Leukemia-associated Antigens Research Articles

Hu8F4-CAR T cells with mutated Fc spacer segment improve target specificity and mediate anti-leukemia activity in vivo

Background aimsHu8F4 is a T-cell receptor–like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor–expressing cells. MethodsWe generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor–expressing cells in vivo. ResultsThe primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. ConclusionsHerein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding–induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Abstract 44: A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells

Abstract Chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies, while promising and efficacious, can also result in a variety of adverse effects. Of note, on-target, off-tumor toxicity is one adverse effect that continues to limit the safety and efficacy of CAR-T cells and has even led to life-threatening or fatal outcomes in patients (Morgan et al. 2010.). To address this, we have developed a unique, codon-based approach by replacing strategically selected codons within CAR non-antigen binding domains with their rare synonymous isoforms. Hoekema et al. (1987) reported the ability of synonymous rare codons to alter gene and protein expression in other model systems. In CAR-T cells, we aim to use this novel, codon-based approach to fine tune and modulate CAR-T cell expression and function to elicit full on-tumor activity while lessening off-tumor toxicity. In our approach, we replaced specific endogenous codons within the non-antigen binding domains of Hu8F4-CAR, a TCR-like 2nd generation CAR developed by our group which targets the leukemia-associated antigen PR1/HLA-A2 (Ma et al. 2016.). Briefly, we created variants of Hu8F4-CAR containing rare codon substitutions in two subtypes: intracellular-based (type 1) and combined extracellular- and intracellular-based (type 2). Each variant subtype (type 1 or 2) had either between 6 and 30 or 27 and 57 rare codons introduced, respectively. Expression profiling and functional assessment of the Hu8F4-CAR variants was assessed in the Jurkat mutant cell line (J.RT3-T3.5) or human primary healthy peripheral blood mononuclear cells (PBMCs) that were enriched for T cells, respectively. Expression profiling results show that surface expression is reduced in J.RT3-T3.5 cells when assessed by flow cytometry by 1-1.5-fold compared to unmodified Hu8F4-CAR. When evaluated by immunoblotting, novel lower molecular weight bands emerge only in rare codon-modified variants, suggesting that rare codons may have altered translational or post-translational processing leading to truncated CAR protein isoforms (30, 45, or 60kDa vs 73kDa for unmodified Hu8F4-CAR), which may contribute to reduced cell surface CAR expression. Furthermore, we have conducted in vitro functional testing of the rare codon-modified variants and show reduced activity against non-malignant off-tumor targets that express PR1 and/or HLA-A2 including healthy donor PBMCs, mobilized hematopoietic cells, and bone marrow. Importantly, in addition to reduced off-tumor activity against healthy myeloid cells, rare codon-modified variants preserve their activity against on-tumor target cells, including the U937-A2+ cell line and primary acute myeloid leukemia. Altogether, these results suggest that our approach can modulate CAR expression and functionality creating a novel methodology to reduce the likelihood of off-tumor adverse effects typically associated with CAR therapies. Citation Format: Rolando A. Vedia, Sijie Lu, Anne V. Philips, Anna Sergeeva, Lisa S. St John, Karen Clise-Dwyer, Gheath Alatrash, Jeffrey J. Molldrem. A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 44.

Blast-Derived Small Extracellular Vesicles in the Plasma of Patients with Acute Myeloid Leukemia Predict Responses to Chemotherapy.

The small extracellular vesicles (sEV) accumulating in acute myeloid leukemia (AML) patients' plasma are mixtures of vesicles produced by leukemic and non-malignant cells. sEV originating from leukemia blasts could serve as potential non-invasive biomarkers of AML response to therapy. To isolate blast-derived sEV from patients' plasma, we developed a bioprinted microarray-based immunoassay using monoclonal antibodies (mAbs) specific for leukemia-associated antigens (LAAs) and mAbs specific for a mix of tetraspanins (CD9, CD63, and CD81). We determined the proportion of LAA+ sEV relative to total plasma sEV (the LAA+/total sEV ratio) in serially collected samples of newly diagnosed AML patients prior to, during, and after chemotherapy. At AML diagnosis, the LAA+/total sEV ratio was significantly higher in patients than in healthy donors (HDs). In patients who achieved complete remission (CR) after induction chemotherapy, the LAA+/total sEV ratios significantly decreased after each chemotherapy cycle to levels seen in HDs. In contrast, the LAA+/total sEV ratios in AML patients with persistent leukemia after therapy remained elevated during and after therapy, as did the percentage of leukemic blasts in these patients' bone marrows. The LAA+/total sEV ratio emerges as a promising non-invasive biomarker of leukemia response to therapy.

