Leukemia Antigen Research Articles

Overview on Immunopathology of Chronic Lymphocytic Leukemia and Tumor-Associated Antigens with Therapeutic Applications

Chronic Lymphocytic Leukemia (CLL) is a clinically and biologically heterogeneous disease with a variable clinical course. The induction of a generalized state of immunosuppression, leading to susceptibility to infections and the failure of anti-tumor immune responses, is a key feature of the clinical course of CLL. In addition to B-cell receptor (BCR) signaling in CLL, several receptor tyrosine kinases (RTKs) have been reported to be constitutively active in leukemic B cells, resulting in promoted survival and resistance to apoptosis induced by chemotherapy. Several treatment options are available for CLL, including a watch-and-wait strategy, chemotherapy, targeted therapies, immunotherapies such as adoptive cellular therapy (CAR T-Cell Therapy), stem cell transplantation (allogeneic transplantation), radiation therapy and surgery. The identification of Tumor-Associated Antigens (TAAs) is the bottleneck of tumor immunology and immunotherapy, serving as promising targets for precise diagnosis, monitoring, or therapeutic approaches. Numerous TAAs have been identified, and their application in immunotherapy holds promise for the treatment of CLL. Furthermore, extensive ongoing research aims to identify new cancer TAAs. In this review, our objective is to provide a comprehensive overview of CLL immunology and recent findings regarding advances in TAAs with therapeutic applications in CLL.

Negative expression of CD117 predicted inferior OS and PFS in acute promyelocytic leukemia.

In recent years, the correlation between CD117 antigen and the prognosis of hematological malignancies has been demonstrated. However, there is limited literature on the clinical significance of CD117 antigen in acute promyelocytic leukemia (APL). The aim of this study was to retrospectively analyze the clinical features and prognostic significance of CD117 in APL. In this study, we retrospectively investigated the clinicopathological characteristics, outcome, and prognostic impact of negative CD117 expression (CD117-) in 169 APL patients treated with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) containing regimen. The median follow-up period was 63.0 months. CD117- was detected in 13 APL patients (7.7%). No significant differences were found in baseline characteristics between CD117+ and CD117- subgroups. However, compared to CD117+ APL, the incidence of early death (ED) was significantly higher in CD117- APL (p = 0.023). By multivariate analysis, CD117- was an independent adverse prognostic factor for overall survival (OS) and progression-free survival (PFS) (p = 0.022 and p = 0.014, respectively). To sum up, CD117- is associated with greater risk of ED and has the statistical power to predict inferior OS and PFS, this marker may be considered to build prognostic scores for risk-adapted therapeutic strategies in APL management.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

Biological Role of Extracellular Vesicles in Myeloid Neoplasms: A Systematic Review of the Current Literature

Background and aim: Extracellular vesicles (EVs) are lipid-bilayer membrane vesicles secreted by a variety of cells that carry bioactive molecules like proteins, lipids and nucleic acids. They are of increasing interest as they play an important biological role in cancer by a multitude of cell-extrinsic and cell-intrinsic mechanisms. However, the investigations of their biology in myeloid malignancies has not been systematically reviewed so far. Therefore, the aim of this review was to summarize the currently published literature in the field and provide an overview of their potential clinical applications. Method: We performed a systematic literature research with defined search strings in the Embase, Medline and PubMed Central databases and used a standardized data-extraction form to collect all relevant information. Results: 63 of 1529 studies met the inclusion/exclusion criteria and could be assigned to seven main investigational categories ( A). They comprised studies on their effect on i) prothrombotic activity (n=13), ii) tumor-microenvironment (n=14), iii) malignant transformation (n=5), iv) tumor progression (n=4), v) chemotherapy-resistance (n=5) as well as their use as vi) biomarkers (n=14) and vii) pharmacotherapeutic carriers (n=3) ( B). The most relevant biological mechanism is the transfer of microRNA (miRNA) by EVs from different clonal and non-clonal cells, which target the microenvironment, vascular system, as well as immune cells. All mechanisms identified in our review were assigned to one of the seven main categories and the most relevant are summarized in C: i) Prothrombotic activity: Leukaemic cells shed prothrombotic EVs containing tissue-factor (TF) and anionic phosphatidylserine (PS), which facilitate the recruitment of coagulation factors and a pro-coagulant state ( Zannoni, 2019). Reduction of EVs from platelets and leucocytes after chemotherapy attenuates this pro-coagulant state ( Van Aalderen, 2011). ii) Tumor microenvironment: EVs derived from MDS cells can disrupt the osteolineage differentiation of bone marrow mesenchymal stromal cells (BM-MSCs) and impair hematopoiesis ( Hayashi, 2022). iii) Malignant transformation: EVs containing BCR-ABL1 mRNA are integrated into BM-MSCs, which induce BCR-ABL1 expression and secretion of TGF-β1 leading to enhanced proliferation of BM-MSCs ( Fu, 2017). iv) Tumor progression: EVs derived from leukemic cells promote tumor progression by reducing apoptosis and tumor immune-surveillance. v) Chemotherapy resistance: AML-cells expel PEGylated liposomal doxorubicin (PLD) through EVs thus increasing their resistance ( Hekmatirad, 2021). vi) Disease biomarkers: AML patients at diagnosis have a higher number of EVs carrying the myeloblast markers CD34, CD117, CD33 and HLA-DR than AML patients in remission or healthy controls ( Tzoran, 2015). Based on differential abundancy and the content of proteins, lipids and nucleid acids EVs may be suitable as biomarkers for diagnosis, prognosis, and disease monitoring. vii) Pharmacotherapeutic role: Cytotoxic T-lymphocytes are more effective in killing the parental leukaemic cells after being exposed to their cell-derived EVs. The mechanism is related to leukemia antigen presentation on EVs to dendritic cells (DCs) using ICAM1 and Hsp70 ( Shen C, 2011). Finally, EVs can be used as carriers for bioactive compounds and may expand their potential use for cell-type specific delivery of drugs. Conclusions: EVs open the window for the future investigation of novel pathophysiological mechanisms that will improve our understanding of their role in the biology of myeloid malignancies, Moreover, they hold promise as potential biomarkers and drug carriers for cell-type specific treatment

