Immunotherapy Of Acute Myeloid Leukemia Research Articles

CARving the Path to Allogeneic CAR T Cell Therapy in Acute Myeloid Leukemia.

Despite advances in the understanding of the genetic landscape of acute myeloid leukemia (AML) and the addition of targeted biological and epigenetic therapies to the available armamentarium, achieving long-term disease-free survival remains an unmet need. Building on growing knowledge of the interactions between leukemic cells and their bone marrow microenvironment, strategies to battle AML by immunotherapy are under investigation. In the current review we describe the advances in immunotherapy for AML, with a focus on chimeric antigen receptor (CAR) T cell therapy. CARs constitute powerful immunologic modalities, with proven clinical success in B-Cell malignancies. We discuss the challenges and possible solutions for CAR T cell therapy development in AML, and examine the path currently being paved by preclinical and clinical efforts, from autologous to allogeneic products.

Monoclonal Antibodies in Acute Myeloid Leukemia—Are We There Yet?

Despite recent advances in the treatment of acute myeloid leukemia (AML), relapses remain high, and long-term survival is poor, emphasizing the need for better treatment options. Development of targeted antibody-based immunotherapeutic agents has been an area of growing research in AML. Target antigens of interest include CD33, CD123, CD47, CD70, FLT3, and CLL-1 because of their high expression on AML blasts and leukemic stem cells. Gemtuzumab ozogamicin, a CD33-directed antibody-drug conjugate, is the only Food and Drug Administration-approved monoclonal antibody (mAb) in AML providing evidence for the potential future role of mAb-based therapies in AML. This article provides an overview of the progress made in targeted immunotherapy in AML, particularly focusing on unconjugated and conjugated mAbs.

Newly Developed Treatments for Acute Lymphoblastic and Acute Myeloid Leukemia

Chemotherapy has been dominating the field of cancer treatment for a long time, however, its limitations have been revealed over time. Therefore, several antigen proteins and chimeric antigen receptorT cells (CAR-T) involved in the immunotherapy of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have been introduced. This paper provides details on the mechanisms, implemented investigations, and drawbacks of the immunotherapy for ALL and AML. Current studies have shown that CAR-T cell therapy can eliminate pediatric ALL relapse along with treating B cell ALL. With the appearance of CAR-T cell therapy, especially CD19-, CD20-, and CD22-directed CAR-T cells, aggressive acute lymphomas involving ALL become treatable. Studies have also shown that AML can be treated with FLT3 inhibitors and immunotherapy including monoclonal antibodies (mbA) and CD33-, CD123-directed CAR-T cells. Anti-CD33 monoclonal antibodies can combine with calicheamicin, a cytotoxic agent in DNA strand cleavage, and monotherapy of gemtuzumab ozogamicin (GO), an antibody-drug conjugate, and this combination has been proved to extend the overall survival of both newly treated patients and R/R AML patients who are unable to tolerate standard chemotherapy.

MAGEA1 and hTERT Peptide Treatment Improves the Potency of The Dendritic Cell- Cytotoxic T Lymphocytes (DC-CTL) Immunotherapy in DAC Treated Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML) is a type of heterogenous malignant hematological disorder. Recently developed immunotherapies such as chimeric antigen receptor T cell (CAR-T) do not demonstrated promising therapeutic results due to the off-target effect. The Dendritic cell-cytotoxic T lymphocyte adoptive immunotherapy (DC-CTL) is one of the recently developed immunotherapies. One of the reasons that DC-CTL does not work well in AML is the lack of antigens with high binding affinity, high antigen presentation potency, and the specificity to AML cells.Methods: DAC was used to treat AML cells to find overexpressed CTAs upon DAC treatment. The overexpression was confirmed at both mRNA and protein level by realtime PCR and western blotting. Peptides was designed by using the NetMHCpan database and EPIP based on the out-screened protein sequences. The peptides were then used to pulse DC-CTL coculture in vitro and tested the cytotoxicity of CTLs in vitro and their cancer inhibition potency in vivo.Results: Two cancer testis antigen (CTA) proteins, MAGEA1 and hTERT, was up-regulated in DAC treated AML cells. DC cells pulsed by the antigen peptides designed based on the sequence of these two proteins demonstrated increased potency to stimulate CTL cells in terms of cytokines secretion. These cytokines included IFN-γ, IL-6, and TNF-α. Moreover, enhanced in vitro cytotoxicity was found in CTL cells treated with peptide pulsed DC cells. AML progress was inhibited by CTA peptides pulsed DC-CTL in a mouse AML model.Conclusions: MAGEA1 and hTERT could possibly serve as specific tumor antigens upon DAC treatment, providing potential targets for the development of immunotherapies for AML in the future.