Kinetics of Neoantigen Specific T Cells in Post-Transplant Relapse

Introduction: In allogeneic stem cell transplantation (alloSCT), donor-derived T cells mediate the graft-versus-leukemia effect (GVL) by recognizing antigens expressed by leukemia cells. These antigens may include either major- or minor-histocompatibility alloantigens, leukemia over-expressed antigens (leukemia-associated antigens), or neoantigens derived from leukemic somatic mutations. In solid tumors, higher somatic mutation burdens are associated with better response to checkpoint inhibitors, which underscores the importance of neoantigen-specific T-cells in cancer immunotherapy. However, mutation burdens are relatively low in acute leukemia, and the role of neoantigens in GVL remains unknown. Here, we screened potential neoantigens and identified neoantigen-reactive T cells in the patients who developed post-transplant relapse. Materials and methods: Leukemic blasts from patients in relapse were FACS-isolated from bone marrow aspirate or peripheral blood samples. Recipient T cells or saliva were isolated from pre-transplant peripheral blood as a germline control, and donor monocyte or CD34-positive cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) at 100X coverage, paired with RNAseq at 40M reads per sample. Somatic mutations were called between recipient blasts and germline controls with mutect and mpileup, followed by annotation with SnpEff. Somatic mutations with variant allele frequency >20% were subjected to pVAC-seq for prediction of neoantigen likely presented on recipient MHC (IC50 < 500nM). 73 SNPs loci associated with publicly known minor histocompatibility antigens (miHAs) were also genotyped to find the miHA disparities between donors and recipients. Serial peripheral blood mononuclear cell samples (PBMCs) were obtained before relapse if available, at the time of relapse, and after treatment for relapse. For HLA-A*02:01-restricted neoantigens, PBMCs were stained with HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65 (control). Purified CD3 cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: Eight patients with relapsed acute leukemia after allo-SCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their transplant donors (4 matched siblings, 1 matched unrelated, 3 haploidentical) were screened for neoantigens. We analyzed samples from 7 subjects with available WES/RNAseq and detected an average of 273 somatic mutations per patient (range 108- 609) on average. Targeted next-generation sequencing was used to detect 2 somatic mutations in one subject. Among these mutations, potential neoantigen candidates were identified in seven out of eight subjects (87.5%), with a median number of 9 candidate neoantigens per subject. In contrast, only 2 subjects (25%) were found with donor:receipient miHAs at tested loci. CD8 T cells derived from UPN8, a responder to DLI, showed dominant CMVpp65-specific T cells at the time of relapse and persistent low frequencyTP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse by HLA-A*02:01 tetramer staining (Figure A). HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these T cell clones during GVHD (Figure B). In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells reactive to neoantigens were detectable by IFN-γ/CD107a production specific to mutant peptides ( SDE2.pK123X and TP53.pR248W respectively). Discussion: Our in-silico analysis suggested the possibility of somatic mutations in acute leukemia to serve as putative neoantigens in alloSCT. We were able to detect neoantigen-specific T cells by tetramer staining and cytokine production assays from a subject who achieved a durable remission after DLI. Although the sample size is very small, we demonstrated the feasibility of studying neoantigen-specific T cells in post-transplant specimens. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with post-relapse outcomes, especially for response to DLI.

Single-Cell Analysis of Immune Recognition in Chronic Myeloid Leukemia Patients Following Tyrosine Kinase Inhibitor Discontinuation

