CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia.

Background: Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. Methods: We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells. Results: Bone marrow composition, in particular the abundance of regulatory T cells and CD25 expressing AML cells, correlated strongly with that of the blood in patients with time-matched samples. In addition, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing a FLT3-ITD mutation or treated with a hypomethylating agent in combination with venetoclax. We adopted a patient-centric approach to study AML clusters with CD25 expression and found it most highly expressed on immature phenotypes. Ex vivo treatment of primary AML patient samples with CD25 Mab, a human CD25 specific glycoengineered IgG1 antibody led to the specific killing of two different cell types, CD25+ AML cells and regulatory T cells, by allogeneic Natural Killer cells. Conclusion: The in-depth characterization of patient samples by proteomic and genomic analyses supported the identification of a patient population that may benefit most by harnessing CD25 Mab's dual mode of action. In this pre-selected patient population, CD25 Mab could lead to the specific depletion of regulatory T cells, in addition to leukemic stem cells and progenitor-like AML cells that are responsible for disease progression or relapse.

Longitudinal expression profiling identifies a poor risk subset of patients with ABC-type diffuse large B-cell lymphoma.

Despite the effectiveness of immuno-chemotherapy, 40% of patients with diffuse large B-cell lymphoma (DLBCL) experience relapse or refractory disease. Longitudinal studies have previously focused on the mutational landscape of relapse but fell short of providing a consistent relapse-specific genetic signature. In our study, we have focused attention on the changes in GEP accompanying DLBCL relapse using archival paired diagnostic/relapse specimens from 38 de novo patients with DLBCL. COO remained stable from diagnosis to relapse in 80% of patients, with only a single patient showing COO switching from activated B-cell-like (ABC) to germinal center B-cell-like (GCB). Analysis of the transcriptomic changes that occur following relapse suggest ABC and GCB relapses are mediated via different mechanisms. We developed a 30-gene discriminator for ABC-DLBCLs derived from relapse-associated genes that defined clinically distinct high- and low-risk subgroups in ABC-DLBCLs at diagnosis in datasets comprising both population-based and clinical trial cohorts. This signature also identified a population of <60-year-old patients with superior PFS and OS treated with ibrutinib-R-CHOP as part of the PHOENIX trial. Altogether this new signature adds to the existing toolkit of putative genetic predictors now available in DLBCL that can be readily assessed as part of prospective clinical trials.

Pharmacodynamics and molecular correlates of response to glofitamab in relapsed/refractory non-Hodgkin lymphoma.

Glofitamab, a novel CD20xCD3, T-cell-engaging bispecific antibody, exhibited single-agent activity in Study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation, and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab's clinical activity, including pharmacodynamic profile, mode of action, and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in Study NP30179 received single-dose obinutuzumab pretreatment (1000 mg) 7 days before IV glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per the clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsy samples. Pharmacodynamic modulation was observed with glofitamab treatment, including dose-dependent induction of cytokines, and T-cell margination, proliferation, and activation in peripheral blood. Gene expression analysis of pretreatment tumor biopsy samples indicated that tumor cell intrinsic factors such as TP53 signaling are associated with resistance to glofitamab, but they may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence regarding glofitamab's mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.

KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas.

Loss-of-function mutations in KMT2D are a striking feature of germinal center (GC) lymphomas, resulting in decreased histone 3 lysine 4 (H3K4) methylation and altered gene expression. We hypothesized that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would reestablish H3K4 methylation and restore the expression of genes repressed on loss of KMT2D. KDM5 inhibition increased H3K4me3 levels and caused an antiproliferative response in vitro, which was markedly greater in both endogenous and gene-edited KMT2D mutant diffuse large B-cell lymphoma cell lines, whereas tumor growth was inhibited in KMT2D mutant xenografts in vivo. KDM5 inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signaling and altered expression of B-cell lymphoma 2 (BCL2) family members, including BCL2 itself. KDM5 inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC lymphomas.

Applications and analysis of targeted genomic sequencing in cancer studies.

Next Generation Sequencing (NGS) has dramatically improved the flexibility and outcomes of cancer research and clinical trials, providing highly sensitive and accurate high-throughput platforms for large-scale genomic testing. In contrast to whole-genome (WGS) or whole-exome sequencing (WES), targeted genomic sequencing (TS) focuses on a panel of genes or targets known to have strong associations with pathogenesis of disease and/or clinical relevance, offering greater sequencing depth with reduced costs and data burden. This allows targeted sequencing to identify low frequency variants in targeted regions with high confidence, thus suitable for profiling low-quality and fragmented clinical DNA samples. As a result, TS has been widely used in clinical research and trials for patient stratification and the development of targeted therapeutics. However, its transition to routine clinical use has been slow. Many technical and analytical obstacles still remain and need to be discussed and addressed before large-scale and cross-centre implementation. Gold-standard and state-of-the-art procedures and pipelines are urgently needed to accelerate this transition. In this review we first present how TS is conducted in cancer research, including various target enrichment platforms, the construction of target panels, and selected research and clinical studies utilising TS to profile clinical samples. We then present a generalised analytical workflow for TS data discussing important parameters and filters in detail, aiming to provide the best practices of TS usage and analyses.