CD155 and CD112 as possible therapeutic targets of FLT3 inhibitors for acute myeloid leukemia.

Acute myeloid leukemia (AML) relapse is considered to be related to escape from antitumor immunity. Changes in the expression of immune checkpoints, including B7 homolog (H)1 and B7-H2, have been reported to contribute to AML progression. Binding of T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) among other immune checkpoints on natural killer (NK) and T cells to CD155/CD112 in tumors is supposed to be inhibitory; however, the mechanism by which changes in CD155 and CD112 expression affect tumor immunity remains unclear. When the increased expression of CD155 and CD112 activates Raf-MEK-ERK pathway and Raf-MEK-ERK pathway is one of the targets of FMS-like tyrosine kinase 3 (FLT3) inhibition. The present study investigated the alterations in CD155 and CD112 expression under FLT3 inhibition (quizartinib and gilteritinib) and studied its effect on NK and T cell cytotoxicity. CD155 and CD112 expression was analyzed using flow cytometry and reverse transcription-quantitative PCR in AML cell lines with or without FLT3 mutation using FLT3 inhibitors. CD155 and CD112 expression was specifically downregulated by FLT3 inhibition in FLT3-mutated cell lines. Direct cytotoxicity and antibody-dependent cellular cytotoxicity against these cells by NK cells were enhanced. However, the cytotoxicity of γδ T cells with low TIGIT expression compared with NK cells was not enhanced in direct cytotoxicity assay using luciferase luminescence. The analysis of clinical trials from The Cancer Genome Atlas (TCGA) revealed that high CD155 and CD112 expression is associated with poor overall survival. The enhanced cytotoxicity of NK cells against CD155- and CD112-downregulated cells following FLT3 inhibition indicated CD155 and CD112 as possible targets of immunotherapy for AML using FLT3 inhibitors.

Impact of NK Cell Activating Receptor Gene Variants on Receptor Expression and Outcome of Immunotherapy in Acute Myeloid Leukemia.

Natural killer cells are important effector cells in the immune response against myeloid malignancies. Previous studies show that the expression of activating NK cell receptors is pivotal for efficient recognition of blasts from patients with acute myeloid leukemia (AML) and that high expression levels impact favorably on patient survival. This study investigated the potential impact of activating receptor gene variants on NK cell receptor expression and survival in a cohort of AML patients receiving relapse-preventive immunotherapy with histamine dihydrochloride and low-dose IL-2 (HDC/IL-2). Patients harboring the G allele of rs1049174 in the KLRK1 gene encoding NKG2D showed high expression of NKG2D by CD56bright NK cells and a favorable clinical outcome in terms of overall survival. For DNAM-1, high therapy-induced receptor expression entailed improved survival, while patients with high DNAM-1 expression before immunotherapy associated with unfavorable clinical outcome. The previously reported SNPs in NCR3 encoding NKp30, which purportedly influence mRNA splicing into isoforms with discrete functions, did not affect outcome in this study. Our results imply that variations in genes encoding activating NK cell receptors determine receptor expression and clinical outcome in AML immunotherapy.

The Role of T Cell Immunotherapy in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease associated with various alterations in T cell phenotype and function leading to an abnormal cell population, ultimately leading to immune exhaustion. However, restoration of T cell function allows for the execution of cytotoxic mechanisms against leukemic cells in AML patients. Therefore, long-term disease control, which requires multiple therapeutic approaches, includes those aimed at the re-establishment of cytotoxic T cell activity. AML treatments that harness the power of T lymphocytes against tumor cells have rapidly evolved over the last 3 to 5 years through various stages of preclinical and clinical development. These include tissue-infiltrated lymphocytes (TILs), bispecific antibodies, immune checkpoint inhibitors (ICIs), chimeric antigen receptor T (CAR-T) cell therapy, and tumor-specific T cell receptor gene-transduced T (TCR-T) cells. In this review, these T cell-based immunotherapies and the potential of TILs as a novel antileukemic therapy will be discussed.