Background: Modern treatment goal for patients with chronic myeloid leukemia (CML) is to maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. The control of residual leukemia cells may occur with immunological mechanisms as several studies have shown that a higher proportion of CD56 dim NK cells before TKI discontinuation is associated with a better probability of TFR. In addition, CD8+ T cell abundances and phenotypes have been associated with responses to TKIs. However, the more detailed phenotypes and mechanisms and how to translate these findings to increased TFR rates have remained unknown. Methods: We analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced (scRNA+TCRαβ-seq) samples from patients with CML ( n=13, N=25), other cancers (AML n=11, CLL n=13, solid tumors n=4), and healthy ( n=7). Additionally, we profiled 90 CML and 786 healthy TCRβ-seq samples, and sorted T cells specific to leukemia-associated antigen PR1 and matched these back to the scRNA+TCRαβ-seq and TCRβ-seq data. To validate and refine our findings, we performed co-culture of primary and expanded NK cells with CML cell lines (K562 and LAMA84) with a multiplexed scRNA-seq readout. Results: In the pan-cancer scRNA+TCRαβ-seq comparison of >250,000 cells, CML was separated from other cancers and healthy by the high number and active phenotype NK cells. The other phenotype more pronounced in CML was CD8+ T EM/T EMRA phenotype. Most NK cells in CML had an active CD56 dim phenotype with high expression of GZMA/B, PRF1, CCL3/4 and IFNG. In the off-TKI samples from patients with TFR, these NK cells upregulated NK cell activation genes such as TNFRSF4/OX40, TNFRSF9/4-1BB, and TNFRSF18/GITR. The most important predicted interactions between NK cells and CD34+ leukemic cells were inhibitory LGALS9- TIM3 and PVR- TIGIT interactions. Following TKI discontinuation, the inhibitory markers HAVCR2/TIM3 and TIGIT decreased in patients with TFR, which was not seen in patients with relapse following TFR. Accordingly, in our co-culture data of NK cells with CML cell lines, we noted activation of NK cells to a similar phenotype as noted in ex vivo off-TKI samples. In co-culture with CML target cells, NK cells gained an activation by upregulation of genes like TNFRSF4/OX40, TNFRSF9/4-1BB, TNFRSF18/GITR and the inhibitory markers HAVCR2/TIM3 and TIGIT. In co-culture with NK cells, CML target cells upregulated the IFN-γ response pathway including elevated HLA-expression and accordingly, upregulation of LGALS9 and PVR. The CD8+ T EM/EMRA cells that were expanded in CML diagnosis were suppressed following TKI treatment and decreased following TKI discontinuation. To track anti-CML T cells, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 from the TCRb-sequenced samples. As a validation of our classifier, we noticed that anti-PR1 T cells were more prevalent in CML than in healthy ( P adj<0.0001) or melanoma ( P adj<0.0001), more expanded than similar anti-viral T cells ( P adj<0.0001) and enriched in bone marrow samples compared to peripheral blood ( P adj<0.0001). From different TKIs, dasatinib expanded anti-PR1 T cells the most ( P adj<0.01). In the scRNA+TCRαβ-seq data, anti-PR1 T cells were enriched in the mature, cytotoxic CD8+ T EMRA phenotype ( P adj<0.0001). In a patient maintaining TFR, anti-PR1 T cells had a highly cytotoxic profile ( GZMB, PRF1) throughout the therapy with upregulated NK receptors like FCGR3A/CD16 and KIR2DL2, even more so than T cells targeting other targets e.g., different viruses. In comparison, the anti-PR1 T cells in a patient with early relapse had an exhausted profile with upregulation of memory marker GZMK and exhaustion markers like LAG3 and HAVCR2/TIM3. Conclusions: With our in-depth analysis of cellular and molecular immune responses in CML, we identified the active NK cells and anti-PR1 T cells that could help maintain TFR in patients discontinuing TKI treatment. Different immune-checkpoint blockade therapies such as anti-TIM3 could be considered as future strategies to improve the TFR rates.

Single-cell analysis of immune recognition in chronic myeloid leukemia patients following tyrosine kinase inhibitor discontinuation

Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαβ-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9–TIM3 and PVR–TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRβ-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.

WT1 and PRAME RNA-loaded dendritic cell vaccine as maintenance therapy in de novo AML after intensive induction chemotherapy.

Intensive induction chemotherapy achieves complete remissions (CR) in >60% of patients with acute myeloid leukemia (AML) but overall survival (OS) is poor for relapsing patients not eligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Oral azacytidine may be used as maintenance treatment in AML in first remission, but can be associated with substantial side effects, and less toxic strategies should be explored. Twenty AML patients in first CR (CR1) ineligible for allo-HSCT were treated with FDC101, an autologous RNA-loaded mature dendritic cell (mDC) vaccine expressing two leukemia-associated antigens (LAAs). Each dose consisted of 2.5-5 × 106 mDCs per antigen, given weekly until week 4, at week 6, and then monthly, during the 2-year study period. Patients were followed for safety and long-term survival. Treatment was well tolerated, with mild and transient injection site reactions. Eleven of 20 patients (55%) remained in CR, while 4 of 6 relapsing patients achieved CR2 after salvage therapy and underwent allo-HSCT. OS at five years was 75% (95% CI: 50-89), with 70% of patients ≥60 years of age being long-term survivors. Maintenance therapy with this DC vaccine was well tolerated in AML patients in CR1 and was accompanied by encouraging 5-year long-term survival.

Immune modulation in chronic myeloid leukaemia patients treated with nilotinib and interferon-alpha.

The addition of interferon to tyrosine kinase inhibitors (TKIs), to improve deep molecular response (DMR) and potentially treatment-free remission (TFR) rates in chronic-phase chronic myeloid leukaemia (CP-CML) patients is under active investigation. However, the immunobiology of this combination is poorly understood. We performed a comprehensive longitudinal assessment of immunological changes in CML patients treated with nilotinib and interferon-alpha (IFN-α) within the ALLG CML11 trial (n = 12) or nilotinib alone (n = 17). We demonstrate that nilotinib+IFN transiently reduced absolute counts of natural killer (NK) cells, compared with nilotinib alone. Furthermore, CD16+ -cytolytic and CD57+ CD62L- -mature NK cells were transiently reduced during IFN therapy, without affecting NK-cell function. IFN transiently increased cytotoxic T-lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) proteinase-3, BMI-1 and PRAME; and had no effect on regulatory T cells, or myeloid-derived suppressor cells. Patients on nilotinib+IFN who achieved MR4.5 by 12 months had a significantly higher proportion of NK cells expressing NKp46, NKp30 and NKG2D compared with patients not achieving this milestone. This difference was not observed in the nilotinib-alone group. The addition of IFN to nilotinib drives an increase in NK-activating receptors, CTLs responding to LAAs and results in transient immune modulation, which may influence earlier DMR, and its effect on long-term outcomes warrants further investigation.