AML Immunotherapy Using a Novel Tcrm-Based Bispecific Antibody That Targets a Leader Sequence Peptide Derived from Cathepsin G

Introduction: Myeloid azurophil granules provide a rich source of intracellular leukemia antigens. Cathepsin G (CG) is a serine protease that has higher expression in AML blasts in comparison to normal myeloid progenitors. Based on the unique biology of HLA-A*0201 (HLA-A2), in which presentation of leader sequence (LS)-derived peptides is favored, we focused on the LS-CG-derived peptide CG1 (FLLPTGAEA). LS peptides are naturally processed and loaded on HLA-A2, providing an abundant source of surface peptide/HLA (pHLA) targets. We eluted CG1 from the surface of primary HLA-A2 + AML blasts and AML cell lines, and demonstrated CG1-targeting immunity in leukemia patients following allogeneic stem cell transplant, hence highlighting the potential for CG1 to be a promising immunotherapeutic target in AML. Numerous platforms have been developed to target cell surface pHLA complexes. T cell receptor (TCR) mimic (m) antibodies are immunotherapeutic antibodies that target pHLA, which are the natural ligands for the TCR. TCRm antibodies have been a breakthrough in immunotherapy, as they: (1) target intracellular antigens expressed on HLA; (2) are engineered to have high affinity for HLA; (3) can elicit their cytotoxic effect independent of the TCR; and (4) can be produced in large quantities providing an off-the shelf-product for patient use. Here we report on the engineering, preclinical efficacy, and safety evaluation of a novel CG1-targeting, T cell-engager, bispecific antibody (CG1xCD3e), which incorporates a novel TCRm construct that recognizes cell surface CG1/HLA-A2 complexes. Methods: To generate the CG1/HLA-A2-binding monoclonal antibodies, H2L2 human transgenic mice were immunized with CG1/HLA-A2 monomers subcutaneously. The resulting paired heavy and light chains were cloned from the antigen-sorted memory B cells into the single pcDNA3.1(+) vector with both human (h) immunoglobulin (Ig)G1 and hKappa for subsequent ExpiCHO high-throughput transient expression. Antibody binding was assessed by Bio-Layer Interferometry (BLI) and T2 cellular binding assays. Xencor anti-CD3e scFv and Knobs-in-holes technology was applied for the bispecific antibody engineering for xMab format. In vitro activity of CG1xCD3e and T cell cytokine secretion was confirmed using flow cytometry-based T cell-dependent cellular cytotoxicity (TDCC). The specificity of CG1xCD3e for leukemia was assessed using colony forming unit assays. In vivo efficacy was evaluated in NSG mice engrafted with AML cell lines and treated with CG1xCD3e with normal donor peripheral blood mononuclear cells (ND-PBMC). Results: Using BLI, binding avidity of the CG1xCD3e to CD3-Fc, KD= 7.52 x 10-10 M, and affinity to CG1/HLA-A2, KD= 1.28 x 10-10 M were calculated. Using flow cytometry, we confirmed high affinity binding of CG1xCD3e to CG1/HLA on the CG1- expressing EM2 HLA-A2 + cell line, and also demonstrated binding of CG1xCD3e to CD3 using jurkat T cell line. After co-culturing GFP/Luciferase + HLA-A2 + AML (U937-A2 and ML-2) or CML (EM2) cell lines with ND-PBMC and different concentrations of CG1xCD3e bispecific antibody for 24 or 48 hours, flow cytometry analysis demonstrated highly specific CG1/HLA-A2 dependent killing of AML cells by T cells cultured with CG1xCD3e bispecific antibody, in comparison with control bispecific antibody with a disabled CG1 binding arm. This correlated with both tumor- and bispecific antibody- dependent T cell activation and cytokine secretion. To study the in vivo anti-leukemia activity of CG1xCD3e, we engrafted NSG mice with the HLA-A2 + ML-2 and U937-A2 AML cell lines, and treated mice after tumor engraftment with ND-PBMC (1 x 107 cells) and weekly intraperitoneal injections of CG1xCD3e bispecific antibody (0.01 mg/Kg, 0.05 mg/Kg, and 0.1 mg/Kg) or PBS. Bioluminescence imaging demonstrated that the mice treated with ND-PBMC + CG1xCD3e had significantly lower levels of leukemia burden compared to the mice treated with ND-PBMC alone (Figure 1). Lastly CFU assays using HLA-A2 + normal donor marrow in a semi-solid matrix of methylcellulose in the presence of T cells and either CG1xCD3e bispecific antibody or isotype antibody, confirmed specificity of CG1xCD3e bispecific antibody for leukemia. Conclusion: Our study provides strong pre-clinical evidence supporting the targeting of LS- derived peptides, specifically CG1, in the setting of AML using a novel TCRm-based bispecific antibody.