CD33-directed immunotherapy with third-generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and CD33-edited acute myeloid leukemia and hematopoietic stem and progenitor cells.

Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.

Management of Acute Myeloid Leukemia: Current Treatment Options and Future Perspectives.

Simple SummaryAML is a genetically heterogeneous disease with a median age of diagnosis between 60 and 70 years. Thus, many AML patients are not eligible for intensive chemotherapy. Often, the disease is accompanied by a poor prognosis due to high-risk genetic features or due to antecedent hematologic disorders (e.g., myelodysplastic syndrome). Therefore, AML treatment remains a challenge; even after intensive chemotherapy and allogeneic stem cell transplantation (alloHSCT), AML relapses are regularly observed. Thus, new concepts of AML therapy, considering tailored treatment approaches after comprehensive molecular diagnostic or implementing new immunotherapeutic strategies, are urgently needed. This review provides a detailed overview of recent developments and current promising concepts to improve the treatment and the outcome of AML patients.Treatment of acute myeloid leukemia (AML) has improved in recent years and several new therapeutic options have been approved. Most of them include mutation-specific approaches (e.g., gilteritinib for AML patients with activating FLT3 mutations), or are restricted to such defined AML subgroups, such as AML-MRC (AML with myeloid-related changes) or therapy-related AML (CPX-351). With this review, we aim to present a comprehensive overview of current AML therapy according to the evolved spectrum of recently approved treatment strategies. We address several aspects of combined epigenetic therapy with the BCL-2 inhibitor venetoclax and provide insight into mechanisms of resistance towards venetoclax-based regimens, and how primary or secondary resistance might be circumvented. Furthermore, a detailed overview on the current status of AML immunotherapy, describing promising concepts, is provided. This review focuses on clinically important aspects of current and future concepts of AML treatment, but will also present the molecular background of distinct targeted therapies, to understand the development and challenges of clinical trials ongoing in AML patients.

Therapeutic Targeting of Mesothelin with Chimeric Antigen Receptor Natural Killer Cell Therapy in Acute Myeloid Leukemia