LYMPHOID ORGANS IN LEUKEMIA AND OTHER PATHOLOGIES IN CATTLE

Using the reaction of anaphylaxis with desensitization on guinea pigs, the antigenic structure of lymphoid organs in leukemia and other pathologies of cattle was studied. It is believed that neoplasia is the result of a distortion of the gene activity program due to the introduction of an oncogenic virus into the cell genome, integration with other biological objects, or gene mutation. All this leads to the emergence, first of all, of quantitative anomalies, expressed in continuous, uncontrolled cell division, cell migration and changes in their biochemical activity. In all likelihood, the essence of the identified differences described in the article is that in the biological fluids of cows with leukemia there are, in addition to BLV antigens, water-soluble leukemia-associated antigens.

Uncovering the Enemy: Single-Cell Transcriptional Profiling of Measurable Residual Disease (MRD) Cells

Albeit novel treatment concepts have entered the therapeutic landscape of AML, relapse rates remain high. This is attributed to treatment-refractory leukemic cells, which can be quantified by flow cytometry and molecular techniques (Measurable Residual Disease, MRD). MRD positivity is a significant prognostic biomarker for relapse and low overall survival rates. Post-remission therapy for the elimination of MRD remains a major challenge. We previously confirmed the prognostic value of early flow MRD assessment during aplasia in patients who achieve complete remission after intensive induction therapy (Koehnke et al., 2019). Here, we hypothesized that single-cell transcriptional profiling of refractory leukemic cells using flow cytometry (flow MRD) during aplasia will help to understand resistance mechanisms to therapy and to identify suitable targets for cellular immunotherapy. Flow MRD detection is based on the definition and characterization of leukemia-associated aberrant immunophenotypes (LAIPs) at the time of initial diagnosis, as described previously (Koehnke et al., 2015). A similar approach was applied to define LAIP+ cells at the time of aplasia (d16-d21 after initiation of induction therapy). For single-cell RNA-seq, LAIP+ cells from paired initial diagnosis and aplasia were sorted and subjected to transcriptional profiling using the mcSCRB-seq (molecular crowding single-cell RNA barcoding and sequencing) protocol (Bagnoli et al., 2018). We included 7 AML patients of different ELN genetic risk group (favorable, n=1; intermediate, n=4; adverse, n=2). In total, 854 LAIP+ cells from paired initial diagnosis-aplasia samples were projected using uniform manifold approximation and projection (UMAP) visualizations. We observed a distinct clustering of LAIP+ cells per patient and time point, revealing high inter-patient heterogeneity (Figure 1A). To identify common transcriptional changes across patients, we performed differential gene expression analysis of LAIP+ cells, comparing aplasia to initial diagnosis. We identified 93 genes significantly upregulated and 41 genes significantly downregulated (p < 0.01, FC >1 or <-1; Figure 1B). Among markedly upregulated genes across patients at the time of aplasia, we found 3 genes related to leukemic cell maintenance. Interestingly, SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, known to be essential for the proliferation and survival of AML cells (Buscarlet et al., 2014; Shi et al., 2013), showed a 1.99-fold increase in expression (p < 0.00). In addition, TRAP1, a candidate target in cancers with antiapoptotic function, was upregulated 1.5-fold (p < 0.00). And lastly, LIN28B, the cancer stem cell and leukemia driver gene, was also found to be upregulated by 0.7-fold in aplasia LAIP+ cells (p < 0.00). In addition, several immune-related genes including DC marker gene, LRP1 (CD91; p < 0.00, FC = 1.3) and the neutrophil/GMP- specific gene, cathepsin G (CTSG; p < 0.01, FC = 0.5) showed a significant up-regulation in aplasia LAIP+ cells. The latter was previously identified as a leukemia-associated antigen and a potential target for immunotherapy (Alatrash et al., 2017). Strikingly, LAIP+ cells at the time of aplasia harbored a LSC gene expression pattern in a patient-specific manner. Lastly, cell type prediction using the annotated dataset (van Galen et al., 2019) assigned the phenotype of LAIP+ cells mainly to GMP-like and cDC-like cells in variable abundance across patients. In summary, our data showed high inter-patient heterogeneity and phenotypic differences, making general statements on chemo-resistance challenging. However, despite this heterogeneity, LAIP+ cells at the time of aplasia harbor remarkable proliferation and survival advantage (stem cell-like phenotype), specifically, due to the upregulation of SMARCA4, TRAP1, and LIN28B genes. This phenotype is accompanied by patient-specific enrichment of LSC signature and upregulation of immune-related genes. Our data open up the path to better understand chemo-resistance and to identify targets that need to be addressed to eradicate MRD and improve relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Humanized T Cell Receptor-like Antibody Hu8F4 Reduces Leukemia By Fc-Mediated Mechanism