Evaluation of Novel Targets, Including CC-Chemokine Receptor 4, in Adult T-Cell Acute Lymphoblastic Leukemia/Lymphoma: A Mayo Clinic Clinical and Pathologic Study.

Unlike B-cell acute lymphoblastic leukemia/lymphoma (ALL/LBL), there have been few therapeutic advances in T-cell ALL (T-ALL)/LBL, an aggressive ALL/LBL subtype. To perform a focused tissue array study to elucidate tumor markers of therapeutic potential in T-ALL/LBL. Using immunohistochemistry, we evaluated expression of leukemic antigens of interest, specifically CC-chemokine receptor 4 (CCR4), among others, on available remnant diagnostic material, including tumor tissue slides obtained from formalin-fixed, paraffin-embedded preserved tissues. Our analysis identified, for the first time, expression of CCR4 in T-ALL/LBL in 11 of 27 cases (40.7%) and confirmed common expression of BCL2, CD38, and CD47, as reported previously. We also identified the expression of CD123 in 4 of 26 cases (15.4%), whereas BCL6 and PDL1 were expressed in a small number of T-ALL/LBL cases. The potential novel target CCR4 was significantly more common in the Pre/Pro-T immunophenotypic subtype, 6 of 9 (66.7%, P = .01). No additional differences in clinical and epidemiologic variables were noted among positive or negative CCR4 cases. These findings support preclinical and clinical testing of therapies targeting CCR4, CD47, BCL2, CD38, and CD123 in T-ALL/LBL, and may help guide the development of targeted clinical trials in T-ALL/LBL, a rare disease in urgent need of novel therapies.

A Novel Tissue Preservation and Transport Solution as a Substitute for Formalin

Formalin, a common laboratory fixative, is a type 1 carcinogen; a biohazard with risks, environmental, disposal, and legal costs; and a chemical modifier of protein epitopes in tissues. A less-toxic tissue preservation method is therefore badly needed. We have developed a novel tissue preservation medium, Amber, composed of low-potassium dextran glucose, 10% honey, and 1% coconut oil. This study investigates Amber as compared with formalin with respect to the following aspects: (1) histologic preservation, (2) epitope integrity with immunohistochemistry (IHC) and immunofluorescence (IF), and (3) integrity of tissue RNA. Rat and human lung, liver, kidney, and heart tissues were collected and stored for 24 hours at 4 °C in Amber or formalin. The tissues were evaluated with hematoxylin and eosin; IHC: thyroid transcription factor, muscle-specific actin, hepatocyte-specific antigen, and common acute lymphoblastic leukemia antigen; and IF: VE-cadherin, vimentin, and muscle-specific actin. RNA quality upon extraction was also assessed. Amber demonstrated superior and/or noninferior performance in rat and human tissue evaluation with respect to standard techniques of histology, IHC, IF, and extracted RNA quality. Amber maintains high-quality morphology without compromising the ability to perform IHC and nucleic acid extraction. As such, Amber could be a safer and superior substitute to formalin for clinical tissue preservation for contemporary pathological examination.

