to parasitic infection, whereas other canine breeds, such as the Boxer, are susceptible to infection. These differences are related to the differentiated activation of the immune response, with the Ibizan hound activating the Th1 immune response, whereas the Boxer breed triggers the Th2 immune response. Cytokine levels and genomic haplotypes of several genes involved in the immune response were analysed in twenty-eight Ibizan hound (resistant canine breed model) and twenty-four Boxer (susceptible canine breed) without clinical signs in the Mediterranean region. Cytokine levels were analysed by ELISA commercial kits and haplotypes were studied using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The results show 126 haplotypes associated with differential immune response in dogs. Specifically, haplotypes in IL12RB1, IL6R, CIITA, THEMIS, NOXA1, HEY2, RAB38, SLC35D2, SLC28A3, RASEF and DAPK1 genes are associated with serum levels of IFN-γ, IL-2, IL-8, and IL-18. These results suggest that the resistance or susceptibility to Leishmania infantum infection could be a consequence of haplotypes in several genes related to immune response. Future studies are needed to elucidate the relationship of these haplotypes with immune response and gene expression regulation.

Electrostatic Complementarities of Glioblastoma-Resident T-Cell Receptors and Cancer Testis Antigens Linked to Poor Outcomes and High Levels of Sphingosine Kinase-2 Expression.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite a growing understanding of glioblastoma pathology, the prognosis remains poor. In this study, we used a previously extensively benchmarked algorithm to retrieve immune receptor (IR) recombination reads from GBM exome files available from the cancer genome atlas. The T-cell receptor complementarity determining region-3 (CDR3) amino acid sequences that represent the IR recombination reads were assessed and used for the generation of chemical complementarity scores (CSs) that represent potential binding interactions with cancer testis antigens (CTAs), which is an approach particularly suited to a big data setting. The electrostatic CSs representing the TRA and TRB CDR3s and the CTAs, SPAG9, GAGE12E, and GAGE12F, indicated that an increased electrostatic CS was associated with worse disease-free survival (DFS). We also assessed the RNA expression of immune marker genes, which indicated that a high-level expression of SPHK2 and CIITA genes also correlated with high CSs and worse DFS. Furthermore, apoptosis-related gene expression was revealed to be lower when the TCR CDR3-CTA electrostatic CSs were high. Adaptive IR recombination reads from exome files have the potential to aid in GBM prognoses and may provide opportunities to detect unproductive immune responses.

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Abstract 4091: Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge

Abstract Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, off-the-shelf availability, and lack of T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced with this approach. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are disrupted and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118).Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR (unmodified).For 3 months persistence studies, allogeneic SGM3 humanized mice were injected with 1 × 106 Luc+ Nalm6 cells and received 7 × 106 control CD19 CAR T cells or HIP CD19 CAR T cells.In the mice treated with either unmodified CD19 CAR T cells and HIP CD19 CAR T cells, tumor control was initially rapidly achieved. However, unmodified CD19 CAR T cells were eventually rejected by the host and the loss of these cells resulted in re-growth of tumor. By contrast, in HIP CD19 CAR T injected mice, tumor control was maintained throughout the study, including following a rechallenge at day 83 with NALM6 cells without further administration of HIP CD19 CAR T cell. Flow cytometry at endpoint from bone marrow and spleen confirmed persistence of HIP CD19 CAR T cells.These findings show that HIP CD19 CAR T cells are immune evasive in allogeneic recipients and data suggest that HIP CD19 CAR T cells are able to persist and maintain efficacy without immunosuppression. Citation Format: Xiaomeng Hu, Pascal Beauchesne, Karl Manner, Corie Gattis, Priscilla Ngo, Rowena De Jesus, Ramya Ankala, Chi Young, Frank Wells, Lindong Weng, Kathy White, William E. Dowdle, Aaron Foster, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge. [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 4091.