8F4, a T cell receptor (TCR) - like antibody which targets the leukemia-associated antigen PR1 (VLQELNVTV) when presented in the context of MHC class I (HLA-A2:01), is a mouse IgG2a monoclonal antibody (mAb) which mediates both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and eliminates AML in PDX (patient derived xenograft) mouse models. Recognizing the potential therapeutic value of 8F4, we produced a humanized IgG1 (Hu8F4) as a first-in-class mAb for preclinical development and clinical use. Characterization of Hu8F4 by ELISA and surface plasmon resonance showed high affinity (KD=6.5 nM) and specificity of Hu8F4 binding to PR1/HLA-A2 monomer. Staining of peptide-pulsed T2 cells confirmed Hu8F4 specificity to PR1, but not to control peptide MART1. To study anti-leukemia activity of Hu8F4 we used HLA-A2-transfected leukemia cell lines U937 (U937-A2) and MV411 (MV411-A2), as well as THP1 (endogenous HLA-A2+) as targets. In contrast to mouse 8F4, Hu8F4 did not induce CDC in vitro. Rather, Hu8F4 mediated both potent antibody-dependent cellular phagocytosis (ADCP) and ADCC of U937-A2 and THP1 cells in NFAT reporter assays, using FcgRI- (EC50 = 0.002 µg/ml), FcgRII- (EC50 = 1.49 µg/ml), and FcgRIII- (EC50 = 0.04 µg/ml) transfected Jurkat cells, as well as pooled human PBMC (EC50 = 0.95 µg/ml) and donor-derived macrophages (EC50 = 1.1 µg/ml), as effectors. Based on the ADCP activity of Hu8F4, we sought to examine the potential synergy of anti-CD47 antibody, a macrophage checkpoint inhibitor, with Hu8F4. Treatment of U937-A2 and THP1 target cells with anti-CD47 F(ab')2 neutralizing antibody made cells susceptible to Hu8F4-mediated phagocytosis by NSG mouse bone marrow derived macrophages (BMDM) in vitro. To confirm the critical role of CD47 in Hu8F4-mediated ADCP we created CD47 KO U937-A2 and THP1 AML lines. Within 96 hours of treatment with Hu8F4 both CD47 KO cell lines were completely phagocytosed by NSG BMDM, thus confirming the role of phagocytosis as a mechanism. To test Hu8F4 activity in vivo, we established xenografts using luciferase-transfected U937-A2 and MV411-A2 cell lines in NSG mice. Hu8F4 was then administered starting on either day 3 (U937-A2) or day 7 (MV411-A2). Treatment with Hu8F4 resulted in elimination of leukemia as measured by FACS and BLI and increased median survival (from 33 days to >85 days for U937-A2, p=0.0075; and from 125 days to >225 days for MV411-A2, p=0.0033). To further test Hu8F4 efficacy, we established PDX in NSG mice using cells from HLA-A2+ refractory/relapsed AML patients. Hu8F4 treatment reduced leukemia by 90.3% to 99.9% when assessed by FACS in four out of five PDX and resulted in increased median survival of the mice (from 41 days to 49 days, p=0.0002) in one PDX tested. To study the mechanism of action of Hu8F4, we produced a 3 aa mutant (Hu8F4 PG LALA), which completely abrogated binding to Fcg receptors. Hu8F4 PG LALA, as well as Hu8F4 F(ab')2, showed no in vivo activity against U937-A2 or MV411-A2 xenografts, confirming that the anti-leukemia activity of Hu8F4 is dependent on its ability to bind Fc receptors. Because FcgR may be expressed on AML we tested fratricide as a possible in vivo mechanism of Hu8F4. We utilized the CRISPR /Cas9 system to edit out both FcgRI and II in the U937-A2 cell line that is naturally deficient in FcgRIII (DKO U937-A2). We then used a retroviral system to stably express FcgRI or FcgRII in U937-A2 to create FcgRI-U937-A2 and FcgRII-U937-A2, respectively, and tested their susceptibility to Hu8F4 in the xenograft model. Compared to FcR-expressing wild-type U937-A2, Hu8F4 showed similar anti-leukemia activity in all three xenografts. There was no significant difference noted in the anti-leukemia potency of Hu8F4 between DKO U937-A2 or single FcR-expressing xenografts compared with FcgRI/FcgRII expressing wild-type U937-A2 xenografts, suggesting that fratricide is an unlikely mechanism in this model. Here we demonstrate high specificity, affinity, and remarkable anti-AML activity of Hu8F4, a first-in-class humanized TCR mimic monoclonal antibody that is currently being tested in a phase I clinical trial for HLA-A2+ refractory/relapsed AML and MDS. This study provides evidence for the importance of FcgR-mediated mechanisms (ADCC and ADCP) in the activity of Hu8F4. In addition, our results support future combination trials with treatments that promote phagocytosis.