Effective immunotherapy for acute myeloid leukemia (AML) has been limited by the lack of AML-restricted targets (expression in AML but silent in normal hematopoiesis). Current immunotherapies targeting lineage markers CD33 and CD123 (if effective) would lead to prolonged myelosuppression or myeloablation, requiring stem cell transplantation to restore hematopoiesis after treatment. In search for AML-restricted targets, we interrogated the AML transcriptome of nearly 3000 cases in pediatric and young adults and contrasting it to normal bone marrow and peripheral blood CD34+ samples. This extensive discovery effort has identified over 200 AML-restricted targets with mesothelin (MSLN) emerged as one of the highest expressing AML-restricted targets and highly enriched in the KMT2A-rearranged AML subtype. We previously validated cell surface expression of MSLN on both AML blasts and leukemic stem cells (Le et. al. 2021). We further showed efficacy targeting MSLN in AML using antibody-drug conjugates (Kaeding et. al. 2021) and chimeric antigen receptor (CAR) T cells (Le et. al. 2021). Given that natural killer (NK) cells are potent immune effector cells and generally have a more favorable toxicity profile than CAR T cells (i.e without cytokine release syndrome), we developed CAR NK cells targeting MSLN and evaluated their efficacy in AML preclinical models.From primary patient samples, we verified MSLN cell surface expression and showed high correlation between cell surface expression (mean fluorescence intensity, MFI) and transcript expression (TPM, R = 0.72, p = 2.1x10 -8, Figure 1A) . Importantly, MSLN expression was restricted to AML blasts and entirely absent in normal lymphocytes and myeloid cells in individual patients (Figure 1B, C). Having confirmed cell surface and AML-restricted expression of MSLN, we developed CAR NK cells targeting MSLN. The VH and VL sequences from immunotoxin SS1P were used to create the single-chain variable fragment domain of the standard CAR (41-BB and CD3Zeta, Figure 1D). CAR NK cells were generated by transducing NK-92 cells with the MSLN CAR construct. Cytotoxicity of CAR NK cells was evaluated against Nomo-1 AML cell line, which expresses endogenous level of MSLN; MV4;11 and Kasumi-1 cell lines engineered to express MSLN with a lentivirus construct (MV4;11 MSLN+ and Kasumi-1 MSLN+).We initially tested the target specificity of MSLN-directed CAR NK cells against MSLN-positive (Nomo-1, MV4;11 MSLN+ and Kasumi-1 MSLN+) and MSLN-negative (Nomo-1 MSLN KO, MV4;11 and Kasumi-1) cells. CAR NK cells exhibited enhanced cytolytic activity against MSLN-positive but not MSLN-negative AML cells after 12 hours of co-incubation at the indicated effector: target (E:T) ratios (Figure 1E). We next assessed the in vivo efficacy of CAR NK cells. Nomo-1 cells transduced with GFP/Luciferase were transplanted into NSG mice at 1x10 6 cells/mouse. Unmodified or CAR NK cells were infused 1 week following leukemic cell injection at 1x10 7 cells/mouse. Monitoring leukemia burden by bioluminescence IVIS imaging showed that after 4 days post NK injection, the leukemia was significantly reduced in Nomo-1 xenograft mice treated with CAR NK cells compared to mice that received unmodified NK cells (Figure 1F), suggesting highly potent anti-leukemia activity of CAR NK cells.In this study, we demonstrate that the cell surface expression of MSLN is restricted to AML blasts but is entirely silent in normal hematopoietic subsets in individual patients. Previous and current clinical trials utilizing NK-92 cells have demonstrated safety and efficacy in variety of cancers, including AML. Here, we demonstrate that NK-92 cells genetically modified with a CAR to redirect their specificity against MSLN-positive AML cells exhibit potent, target-dependent anti-leukemia activity in vitro and in vivo. These results provide compelling data to evaluate MSLN-directed CAR NK cell therapy in clinical trials for refractory/relapsed AML, especially for high-risk KMT2A-rearranged leukemia where majority of patients express MSLN at diagnosis and relapse. [Display omitted] DisclosuresPardo: Hematologics, Inc.: Current Employment.

Combinatorial Anti-Tumor Activity in Animal Models of a Novel CD123 x CD3 Bispecific Dart® Molecule (MGD024) with Cytarabine, Venetoclax or Azacitidine Supports Combination Therapy in Acute Myeloid Leukemia

Combinatorial Anti-Tumor Activity in Animal Models of a Novel CD123 x CD3 Bispecific Dart® Molecule (MGD024) with Cytarabine, Venetoclax or Azacitidine Supports Combination Therapy in Acute Myeloid Leukemia