The relevance of HLA class II genes in JAK2 V617F-positive myeloproliferative neoplasms

In the present study we analyzed the relevance of HLA class II in JAK2 V617F-positive (JAK2 V617F+) myeloproliferative neoplasms (MPNs) focusing on genotype diversity, associations with specific alleles and haplotypes and the level of gene expression. One hundred and thirty-nine JAK2 V617F+ MPN patients and 1083 healthy controls, typed by Next generation sequencing (NGS) were included in the study. Multivariate generalized linear models with age as a covariate were applied for analysis of HLA-II allele and haplotype associations. Publicly available gene expression datasets were used to analyze HLA-II pathway genes expression in CD34+ stem cells (SCs) from MPN patients and healthy controls. We did not observe differences in HLA evolutionary divergence (HED) between JAK2 V617F+ MPNs and healthy controls. Two alleles: HLA-DPB1*03:01, DQB1*04:02 and 4 haplotypes: DPB1*02:01-DQA1*05:05-DQB1*03:01-DRB1*11:01, DPB1*04:02-DQA1*05:05-DQB1*03:01-DRB1*11:03, DPB1*02:01-DQA1*01:04-DQB1*05:03-DRB1*14:04, and DPB1*04:01-DQA1*03:01-DQB1*03:02-DRB1*04:01 had significantly lower frequency in MPN patients compared to controls. Additionally, we observed HLA-II alleles and haplotypes with statistically higher frequencies in JAK2 V617F+ patients. Differential gene expression analysis showed down-regulation of HLA-DRB1, -DRA, -DMA, -DMB, -DOA,-DRB4, CIITA, and CD74 genes in JAK2 V617F+ MPN CD34+ SCs as compared to normal CD34 + SCs. In conclusion, this study provides evidence for the pleiotropic effects of HLA-II genes in JAK2 V617F-driven MPNs.

Efficient and Specific Multi-Locus Editing of Allogeneic CAR T Cells for Hypoimmunity during Large Scale Manufacture Using Cas12b

Introduction: Allogeneic CAR T cell therapies may offer key advantages over autologous therapies including choice of starting cells, better manufacturing control, and lower cost. However, the manufacture of these cells imposes a high demand on genome engineering specificity and efficiency, given the dual needs for product safety and yield. This challenge is amplified for newer generations of allogeneic CAR T cell designs that may offer improved performance but require more edits to produce, which may increase the potential of unintended genome modification and insufficient yield of fully engineered product. Here we have tested a novel editing platform, Cas12b, for the ability to mediate efficient and specific disruption of three genes (B2M, CIITA, and TRAC) during large scale manufacturing of CAR T cells with immune evasive properties and abrogated graft-vs-host response. We have characterized the resulting cell product for on-target modification efficiency, type and levels of unintended genome modification, and potency for tumor clearance in a Nalm6 humanized mouse model, with the aim of demonstrating suitability for translation to human studies. Methods: Our manufacturing strategy to generate hypoimmune CAR T cells introduces five genetic modifications, using Cas12b for disruption of B2M, CIITA, and TCR expression, and lentiviral transduction to overexpress CD47 and introduce a CD19 CAR. T cells obtained from healthy donors were first transduced with lentiviral vector and then edited simultaneously to disrupt the B2M, CIITA and TRAC genes via electroporation of Cas12b mRNA and appropriate guides. Edited and purified cells were characterized via flow cytometry for efficiency of each genetic modification. Cells were further characterized for knockout levels via next generation sequencing (NGS) as well as vector copy number per diploid genome (VCN). Cells were characterized for viability, proliferation, and efficacy in a Nalm6 mouse model of tumor clearance. An initial assessment of chromosomal translocations was performed by G banding karyotype analysis. To assess editing specificity, each Cas12b guide was evaluated by Cas-OFFinder and an unbiased oligonucleotide duplex capture assay in T cells from multiple donors under conditions of high on-target modification. Candidate off-target sites were then examined for evidence of cleavage via PCR amplification from the manufactured cell product followed by NGS. Results: Our manufacturing process yielded clinically relevant scales of cell product having > 85% B2M knockout, > 90% CIITA knockout, and < 1% of residual TCR expressing cells. VCN levels were < 5. Cell viability and proliferation were minimally impacted by the gene editing process. Cells exhibited robust anti-tumor properties with doses of 7 x 106 hypoimmune CD19 CAR T cells (per animal) providing efficient killing in a Nalm6 mouse model. Initial karyotype analysis identified low levels of translocation between on-target sites with overall translocation levels otherwise not significantly affected compared to unedited cells. Our characterization of Cas12b in edited T cells across multiple donors revealed high specificity, consistent with previous reports (Nat Commun 10 212), with studies to date having yielded no evidence of cleavage at any candidate off-target site. Summary: Cas12b-mediated multi-locus editing of allogeneic CAR T cells during large scale manufacture resulted in a high yield product with potent tumor clearance capabilities. Initial analysis of editing specificity across multiple donors and engineering runs identified no off-target sites, and early karyotype assessments indicated an overall level of chromosomal translocations consistent with prior experiences in the field. In companion in vivo studies, these hypoimmune CD19 CAR T cells have been shown to efficiently evade host immune response (see posters by Hu and Duback). Together, these data demonstrate a profile for potency, genome integrity, and manufacturing yield that supports progressing these cells into human studies.