Individualized Antigen Expression in Precursor T-Cell Acute Lymphoblastic Leukemia

BACKGROUND: In T-acute lymphoblastic leukemia (T-ALL), multi-parametric flow cytometry can serve to detect minimal residual disease (MRD) by using immature or aberrant antigens expression as well as the altered expression of T-cell antigens. The latter approach has been specifically introduced to overcome the absence of leukemia-associated antigens. However, there is no agreed-upon method for the use of T-cell antigens in T-ALL MRD testing. AIMS AND OBJECTIVES: To compare the expression of classic T-cell antigens on T-lymphoblasts and T-lymphocytes to establish a protocol for their use in MRD analysis. MATERIALS AND METHODS: Flow cytometric data of PB or BM samples from 63 adults with T-ALL were collected. We assessed the frequency and degree of brightness or dimness of each T-cell marker, in addition to studying the uniformity of the events scatter of a total of 287 follow-up BM samples from 50 patients. RESULTS: Significant differences in expression intensity of T-cell markers were found between T-lymphoblasts and T-lymphocytes; they were reasonably stable on blasts in follow up samples. This detailed study has nominated the conjoint use sCD3neg/dim and CD5dim/neg in the identification of residual cells, to be supported by other T-cell markers. CONCLUSION: The suggested gating sequence showed an acceptable level of accuracy in detecting residual leukemia, supporting their use in T-ALL MRD especially when other distinguishing markers might be absent in the diagnosis sample, or susceptible to be lost with induction therapy.

Enhanced stimulation of antigen-specific immune responses against nucleophosmin 1 mutated acute myeloid leukaemia by an anti-programmed death 1 antibody.

Nucleophosmin1 (NPM1) is one of the most commonly mutated genes in AML and is often associated with a favourable prognosis. Immune responses play an increasing role in AML treatment decisions; however, the role of immune checkpoint inhibition is still not clear. To address this, we investigated specific immune responses against NPM1, and three other leukaemia-associated antigens (LAA), PRAME, Wilms' tumour 1 and RHAMM in AML patients. We investigated T cell responses against leukaemic progenitor/stem cells (LPC/LSC) using colony-forming immunoassays and flow cytometry. We examined whether immune checkpoint inhibition with the anti-programmed death 1 antibody increases the immune response against stem cell-like cells, comparing cells from NPM1 mutated and NPM1 wild-type AML patients. We found that the anti-PD-1 antibody, nivolumab, increases LAA stimulated cytotoxic T lymphocytes and the cytotoxic effect against LPC/LSC. The effect was strongest against NPM1mut cells when the immunogenic epitope was derived from the mutated region of NPM1 and these effects were enhanced through the addition of anti-PD-1. The data suggest that patients with NPM1 mutated AML could be treated with the immune checkpoint inhibitor anti-PD-1 and that this treatment combined with NPM1-mutation specific directed immunotherapy could be even more effective for this unique group of patients.

Neutrophil extracellular traps arm DC vaccination against NPM-mutant myeloproliferation.

Neutrophil extracellular traps (NETs) are web-like chromatin structures composed by dsDNA and histones, decorated with antimicrobial proteins. Their interaction with dendritic cells (DCs) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and the induction of a CD8+ T-cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow (BM) chimeras in a competitive BM transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an antileukemia CD8 T-cell memory response.

Increasing Role of Targeted Immunotherapies in the Treatment of AML.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The standard of care in medically and physically fit patients is intensive induction therapy. The majority of these intensively treated patients achieve a complete remission. However, a high number of these patients will experience relapse. In patients older than 60 years, the results are even worse. Therefore, new therapeutic approaches are desperately needed. One promising approach in high-risk leukemia to prevent relapse is the induction of the immune system simultaneously or after reduction of the initial tumor burden. Different immunotherapeutic approaches such as allogenic stem cell transplantation or donor lymphocyte infusions are already standard therapies, but other options for AML treatment are in the pipeline. Moreover, the therapeutic landscape in AML is rapidly changing, and in the last years, a number of immunogenic targets structures eligible for specific therapy, risk assessment or evaluation of disease course were determined. For example, leukemia-associated antigens (LAA) showed to be critical as biomarkers of disease state and survival, as well as markers of minimal residual disease (MRD). Yet many mechanisms and properties are still insufficiently understood, which also represents a great potential for this form of therapy. Therefore, targeted therapy as immunotherapy could turn into an efficient tool to clear residual disease, improve the outcome of AML patients and reduce the relapse risk. In this review, established but also emerging immunotherapeutic approaches for AML patients will be discussed.