Unraveling Subcellular Localization of CD33 Splicing Polymorphism Variant

Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcome. The standard AML therapy has been intense chemotherapy and stem-cell transplantation (SCT). However, the addition of targeted immunotherapeutic agents such as gemtuzumab ozogamicin (GO) to this regimen has shown promise in trials, such as AAML03P1 (NCT00070174) and AAML0531 (NCT00372593) (Cooper et al., 2012; Gamis et al., 2014). Since these trials, GO has been added to the National Comprehensive Cancer Network (NCCN) practice guidelines for use in AML therapy for newly diagnosed and relapse patients, both in combination with chemotherapy and as monotherapy (NCCN., 2018). GO is a humanized monoclonal antibody conjugated to calicheamicin and targets the cell surface antigen CD33, observed on most AML blasts (Bernstein, 2002; Walter et al., 2012). Upon binding to GO, CD33 is internalized and the subsequent release of calicheamicin mediates cytotoxicity. CD33 is a member of the SIGLEC family containing two extracellular domains: IgV and IgC (Andrews et al., 1983). The IgV domain is coded by CD33 exon 2 and is the binding site for GO. A modulatory splicing single nucleotide polymorphism (SNP) rs12459419 (C>T; A14V) in exon 2 results in a shorter isoform (CD33-D2) and has been previously reported to be significantly associated with CD33 abundance on cell surface as well as clinical benefit from GO addition to standard chemotherapy. Patients with CC genotype (~50% of the study cohort) expressed full length CD33 (CD33-FL) and benefitted significantly from GO. However, heterozygous CT patients did not derive the same benefit and were shown to have intermediate CD33 levels (Lamba et al., 2017). The biological rationale for this discrepancy is poorly understood, with a major barrier being the lack of antibodies binding the IgC domain, and by extension the only extracellular domain of CD33-D2. To address this, we generated novel antibodies directed against the IgC domain and have recently shown that CD33-D2 is variably expressed on the surface of AML cell lines as well as primary cells (Gbadamosi et al., 2021). In this study, we have further explored the subcellular localization of both CD33-D2 and CD33-FL. Using pMXs retroviral particles encoding CD33-FL or CD33-D2, we engineered CRSIPR/Cas mediated CD33 KO AML cell lines to overexpress either of the two CD33 isoforms. Cells were then screened and confirmed for expression using either P67.6 or HL2541, which recognizes CD33-D2 (Fig. 1). We used immunofluorescence microscopy to determine subcellular localization of CD33 isoforms in these cell lines with the following organelles and their specific markers: endoplasmic reticulum (calreticulin), peroxisomes (PMP70), or lysosomes (LAMP1). Maximum projections from Z-stacks show that both CD33-FL and CD33-D2 localize only to peroxisomes in AML cells under normal, unperturbed conditions (Fig. 2). With this, our group has shown the cell surface and subcellular localization of CD33-D2 for the first time in AML cells. Ongoing studies are focused on determining the differences in CD33 glycosylation patterns as well cytokine signaling in AML cells expressing these variants. Our finding adds to the growing knowledge of CD33-D2 biology and sets the platform for further functional studies to gain mechanistic insights into its role in response to AML immunotherapy. The study was supported by Leukemia Lymphoma Society and St Baldrick's Foundation. [Display omitted] DisclosuresHylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company.

Hepcidin‐Based Nanocomposites for Enhanced Cancer Immunotherapy by Modulating Iron Export‐Mediated N6‐Methyladenosine RNA Transcript

AbstractDespite recent advances in targeted therapies and immunotherapies in acute myeloid leukemia, patient prognosis is still dismal given the high frequency of refractory cases or post‐remission relapse. Ferrotherapy, a novel anti‐cancer treatment strategy using iron‐based compounds, can mechanistically bypass the chemoresistance of cancer cells. However, cancer cells often overexpress the transmembrane iron exporter ferroportin that expels accumulated iron into the plasma, which can potentially limit the efficacy of ferrotherapy and lead to systemic toxic effects. Here, a ferro‐therapeutic nanoplatform is designed using fusing hepcidin and leukemia cell‐membrane vesicles on gold nanoparticles (AuNPs)‐loaded hollow mesoporous Prussian Blue (AuPB@LMHep). AuPB@LMHep specifically targeted leukemia cells through ferroportin and is subsequently internalized and disintegrated into iron and AuNPs through autophagy. The accumulation of iron due to blockade of ferroportin‐mediated iron efflux and the depletion of glutathione by the AuNPs triggered ferroptosis. Furthermore, iron accumulation inactivated the endogenous iron‐dependent m6A demethylase, thereby increasing global m6A RNA modification and suppressing multiple oncogenes. Finally, AuPB@LMHep enhanced the immune response to anti‐programmed death ligand1 treatment via increasing cytotoxic tumor‐infiltrating T cells. Pre‐clinical findings provide the proof‐of‐concept of hepcidin‐based nanocomposite as an “epigenetic drug” and immunotherapeutic agent for treating leukemia.

An IDO1-related immune gene signature predicts overall survival in acute myeloid leukemia