Engineered Hypoimmune CAR T Cells Provide Lasting Tumor Control in Fully Immunocompetent Allogeneic Humanized Mice

Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, avoidance of malignant contamination and T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced with this approach. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are disrupted and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR. When transplanted into allogeneic humanized mice, hypoimmune CAR T cells evade immune recognition by T cells using ELISPOT analysis. In contrast, transplantation of control CD19 CAR T cells generated from the same human donor resulted in a significant T cell activation (p<0.0001 unpaired t-test). Innate immune cell assays show that CD47 overexpression protects HLA-I/II deficient CD19 CAR T cells from NK cell and macrophage killing in vitro and blocking of CD47 results in killing by innate allogeneic immune cells. For persistence studies, allogeneic humanized mice were injected with 1´106 Luc+ Nalm6 cells and received 7´106 control CD19 CAR T cells or HIP CD19 CAR T cells. However, CD19 CAR T cells are recognized and rejected by the allogeneic immune system of the humanized mice, leading to tumor grow in this group. Flow cytometry on Day 56 from bone marrow and spleen confirms persistence of HIP CD19 CAR T cells, but not control CD19 CAR T cells (see Figure). These findings show that HIP CD19 CAR T cells are functionally immune evasive in allogeneic recipients with cytotoxic anti-tumor capacity and suggest they could provide universal HIP CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that HIP CD19 CAR T cells can be used for re-dosing strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune and Genomic Analysis of Boxer Dog Breed and Its Relationship with Leishmania infantum Infection.

Leishmaniosis, one of the most important zoonoses in Europe, is caused by Leishmania infantum, an intracellular protozoan parasite. This disease is endemic in the Mediterranean area, where the main reservoir is the dog. Several studies indicate a possible susceptibility to L. infantum infection with clinical signs in some canine breeds. One of them is the boxer breed, which shows a high prevalence of disease. In this study, immunological and genomic characterization of serum samples from boxer dogs living in the Mediterranean area were evaluated to analyze the immune response and the possible genetic explanation for this susceptibility. Serum levels of cytokines IFN-γ, IL-2, IL-6, IL-8, and IL-18 were determined by ELISA commercial tests, while the genotyping study was performed using the CanineHD DNA Analysis BeadChip. The results show relevant differences in the serum levels of cytokines compared to published data on other canine breeds, as well as sequence changes that could explain the high susceptibility of the boxer breed to the disease. Concretely, polymorphic variants in the CIITA, HSF2BP, LTBP1, MITF, NOXA1, PKIB, RAB38, RASEF, TLE1, and TLR4 genes were found, which could explain the susceptibility of this breed to L. infantum infection.