Donor-derived multiple leukemia antigen–specific T-cell therapy to prevent relapse after transplant in patients with ALL

AbstractHematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk acute lymphoblastic leukemia (ALL), but relapse remains a major cause of treatment failure. To prevent disease relapse, we prepared and infused donor-derived multiple leukemia antigen–specific T cells (mLSTs) targeting PRAME, WT1, and survivin, which are leukemia-associated antigens frequently expressed in B- and T-ALL. Our goal was to maximize the graft-versus-leukemia effect while minimizing the risk of graft-versus-host disease (GVHD). We administered mLSTs (dose range, 0.5 × 107 to 2 × 107 cells per square meter) to 11 patients with ALL (8 pediatric, 3 adult), and observed no dose-limiting toxicity, acute GVHD or cytokine release syndrome. Six of 8 evaluable patients remained in long-term complete remission (median: 46.5 months; range, 9-51). In these individuals we detected an increased frequency of tumor-reactive T cells shortly after infusion, with activity against both targeted and nontargeted, known tumor-associated antigens, indicative of in vivo antigen spreading. By contrast, this in vivo amplification was absent in the 2 patients who experienced relapse. In summary, infusion of donor-derived mLSTs after allogeneic HSCT is feasible and safe and may contribute to disease control, as evidenced by in vivo tumor-directed T-cell expansion. Thus, this approach represents a promising strategy for preventing relapse in patients with ALL.

Combinatorial antigen targeting strategies for acute leukemia: application in myeloid malignancy

Background aimsEfforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse. MethodsFor co-transduction with the authors’ previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens. ResultsThe in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors. ConclusionsOverall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.

Aspartate β-Hydroxylase (ASPH) Expression in Acute Myeloid Leukemia: A Potential Novel Therapeutic Target.

BackgroundAspartate β-hydroxylase (ASPH) is an embryonic transmembrane protein aberrantly upregulated in cancer cells, associated with malignant transformation and, in some reports, with poor clinical prognosis.ObjectiveTo report the expression patterns of ASPH in acute myeloid leukemia (AML).MethodsCell surface expression of ASPH was measured via 8-color multiparameter flow cytometry in 41 AML patient samples (31 bone marrow, 10 blood) using fluorescein isothiocyanate (FITC)-conjugated anti-ASPH antibody, SNS-622. A mean fluorescent intensity (MFI) of 10 was used as a cutoff for ASPH surface expression positivity. Data regarding patient and disease characteristics were collected.ResultsASPH surface expression was found on AML blasts in 16 samples (39%). Higher ASPH expression was seen in myeloblasts of African American patients (p=0.02), but no correlation was found between ASPH expression and other patient or disease characteristics. No association was found between ASPH status and CR rate (p=0.53), EFS (p=0.87), or OS (p=0.17).ConclusionsASPH is expressed on blasts in approximately 40% of AML cases, and may serve as a new therapeutically targetable leukemia-associated antigen.

A Novel Humanized Murine Model to Identify Neoantigen-Specific T Cells in CBFA2T3-GLIS2 Positive Acute Megakaryoblastic Leukemia

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p<0.001), indicating a potential leukemia-directed cytokine lymphocyte response. Single cell TCR repertoire analysis of CD8+ T cells from day 40 revealed a decrease in alpha beta TCR diversity when exposed to PDX cells compared to the PBMC only control, consistent with a clonal expansion of neoantigen-directed TCRs. Single TCR expressing stable cell lines have been generated for the top expanded clones. In ongoing experiments, we are utilizing these cell lines to assess leukemia clearance in our model and measuring specificity and functionality for the putative neoantigens through tetramer staining and peptide stimulation assays, respectively. For the latter studies, we have taken two approaches for the identification of potential neoantigens that may be directing this observed clonal expansion. First, to interrogate potential neoantigens spanning the fusion junction we used the algorithm from NetMHCcons which identified two peptides with intermediate binding affinity (defined by an IC50 of 150 - 500 nM). Second, we utilized immunopeptidomics to identify HLA class I presented peptides on the surface of the PDX cells. One of our top significant hits was a peptide that corresponds to the 5‘ untranslated region (UTR) of bone morphogenetic protein 2 (BMP2). This peptide fragment was not present in the human Uniprot database, suggesting the 5‘ UTR is not expressed in normal tissues. Although its role in leukemia is not well described, BMP2 is aberrantly upregulated in CBFA2T3-GLIS2 positive AMKL and required for serial replating in colony formation unit assays; therefore, this peptide is of great interest as a candidate leukemia-associated antigen. In summary, our novel humanized PDX model sheds light on the immunogenicity of CBFA2T3-GLIS2 mediated AMKL and the potential for TCR-T immunotherapy in the setting of minimal residual disease for this chemotherapy resistant malignancy. Studies are ongoing to identify the TCR clone(s) responsible for the anti-leukemic response seen in our model and the leukemia-associated antigens that are mediating this response. DisclosuresGruber: Kura Oncology: Consultancy.