CD38 as a pan-hematologic target for chimeric antigen receptor T cells

AbstractMany hematologic malignancies are not curable with chemotherapy and require novel therapeutic approaches. Chimeric antigen receptor (CAR) T-cell therapy is 1 such approach that involves the transfer of T cells engineered to express CARs for a specific cell-surface antigen. CD38 is a validated tumor antigen in multiple myeloma (MM) and T-cell acute lymphoblastic leukemia (T-ALL) and is also overexpressed in acute myeloid leukemia (AML). Here, we developed human CD38-redirected T cells (CART-38) as a unified approach to treat 3 different hematologic malignancies that occur across the pediatric-to-adult age spectrum. Importantly, CD38 expression on activated T cells did not impair CART-38 cells expansion or in vitro function. In xenografted mice, CART-38 mediated the rejection of AML, T-ALL, and MM cell lines and primary samples and prolonged survival. In a xenograft model of normal human hematopoiesis, CART-38 resulted in the expected reduction of hematopoietic progenitors, which warrants caution and careful monitoring of this potential toxicity when translating this new immunotherapy into the clinic. Deploying CART-38 against multiple CD38-expressing malignancies is significant because it expands the potential for this novel therapy to affect diverse patient populations.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors.

CLASH of the Titans: How CAR-T Cells Can Triumph Over Tumors.

A stem cell epigenome is associated with primary nonresponse to CD19 CAR T cells in pediatric acute lymphoblastic leukemia

AbstractCD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre–B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children’s Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin–sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.

The Latest Breakthroughs in Immunotherapy for Acute Myeloid Leukemia, with a Special Focus on NKG2D Ligands.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of stem and myeloid progenitor cells. Immunotherapy has revolutionized the care for other cancers such as solid tumors and lymphomas, and has the potential to effectively treat AML. There has been substantial progress in the developments of immunotherapeutic approaches for AML over the last several years, including the development of antibodies that further increase the innate immunogenicity of leukemia cells by the inhibition of NKG2D ligand-particularly MICA and MICB-shedding, chimeric proteins such as IL-15 superagonist that expand natural killer (NK) cells, blockers of immunologic checkpoints such as NKG2A, and chemicals that indirectly increase expression of immune stimulatory proteins in leukemia stem cells. Furthermore, cellular therapies have been designed to enable alloreactive immunity by allogeneic NK cells or target leukemia antigens such as mutated NPM1. These immunotherapeutic approaches have demonstrated remarkable efficacies in preclinical studies and have successfully transitioned to early phase clinical trials, to establish safety and initial signal of clinical activity. Here, we briefly discuss some of the most recent and impactful developments in the AML immunotherapy field and provide our perspectives for the future directions of this exciting and new therapeutic opportunity.

Development of a mRNA Lipid Nanoparticle (mRNA-LNP) Cancer Vaccine to Prevent Leukemia Relapse after Stem Cell Transplant

Introduction The most common acute leukemia in adults is acute myeloid leukemia (AML). Approximately 20,050 new cases and 11,540 deaths are expected for 2022 in the US. Allogeneic stem cell transplant (alloSCT) remains the only cure for high-risk AML. AlloSCTs leverage the immune system to kill leukemic cells through a graft-versus-leukemia (GVL) response mediated by donor CD4+ and CD8+ T cells that recognize recipient leukemic minor histocompatibility antigens (mHAs) as foreign. Unfortunately, transplantation failure related to relapse is common. We postulate that proper targeting of mHAs could enhance leukemia-specific immune responses without off-target effects, improving relapse rates. Using computational modeling, we predicted a series of GVL mHAs, confirmed our first GVL mHA (UNC-GRK4-V) through targeted mass spectrometry, and generated a high-affinity T cell receptor (TCR) against it. Using UNC-GRK4-V as our target, we developed a novel mHA mRNA lipid nanoparticle (LNP) vaccine that we hypothesized could induce an antigen-specific immune response. Methods A custom mRNA was ordered from TriLink that encoded the UNC-GRK4-V peptide linked to an MHC Class I trafficking domain (MITD) and was modified with a 5' cap and polyadenylated tail. We created mRNA LNPs by mixing an ethanol lipid solution made of ionizable lipid (SS-OP, SM-102, or MC3)/ DOPE/Cholesterol/DMG-PEG 2000 with an aqueous solution of mRNA (eGFP or UNC-GRK4-V mRNA) dissolved in a citrated buffer solution. We measured mRNA LNP sizes with a Malvern ZS90 and quantified encapsulation rates using a Quant-it RiboGreen RNA assay kit. Immature monocyte-derived dendritic cells (mo-DCs) were differentiated from monocytes collected from fresh blood and transfected with mRNA LNPs. As a control, we measured eGFP mRNA LNP transfection rates of mo-DCs using flow cytometry. CD8+ T cells were transduced with a retroviral supernatant containing a high-affinity UNC-GRK4-V T-cell receptor and stimulated. We co-cultured mRNA LNP transfected mo-DCs with transduced CD8+ T cells. IFN-g was measured from the co-culture supernatant using IFN-g ELISA assays. We used flow cytometry to determine the cytotoxic killing of transfected mo-DCs by transduced CD8+ T cells. Results To identify the most efficient ionizable lipid platform for mRNA delivery and transfection, we tested three commercially available ionizable lipids: SS-OP, SM-102, and MC3. We found that all three ionizable lipids were easily reproducible with SS-OP 75.2nm ± SD 14.8, MC3 97.0nm ± SD 5.4, SM-102 102.1nm ± SD 0.651. The encapsulation rates of mRNA were 75.4% ± SD 6.3% for SS-OP, 70.5% ± SD 2.6% for MC3, and 73.7% ± SD 0.1% for SM-102. The transfection rate of mo-DCs using eGFP mRNA LNPs were 0.48%, 19.3%, and 62.5% for SS-OP, MC3, and SM-102, respectively. Based on poor transfection rates with SS-OP, we used MC3 and SM-102 LNP formulations to study cytokine expression and cytotoxic killing using a co-culture model of transfected mo-DC with transduced CD8+ T cells. We found a 16.8-fold increase in IFN-g release for the SM-102 based LNPs compared to control empty LNPs (p < 0.0001) and a 4.1-fold increase in IFN-g release for the MC3 based LNPs compared to control empty LNPs (p < 0.0001). We then evaluated SM-102 LNPs for cytotoxic killing as they demonstrated the highest IFN-g release. SM-102 demonstrated increased cell death of 13.5% versus 3.5% for control (empty SM-102) after being normalized for background mo-DC death. Conclusions The SM-102 based UNC-GRK4-V mRNA LNP vaccine elicits robust, antigen-specific T-cell responses and could be a potent therapeutic for decreasing relapse rates post-alloSCT. Furthermore, advances in alloSCT have created regimens that eliminate traditional long-term calcineurin inhibitor use in post-alloSCT, allowing therapeutic cancer vaccines to be given much earlier after transplant.

