Immunotherapy as a turning point in the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant disease of hematopoietic precursors at the earliest stage of maturation, resulting in a clonalproliferation of myoblasts replacing normal hematopoiesis. AML represents one of the most common types of leukemia, mostly affecting elderly patients. To date, standard chemotherapy protocols are only effective in patients at low risk of relapse and therapy-related mortality. The average 5-year overall survival (OS) is approximately 28%. Allogeneic hematopoietic stem cell transplantation (HSCT) improves prognosis but is limited by donor availability, a relatively young age of patients, and absence of significant comorbidities. Moreover, it is associated with significant morbidity and mortality. However, increasing understanding of AML immunobiology is leading to the development of innovative therapeutic strategies. Immunotherapy is considered an attractive strategy for controlling and eliminating the disease. It can be a real breakthrough in the treatment of leukemia, especially in patients who are not eligible forintensive chemotherapy. In this review, we focused on the progress of immunotherapy in the field of AML by discussing monoclonal antibodies (mAbs), immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T cells), and vaccine therapeutic choices

CD16-158-valine chimeric receptor T cells overcome the resistance of KRAS-mutated colorectal carcinoma cells to cetuximab

KRAS mutations hinder therapeutic efficacy of epidermal growth factor receptor (EGFR)-specific monoclonal antibodies cetuximab and panitumumab-based immunotherapy of EGFR+ cancers. Although cetuximab inhibits KRAS-mutated cancer cell growth in vitro by natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), KRAS-mutated colorectal carcinoma (CRC) cells escape NK cell immunosurveillance in vivo. To overcome this limitation, we used cetuximab and panitumumab to redirect Fcγ chimeric receptor (CR) T cells against KRAS-mutated HCT116 colorectal cancer (CRC) cells. We compared four polymorphic Fcγ-CR constructs including CD16158F-CR, CD16158V-CR, CD32131H-CR, and CD32131R-CR transduced into T cells by retroviral vectors. Percentages of transduced T cells expressing CD32131H-CR (83.5 ± 9.5) and CD32131R-CR (77.7 ± 13.2) were significantly higher than those expressing with CD16158F-CR (30.3 ± 10.2) and CD16158V-CR (51.7 ± 13.7) (p < 0.003). CD32131R-CR T cells specifically bound soluble cetuximab and panitumumab. However, only CD16158V-CR T cells released high levels of interferon gamma (IFNγ = 1,145.5 pg/ml ±16.5 pg/ml, p < 0.001) and tumor necrosis factor alpha (TNFα = 614 pg/ml ± 21 pg/ml, p < 0.001) upon incubation with cetuximab-opsonized HCT116 cells. Moreover, only CD16158V-CR T cells combined with cetuximab killed HCT116 cells and A549 KRAS-mutated cells in vitro. CD16158V-CR T cells also effectively controlled subcutaneous growth of HCT116 cells in CB17-SCID mice in vivo. Thus, CD16158V-CR T cells combined with cetuximab represent useful reagents to develop innovative EGFR+KRAS-mutated CRC immunotherapies

In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab

Cetuximab and panitumumab bind the human epidermal growth factor receptor (EGFR). Although the chimeric cetuximab (IgG1) triggers antibody-dependent-cellular-cytotoxicity (ADCC) of EGFR positive target cells, panitumumab (a human IgG2) does not. The inability of panitumumab to trigger ADCC reflects the poor binding affinity of human IgG2 Fc for the FcγRIII (CD16) on natural killer (NK) cells. However, both human IgG1 and IgG2 bind the FcγRII (CD32A) to a similar extent. Our study compares the ability of T cells, engineered with a novel low-affinity CD32A131R-chimeric receptor (CR), and those engineered with the low-affinity CD16158F-CR T cells, in eliminating EGFR positive epithelial cancer cells (ECCs) in combination with cetuximab or panitumumab. After T-cell transduction, the percentage of CD32A131R-CR T cells was 74 ± 10%, whereas the percentage of CD16158F-CR T cells was 46 ± 15%. Only CD32A131R-CR T cells bound panitumumab. CD32A131R-CR T cells combined with the mAb 8.26 (anti-CD32) and CD16158F-CR T cells combined with the mAb 3g8 (anti-CD16) eliminated colorectal carcinoma (CRC), HCT116FcγR+ cells, in a reverse ADCC assay in vitro. Crosslinking of CD32A131R-CR on T cells by cetuximab or panitumumab and CD16158F-CR T cells by cetuximab induced elimination of triple negative breast cancer (TNBC) MDA-MB-468 cells, and the secretion of interferon gamma and tumor necrosis factor alpha. Neither cetuximab nor panitumumab induced Fcγ-CR T antitumor activity against Kirsten rat sarcoma (KRAS)-mutated HCT116, nonsmall-cell-lung-cancer, A549 and TNBC, MDA-MB-231 cells. The ADCC of Fcγ-CR T cells was associated with the overexpression of EGFR on ECCs. In conclusion, CD32A131R-CR T cells are efficiently redirected by cetuximab or panitumumab against breast cancer cells overexpressing EGFR

Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.Johannes Foßelteder, Gabriel Pabst, Tommaso Sconocchia, Angelika Schlacher, Lisa Auinger, Karl Kashofer, Christine Beham-Schmid, Slave Trajanoski, Claudia Waskow, Wolfgang Schöll, Heinz Sill, Armin Zebisch, Albert Wölfler, Daniel Thomas, and Andreas Reinisc

High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor

BACKGROUND Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). METHODS A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. RESULTS MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO-. CONCLUSIONS High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC

Natural humoral immune response to ribosomal P0 protein in colorectal cancer patients

Tumor associated antigens are useful in colorectal cancer (CRC) management. The ribosomal P proteins (P0, P1, P2) play an important role in protein synthesis and tumor formation. The immunogenicity of the ribosomal P0 protein in head and neck, in breast and prostate cancer patients and the overexpression of the carboxyl-terminal P0 epitope (C-22 P0) in some tumors were reported