AbstractThe contribution of the bone marrow (BM) immune microenvironment to acute myeloid leukemia (AML) development is well-known, but its prognostic significance is still elusive. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is an interferon-γ-inducible mediator of immune tolerance. With the aim to develop a prognostic IDO1-based immune gene signature, biological and clinical data of 982 patients with newly diagnosed, nonpromyelocytic AML were retrieved from public datasets and analyzed using established computational pipelines. Targeted transcriptomic profiles of 24 diagnostic BM samples were analyzed using the NanoString's nCounter platform. BIN1 and IDO1 were inversely correlated and individually predicted overall survival. PLXNC1, a semaphorin receptor involved in inflammation and immune response, was the IDO1-interacting gene retaining the strongest prognostic value. The incorporation of PLXNC1 into the 2-gene IDO1-BIN1 score gave rise to a powerful immune gene signature predicting survival, especially in patients receiving chemotherapy. The top differentially expressed genes between IDO1lowand IDO-1high and between PLXNC1lowand PLXNC1high cases further improved the prognostic value of IDO1 providing a 7- and 10-gene immune signature, highly predictive of survival and correlating with AML mutational status at diagnosis. Taken together, our data indicate that IDO1 is pivotal for the construction of an immune gene signature predictive of survival in AML patients. Given the emerging role of immunotherapies for AML, our findings support the incorporation of immune biomarkers into current AML classification and prognostication algorithms.

Trial Watch: Adoptive TCR-Engineered T-Cell Immunotherapy for Acute Myeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is a type of blood cancer with an extremely grim prognosis. This is due to the fact that the majority of patients will relapse after frontline treatment. Overall survival of relapsed AML is very low, and treatment options are few. T lymphocytes harnessed with antitumor T-cell receptors (TCRs) can produce objective clinical responses in certain cancers, such as melanoma, but have not entered the main road for AML. In this review, we describe the current status of the field of TCR-T-cell therapies for AML.Despite the advent of novel therapies, acute myeloid leukemia (AML) remains associated with a grim prognosis. This is exemplified by 5-year overall survival rates not exceeding 30%. Even with frontline high-intensity chemotherapy regimens and allogeneic hematopoietic stem cell transplantation, the majority of patients with AML will relapse. For these patients, treatment options are few, and novel therapies are urgently needed. Adoptive T-cell therapies represent an attractive therapeutic avenue due to the intrinsic ability of T lymphocytes to recognize tumor cells with high specificity and efficiency. In particular, T-cell therapies focused on introducing T-cell receptors (TCRs) against tumor antigens have achieved objective clinical responses in solid tumors such as synovial sarcoma and melanoma. However, contrary to chimeric antigen receptor (CAR)-T cells with groundbreaking results in B-cell malignancies, the use of TCR-T cells for hematological malignancies is still in its infancy. In this review, we provide an overview of the status and clinical advances in adoptive TCR-T-cell therapy for the treatment of AML.

Parallel evaluation of cell‑based phage display panning strategies: Optimized selection and depletion steps result in AML blast‑binding consensus antibodies.

Phage display technology (PD) is a powerful technique for the generation of tumor-targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell-based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell-based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast-binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor-targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML-binding phages during panning. Furthermore, suggesting a common design-related mechanism using a ‘single-pot’ PD library, such as the well-known Tomlinson single-chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast-binding phage particles were isolated and soluble scFv-Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv-Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.

Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design

BackgroundSuccessful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent...

Paving the Way for Immunotherapy in Pediatric Acute Myeloid Leukemia: Current Knowledge and the Way Forward.

Simple SummaryImmunotherapy may be an attractive treatment option to increase survival, and to reduce treatment-related side effects, for children with acute myeloid leukemia (AML). While immunotherapies have shown successes in many cancer types, the development and subsequent clinical implementation have proven difficult in pediatric AML. To expedite the development of immunotherapy, it will be crucial to understand which pediatric AML patients are likely to respond to immunotherapies. Emerging research in solid malignancies has shown that the number and phenotype of immune cells in the tumor microenvironment is predictive of response to several types of immunotherapies. Such a predictive model may also be applicable for AML and, thus, knowledge on the immune cells infiltrating the bone marrow environment is needed. Here, we discuss the current state of knowledge on these infiltrating immune cells in pediatric AML, as well as ongoing immunotherapy trials, and provide suggestions concerning the way forward.Immunotherapeutic agents may be an attractive option to further improve outcomes and to reduce treatment-related toxicity for pediatric AML. While improvements in outcome have been observed with immunotherapy in many cancer types, immunotherapy development and implementation into patient care for both adult and pediatric AML has been hampered by an incomplete understanding of the bone marrow environment and a paucity of tumor-specific antigens. Since only a minority of patients respond in most immunotherapy trials across different cancer types, it will be crucial to understand which children with AML are likely to respond to or may benefit from immunotherapies. Immune cell profiling efforts hold promise to answer this question, as illustrated by the development of predictive scores in solid cancers. Such information on the number and phenotype of immune cells during current treatment regimens will be pivotal to generate hypotheses on how and when to intervene with immunotherapy in pediatric AML. In this review, we discuss the current understanding of the number and phenotype of immune cells in the bone marrow in pediatric AML, ongoing immunotherapy trials and how comprehensive immune profiling efforts may pave the way for successful clinical trials (and, ultimately, implementation into patient care).