Modeling Flotetuzumab-Associated CRS in AML Using inVitro and inVivo Preclinical Models

Introduction: Flotetuzumab (FLZ) is a bispecific CD123 x CD3 DART® molecule that is being investigated for the treatment of relapsed and refractory acute myeloid leukemia (AML). FLZ induces artificial immunological synapses between CD3 on cytotoxic T-cells and CD123, which is expressed on AML blasts along with off-target hematopoietic and non-hematopoietic tissues. T-cell activation and expansion requires cytokine release from both innate and adaptive immune cells, which when excessive, can result in cytokine release syndrome (CRS), a potentially life-threatening toxicity that manifests with fever, hypotension, organ dysfunction, respiratory failure, and/or coagulopathy. FLZ-mediated anti-leukemia efficacy is often limited by CRS. Therefore, developing pre-clinical models by which to better understand the mechanisms of CRS induction, including collaborating cell types that produce IL-6 and other inflammatory cytokines, is essential to understanding the biology of CRS and developing mechanistically plausible preventative and treatment strategies. MacroGenics has developed a murine genetic knock-in model that expresses a chimeric murine CD3 epsilon (CD3e) chain bearing the human CD3e epitope recognized by FLZ (hCD3e). These mice are immune competent, maintain normal signaling through the T-cell receptor and expand and secrete cytokines upon binding to FLZ and human CD123. To validate this model, we explore whether T-cells harvested from the spleens of immune competent C57Bl/6 hCD3e mice will bind FLZ and kill human CD123-expressing target cells in vitro. Methods: A murine AML cell line (AML1-GFP FLT3-ITD, DNMT3AR878H) was transduced with human CD123 (hCD123) using a lentiviral vector construct and single cell clones expressing high levels of hCD123 were expanded (AMLGFP-hCD123). Spleens from hCD3eHOM C57Bl/6 mice were harvested for isolation of T-lymphocytes via negative immunomagnetic selection on an AutoMACS machine. In vitro killing assays were set up using purified C57Bl/6 hCD3eHOM T-cells and two clones of AMLGFP-hCD123 cells. The effector to target ratio was 1:1, with 2.5x105 cells per well in a 24-well plate. Flow cytometry/FACS was then performed at 24 hours to assess killing of AMLGFP-hCD123 clones 4 and 14 and T-cell subset number and phenotype. Antibodies to the following antigens were used for FACS: mCD4, hCD3 (epitope SP38), mCD25, mCD8, mCD44, mCD62L, hCD123, mCD3, mCD127, mCD69, and mCD45, in addition to endogenous GFP. Results: At 24 hours, target cell killing of clones 4 and 14 was noted (Figure 1). Compared to the controls treated with FLZ or T-cells alone, targets treated with T-cells + FLZ showed a 2.3- and 2.5-fold decrease in the number of AMLGFP-hCD123 targets in clones 4 and 14, respectively, at 24 hours (Figure 1). In terms of T-cell phenotypic changes, the percentage and MFI expression of activation markers, namely CD25 and CD69, increased for CD4+ and CD8+ subsets in the treatment group compared to controls (data not shown). T-cell subsets were characterized as naïve (CD62L+/CD44-), central memory (CD62L+/CD44+), and effector memory (CD62L-/CD44+), as shown in Figure 2. For CD4+ and CD8+ subsets co-cultured with targets, the addition of FLZ induced phenotypic changes away from naïve and towards central and effector memory subsets (Figure 2). In the two control groups, naïve T-cells predominated at the 24-hour time point (Figure 2). Conclusions: The in vitro killing assay shows that T-cells from the spleens of immune competent C57Bl/6 hCD123eHOM mice recognize FLZ and hCD123 on transduced murine leukemia cells. Co-culture with T-cells + target cells + FLZ showed killing at 24 hours compared to controls. T-cell activation and proliferation were noted based on cell surface markers. The shift in phenotype from naïve to central and effector memory subsets suggests T-cell expansion upon leukemia antigen recognition and portends killing of target cells. Pending in vivo studies will evaluate if FLZ can activate endogenous T cells, induce CRS, and kill AMLGFP-hCD123 targets in the huCD3eHOM mice. If successful, we will use the huCD3eHOM model to test the efficacy of various therapies to mitigate CRS and preserve FLZ-mediated T-cell cytotoxicity against AMLGFP-hCD123 targets in huCD3eHOM mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD70 CAR T cells in AML: Form follows function