Pembrolizumab in Combination with Antigen-Specific Cytotoxic T Lymphocytes Enhances Killing of Acute Myeloid Leukemia

BackgroundPembrolizumab, an antibody that blocks programmed cell death protein 1 (PD-1), has been FDA approved for several solid tumors and hematologic malignancies. Currently, pembrolizumab is under investigation for acute myeloid leukemia (AML) in combination with hypomethylating agents. It is established that AML is highly responsive to immunotherapy, as seen with the anti-leukemic effect of allogeneic hematopoietic stem cell transplantation (alloHSCT). However, response to cytotoxic T lymphocytes (CTLs) that target leukemia-associated antigens (LAAs) has been less reliable in eradicating disease. This insufficient response to LAA-specific CTLs is likely partially accounted for by the immune dysregulation seen in AML. Because of promising murine data that blockade of the PD1/PD-L1 pathway enhances the graft versus leukemia effect of alloHSCT, we investigated if adding pembrolizumab to CTLs that target the two LAAs CG1 and PR1 will enhance CTL antileukemia activities. CG1 and PR1are two HLA-A2 restricted nonameric peptides that we validated as promising AML targets derived from cathepsin G (i.e. CG1), and proteinase 3 (P3) and neutrophil elastase (NE) (i.e. PR1). We hypothesized that pembrolizumab added to CG1-CTLs and PR1-CTLs, will enhance their anti-leukemic effects with minimal off target toxicities.MethodsUsing a standard calcein AM in vitro cytotoxicity assay, we co-cultured AML targets, including U937 HLA-A2 + (U937-A2) AML cell line and primary patient HLA-A2 + AML samples, with CG1-CTL and PR1-CTL. AML cells were loaded with calcein AM and then incubated with CG1- and PR1-CTLs at increasing effector to target ratios. Pembrolizumab or isotype antibody were added to the cultures. After 4 hours, calcein AM was measured to determine cell viability. T2 cells pulsed with PR1 or CG1, were used as a positive control and non-pulsed T2 cells were used as negative control cells. For in vivo experiments, we used a human established AML xenograft mouse treatment model to determine the effect of pembrolizumab when added to CG1- or PR1- CTL in vivo. CG1-CTL and PR1-CTLs were expanded from the same donor and used as the effector cells. NOD/SCID gamma (NSG) mice (4-6-week-old females) were engrafted with AML samples by tail vein injection. After confirming engraftment, CG1- and/or PR1-CTL were administered to mice and pembrolizumab or isotype antibody [100ug/mouse] were given three times over two weeks. Mice were monitored for clinical GVHD and AML three times/week. Mice were sacrificed at approximately two weeks following treatment. To assess for GVHD, mouse tissues including spleen, liver, kidney, BM, intestine, brain, heart, and lung were harvested and fixed in 10% formalin. The fixed tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to histologic examination. Bone marrow (BM) was processed using standard methodology and analyzed for residual AML by flow cytometry.Results:In vitro our data demonstrate that U937-A2 and two primary patient HLA-A2 AML samples, have enhanced cell lysis when treated with pembrolizumab (CTL+ pembrolizumab) in comparison with isotype (Iso) + CTL, pembrolizumab only, iso only or CTL only groups (Figure 1). In vivo data show a decrease in U937-A2 disease burden and primary patient AML after treatment with CG1and/or PR1-CTL (CTL-treated) . This decrease was enhanced when pembrolizumab was added to the CTL-treated mice (CTL+ pembrolizumab) in comparison with mice treated with pembrolizumab only (pembrolizumab-treated), or CTL only (CTL-treated) (Figure 2). Pathology data to assess toxicity in mice treated with CTL +/- pembrolizumab showed an enhanced pulmonary (perivascular and parabronchial) lymphocytic infiltration in mice treated with the combination of CTL and pembrolizumab. The other organs investigated, showed no change when combination therapy was used.Conclusion:We have validated in vitro and in vivo the enhanced killing of AML (cell lines and primary patient samples) by CG1-CTL and/or PR1-CTL after addition of pembrolizumab. Toxicity data show mild enhancement of lymphocytic infiltrate in the lungs after addition of pembrolizumab to CTL. Our data suggest that the strategy of combining LAA-specific CTL with immune checkpoint blockade could prove beneficial in the setting of adoptive T cell therapy and allogeneic stem cell transplantation for AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.