Siglec-7 is an indicator of natural killer cell function in acute myeloid leukemia

Immune dysfunction is an established risk factor in acute myeloid leukemia (AML). The cytotoxicity of natural killer (NK) cells is greatly impaired in AML, and the profile of NK cell receptors is markedly altered in AML; however, this is not yet well characterized. In this study, we found the downregulation of Siglec-7 could be utilized as a potential marker of NK cell dysfunction in AML. The absolute numbers and percentages of NK cells were declined in the peripheral blood of patients with AML, and the levels of activating receptors NKG2D, NKp46, and NKp30 were reduced in NK cells from patients with AML compared with healthy controls. In contrast, the levels of inhibitory receptors TIM-3, ILT-4, ILT-5, and PD-1 were increased in NK cells from patients with AML. Of note, the level of Siglec-7 in NK cells from patients with AML was significantly lower than that in NK cells from healthy controls, and Siglec-7+ NK cells displayed higher levels of activating receptors and stronger cytotoxicity when compared with Siglec-7– NK cells. Our data indicate that decreased Siglec-7 level may predict NK cell dysfunction in AML, and NK cells may be promising targets of immunotherapy for AML.

Abstract 525: Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies

Abstract Purpose: The medical need due to high mortality in acute myeloid leukemia (AML) remains high, and the treatment of relapsed or refractory AML continues to be therapeutically challenging. MYLOTARG, the only approved anti-CD33 antibody drug conjugate (ADC), has provided proof-of-concept for targeted immunotherapies in AML. Currently, a plethora of ADCs and T-cell engager (TCE) therapies have entered clinical development in AML, but those therapies are often accompanied by dose limiting toxicities, preventing dose escalation to desired anti-tumor efficacy. The biggest challenges seem to be limited target specificity and hyperstimulation of the immune system leading to e.g. myelotoxicities and cytokine release syndrome, respectively. Therefore, more selective therapies are needed to allow for robust anti-tumor activity with a more acceptable safety profile. Experimental design: To address the selectivity challenge, we have generated multi-specific T-cell engaging DARPin® molecules, targeting two different tumor associated antigens (TAAs) with optimized affinity for their targets. In order to find the right target combination, the optimal affinity to increase tumor specificity via avidity, as well as the best molecular architecture, we took advantage of our unique modular DARPin® platform and screened 1000s of combinations of multi-specific DARPin® molecules, binding simultaneously to multiple TAAs in conjunction with our CD3-binding DARPin® molecule. Results: We constructed multi-specific TCEs targeting two different AML antigens with optimized affinity leading to a substantial avidity gain when both targets are co-expressed on tumor cells. The avidity gain resulted in strongly enhanced in vitro potency as shown by activation of both CD8+ and CD4+ T cells and subsequent killing of AML tumor cells, with bioactivities in the range of established TCE benchmark formats (e.g. BiTE® and DART®). In contrast, in an ex vivo whole blood assay the multi-specific DARPin® constructs induced profoundly less cytokine release as compared to benchmark molecules indicating an improved therapeutic window. Finally, we also demonstrated tumor regression in PMBC humanized mouse models bearing MOLM-13 tumors, using both half-life extended (HLE) and non-HLE lead constructs. In conclusion, we have generated TCEs based on multi-specific DARPin® constructs with high potency, selectivity and ultimately with the potential for an improved therapeutic window for the treatment of AML. Citation Format: Nina Reschke, Thamar Looser, Jennifer Krieg, Matteo Bianchi, Patricia Schildknecht, Nicole Bassler, Yvonne Gruebler, Sebastian Grimm, Laura Jeanbart, Tanja Hospodarsch, Alexandra Neculcea, Daniel Steiner, Bernd Schlereth, Christian Reichen. Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 525.