Using a multimodal approach toward developing a new CD70-targeted Chimeric antigen receptor (CAR) T cell in acute myeloid leukemia, Leick et al.1 report on their synergetic strategy, which incorporates both CAR T cell construct modifications with enhancement of leukemia antigen expression to improve CAR T cell functionality.

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.

Quantification and Phenotypic Characterization of Extracellular Vesicles from Patients with Acute Myeloid and B-Cell Lymphoblastic Leukemia.

Simple SummaryExtracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular space. They play an important role in cell-to-cell communication by transporting diverse messenger molecules, including DNA, RNA, lipids, and proteins. Cancer cells in patients with acute leukemia also produce EVs that can influence cells from the surrounding bone marrow microenvironment and play a role in disease progression. In our study, we applied several methods such as cryo-electron microscopy, fluorescence triggering flow cytometry, and nanoparticle tracking analysis to quantify and phenotypically characterize EVs from a series of acute leukemia patient blood samples. Our results show how these methods can be used to study the functional role of EVs in leukemia development and to evaluate their potential as targets for therapy and as biomarkers of the disease.Extracellular vesicles (EVs) act in cell-to-cell communication, delivering cargo from donor to recipient cells and modulating their physiological condition. EVs secreted by leukemic blasts in patients with leukemia have been shown to influence the fate of recipient cells in the bone marrow microenvironment. Methods to quantify and to characterize them phenotypically are therefore urgently needed to study their functional role in leukemia development and to evaluate their potential as targets for therapy. We have used cryo-electron microscopy to study morphology and size of leukemic EVs, and nanoparticle tracking analysis and fluorescence triggering flow cytometry to quantify EVs in platelet-free plasma from a small cohort of leukemia patients and healthy blood donors. Additional studies with a capture bead-based assay allowed us to establish phenotypic signatures of leukemic EVs from 17 AML and 3 B-ALL patients by evaluating the expression of 37 surface antigens. In addition to tetraspanins and lineage-specific markers we found several adhesion molecules (CD29, and CD146) to be highly expressed by EVs from B-ALL and several leukemic stem cell antigens (CD44, CD105, CD133, and SSEA-4) to be expressed by EVs from AML patients. Further improvements in analytical methods to study EVs are needed before potentially using them as biomarkers for leukemia prognosis and follow-up.

In Vitro Generated Dendritic Cells of Leukemic Origin Predict Response to Allogeneic Stem Cell Transplantation in Patients With AML and MDS.

Allogeneic stem cell transplantation (alloSCT) is the treatment of choice for many patients with acute myeloid leukemia (AML) and myelodysplastic syndrome. The presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. Conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (alloSCT). Our purpose was to test, whether the ability of malignant blasts to differentiate in vitro toward dendritic cells of leukemic origin (DCleu) is associated with clinical outcome. We isolated leukemic blasts from peripheral blood or bone marrow of AML and myelodysplastic syndrome patients before alloSCT (n=47) or at relapse after alloSCT (n=22). A panel of 6 different assays was used to generate DCleu in vitro. Results were correlated with clinical outcome. DCleu could be generated from all 69 samples. Significantly higher mean frequencies of DCleu were found in clinical long-term responders versus nonresponders to SCT (76.8% vs. 58.8%, P=0.006). Vice versa, the chance for response to SCT was significantly higher, if a DCleu+/dendritic cells (DC) ratio of >50% could be reached in vitro (P=0.004). Those patients were characterized by a longer time to relapse (P=0.04) and by a higher probability for leukemia-free survival (P=0.005). In vitro generation of DC and DCleu from leukemic blasts correlated with the clinical outcome. This observation may support a role of leukemic antigen presentation by "leukemia-derived DC" for the stimulation of an allogeneic immune response in AML.

CD45RO+ T-Cell Add Back and Prophylactic Blinatumomab Administration Post Tcrαβ/CD19-Depleted Haploidentical Transplantation in Pediatric Patients with High Risk Acute Leukemia

For patients receiving haploidentical donor (haplo) hematopoietic cell transplant (HCT), depletion of TCRαβ T cells from the haplo-graft allows for excellent outcomes and low rates of graft-versus-host disease (GVHD), albeit with a significant delay in immune reconstitution (IR). The adoptive transfer of donor T-cells has been used to optimize IR but increases the risk of GVHD. CD45RA-depleted (memory) T-cells are associated with low rates of alloreactivity and thereby GVHD but retain specificity for leukemic and viral antigens. We implemented a prospective trial utilizing escalating doses of CD45RA-depeleted T-cell as addback following TCRαβ/CD19-depleted haploHCT to improve IR. Patients with acute lymphoblastic leukemia (ALL) also received prophylactic Blinatumomab (Blina) following infusion of CD45RA-depeleted T-cell to overcome the risk immune escape secondary to HLA-loss and relapse (NCT03849651).Between 2019 to 2020, 30 pediatric patients (9 males, 21 females) with high-risk acute leukemia were enrolled. Median age at HCT was 8.7 years (range 0.9-18.8). Nineteen patients had ALL, 11 patients had AML. Ten patients were in CR1, 13 in CR2 and 7 in CR3/>. Five patients received prior CD19-CAR T-cell therapy. The donors used were mothers (n=15), fathers (n=13), or sibling/others (n=2). All patients received a reduced intensity preparative regimen consisting of Fludarabine, Melphalan, Cyclophosphamide and Thiotepa. ATG was given on days -5, -4 and -3. Mobilized peripheral blood graft were infused on day 0 with a median number of CD34+ cells, γδ+ T-cells, αβ+ T cells, and B cells of 14.8, 22.7, 0, and 0.09x 10 6/kg, respectively. No GVHD prophylaxis was used post-HCT. Two weeks following engraftment, patients received CD45RA-depleted T-cell addback in 3 escalating doses (DL1: 1x10 5cells/kg, DL2: 1x10 6 cells/kg, DL3:1x10 7 cells/kg).All 30 patients engrafted with a median time for neutrophil and platelet engraftment of 10 (range: 9-11) and 15 (range: 13-20) days, respectively. Infusion of escalating doses of CD45RA-depleted T-cells was well tolerated. One month post infusion, there was a significant increase in the median number of CD3 T-cells, including CD8 and CD45RO+ T-cell subsets (**p<0.01, ***p<0.001, Fig 1A). There was also significant expansion of virus-specific T-cells (VSTs) directed towards Cytomegalovirus (CMV), Adenovirus (AdV), BK, or HHV-6 as shown by Elispot assays (**p<0.01, Fig. 1B). TCR repertoire, as assessed by Vb spectratyping, was broad and comparable to the donor by month 6 post-HCT. The incidence of CMV, AdV, and HHV-6 viremia was 60%, 6.7%, and 16.7% respectively. The median duration of viremia was 4 weeks for CMV (range: 1-13), 3 weeks for AdV (range: 2-13) and 2.5 weeks for HHV-6 (range: 2-20). There were 11 episodes of viral disease (7 colitis, 3 pneumonitis, 1 lymphadenitis). All episodes of viral disease resolved, except 2 that were ongoing at the time of death. The incidence of acute GVHD within 28 days post-infusion after dose level 1, 2 and 3 was 0%, 20% and 10% respectively (p=NS). The cumulative incidence of aGVHD and grade III-IV aGVHD for the entire cohort was 26.7% (12.4-43.3%) and 13.3% (4.1-28.1%) respectively. There was no chronic GVHD; however, follow up is short. Sixteen patients received and tolerated prophylactic Blina infusions. The median time to receiving Blina after CD45RO+ infusion was 29 days (range 15-56). Four of the 16 patients who received Blina relapsed: 3 with CD19+ disease and 1 with CD19-negative disease. With a median follow up of 12.7 months (range 3.5-24.5), 1 year OS and LFS for the cohort was 86.3% (74.6-99.7%) and 69.8% (55.2-88.4%) respectively. The cumulative incidence of relapse was 31.8% (15.3-49.8%) and of non-relapse mortality was 3.3% (0.2-14.8%) respectively.In this interim analysis, addback of CD45RA- depleted T-cells following TCRαβ/CD19-depleted haplo HCT was safe and led to enhanced functional immune reconstitution. Prophylactic infusion of Blina is well tolerated and its use post-transplant warrants further investigation. Analyses into the effect of ATG on immune reconstitution are underway. [Display omitted] DisclosuresSharma: CRISPR Therapeutics: Other, Research Funding; Novartis: Other: Salary support paid to institution; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Medexus Inc: Consultancy; Vindico Medical Education: Honoraria. Gottschalk: Catamaran Bio: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Novartis: Consultancy; Tidal: Consultancy; Tessa Therapeutics: Consultancy. Triplett: Miltenyi: Other: Travel, meeting registration.

Understanding the CD8+ CAR T Cell Response to Antigen-Low Leukemias

Chimeric antigen receptor (CAR) T cell therapy has emerged as a highly efficacious treatment for B-lineage acute lymphoblastic leukemias (B-ALL). However, downregulation of the CAR-targeted antigen on leukemia cells, predicted to reduce cellular avidity, is associated with post-CAR T cell leukemic relapse following CD22 CAR treatment (Fry et al., Nat. Med., 2017). We have observed reduced function of human CAR T cells against low target antigen site density (Ag Lo) human leukemia in immunodeficient mouse models, relative to CARs responding to high-antigen expressing leukemia. Thus, a better understanding of CAR responses to Ag Lo leukemia could help to increase the durability of remissions. We set out to develop a model system in which we could further interrogate the consequences of low-avidity interactions on CAR immunobiology, generating variants of a murine B-ALL driven by the E2A-PBX fusion protein (E2A) with different levels of target antigen to use in an immunocompetent syngeneic mouse model.We observed impaired expansion (p<0.0001) and tumor clearance (p<0.001) of CAR T cells responding to low-antigen variants of E2A (E2A-Ag Lo) as compared to wildtype E2A expressing high levels of antigen (E2A-WT). While CD8+ CAR T cell (CAR8) transcription factor (TF) expression in response to E2A-Ag Lo versus E2A-WT was largely similar early after CAR infusion, by day 9 post-CAR, CAR8s responding to E2A-Ag Lo exhibited decreased expression of multiple TFs, with Eomes (p<0.01), Irf4 (p<0.001) and Blimp1 (p<0.01) showing the largest magnitude change relative to CAR8s responding to E2A-WT. Additionally, CAR8s from mice bearing E2A-Ag Lo became enriched for cells of a “terminally exhausted” phenotype (Eomes+/PD1 Hi/TOX Hi) by day 11 post-CAR, and negatively-enriched for the “progenitor exhausted” (Tcf1+/PD1 Int) phenotype which can be functionally rescued by anti-PD1 therapy (p<0.0001, p<0.01). These data suggest that continual stimulation by low density antigen leads to a gradual reduction in the ability of CAR8s to mount an effector response, and eventually to T cell states with sub-optimal anti-tumor efficacy.Following in vitro stimulation of human CD22 CARs across a range of leukemic antigen densities, we saw that the percentage of CAR+ cells capable of producing IFNγ and IL2 corresponded to target antigen density (p<0.01, p<0.001). As human CARs are commonly manufactured from heterogenous bulk donor T cells, we hypothesized that antigen sensitivity is impacted by the prior antigen-experience of a given T cell. We predicted that T cells which had encountered cognate antigen through their TCR prior to CAR manufacturing (CAR8 AgEx) would have enhanced capacity to respond to low-avidity stimulation compared to CARs manufactured from naïve CD8+ T cells (CAR8 Naïve). We used a well-characterized ovalbumin vaccination model with OT-I TCR-transgenic T cells, allowing defined control of T cell antigen experience, to generate CAR8 AgEx. We found that CAR8 AgEx were highly antigen-sensitive relative to CAR8 Naïve, showing almost no reduction in numbers of cells capable of producing IFNγ and TNFα in vitro against E2A-Ag Lo as compared to E2A-WT. In vivo, CAR8 AgEx showed near complete depletion of E2A-Ag Lo in bone marrow by day 11 post-CAR, while mice treated with CAR8 Naïve maintained a substantial tumor burden (p<0.01). To test our hypothesis in human cells, we manufactured CD22 CAR T cells from naïve (CD45RO-) versus non-naïve (CD45RO+) starting T cell populations, and again found that CAR AgEx outperformed CAR Naïve against Ag Lo leukemia in production of IFNγ and IL2 in vitro (p<0.001, p<0.01) and in early leukemic clearance in vivo (p<0.0001, day 13).In conclusion, we have established a model to study the immunobiology of the CAR T cell response to Ag Lo B-ALL in an intact host. Preliminary findings indicate impaired expansion and tumor clearance of Ag Lo leukemia, associated with altered CAR T cell transcriptional profiles and features of T cell exhaustion. Furthermore, T cell history prior to CAR manufacturing has a drastic impact on the capacity to respond to Ag Lo leukemia. Future studies with this model will expand our characterization of CAR T cells responding to Ag Lo leukemia, with the goal of optimizing antigen sensitivity.We expect that advancing our understanding on the interplay of antigen density and CAR differentiation status will prove useful in developing more effective iterations of this therapy. DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.