Correction: Genomic profiling reveals spatial intra-tumor heterogeneity in follicular lymphoma.

In the original version of this article the authors noted an omission in the author affiliations where the university details: Queen Mary University of London was not included in the original affiliation for the majority of the authors. The correct affiliations are as follows1. Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK3. Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK6. Evolution and Cancer Laboratory, Barts Cancer Institute, Queen Mary University of London, London, UK.

Precision medicine and lymphoma.

PURPOSE OF REVIEW: The treatment of the germinal center lymphomas, diffuse large B cell (DLBCL) and follicular lymphoma, has changed little beyond the introduction of immunochemotherapies. However, there exists a substantial group of patients within both diseases for which improvements in care will involve appropriate tailoring of treatment. RECENT FINDINGS: DLBCL consists of two major subtypes with striking differences in their clinical outcomes paralleling their underlying genetic heterogeneity. Recent studies have seen advances in the stratification of germinal center lymphomas, through comprehensive profiling of 1001 DLBCLs alongside refinements in the identification of high-risk follicular lymphoma patients using m7-FLIPI and 23G models. A new wave of novel therapeutic agents is now undergoing clinical trials for germinal center lymphomas, with BCR and EZH2 inhibitors demonstrating preferential benefit in subgroups of patients. The emergence of cell-free DNA has raised the possibility of dynamic disease monitoring to potentially mitigate the complexity of spatial and temporal heterogeneity, whilst predicting tumor evolution in real time. SUMMARY: Altogether knowledge of the genomic landscape of germinal center lymphomas is offering welcome opportunities in patient risk stratification and therapeutics. The challenge ahead is to establish how best to combine upfront or dynamic prognostication with precision therapies, while retaining practicality in clinical trials and the real-world setting.

Follicular lymphoma, a B cell malignancy addicted to epigenetic mutations.

While follicular lymphoma (FL) is exquisitely responsive to immuno-chemotherapy, many patients follow a relapsing remitting clinical course driven in part by a common precursor cell (CPC) population. Advances in next generation sequencing have provided valuable insights into the genetic landscape of FL and its clonal evolution in response to therapy, implicating perturbations of epigenetic regulators as a hallmark of the disease. Recurrent mutations of histone modifiers KMT2D, CREBBP, EP300, EZH2, ARIDIA, and linker histones are likely early events arising in the CPC pool, rendering epigenetic based therapies conceptually attractive for treatment of indolent and transformed FL. This review provides a synopsis of the main epigenetic aberrations and the current efforts in development and testing of epigenetic therapies in this B cell malignancy.

CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression.

Targeting the fusion oncoprotein BCR-ABL with tyrosine kinase inhibitors has significantly affected chronic myeloid leukemia (CML) treatment, transforming the life expectancy of patients; however the risk for relapse remains, due to persistence of leukemic stem cells (LSCs). Therefore it is imperative to explore the mechanisms that result in LSC survival and develop new therapeutic approaches. We now show that major histocompatibility complex (MHC)-II and its master regulator class II transactivator (CIITA) are downregulated in CML compared with non-CML stem/progenitor cells in a BCR-ABL kinase-independent manner. Interferon γ (IFN-γ) stimulation resulted in an upregulation of CIITA and MHC-II in CML stem/progenitor cells; however, the extent of IFN-γ-induced MHC-II upregulation was significantly lower than when compared with non-CML CD34+ cells. Interestingly, the expression levels of CIITA and MHC-II significantly increased when CML stem/progenitor cells were treated with the JAK1/2 inhibitor ruxolitinib (RUX). Moreover, mixed lymphocyte reactions revealed that exposure of CD34+ CML cells to IFN-γ or RUX significantly enhanced proliferation of the responder CD4+CD69+ T cells. Taken together, these data suggest that cytokine-driven JAK-mediated signals, provided by CML cells and/or the microenvironment, antagonize MHC-II expression, highlighting the potential for developing novel immunomodulatory-based therapies to enable host-mediated immunity to assist in the detection and eradication of CML stem/progenitor cells.

Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition.

A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. SIGNIFICANCE: In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197.

Advances in the molecular diagnosis of diffuse large B-cell lymphoma in the era of precision medicine.

INTRODUCTION: The adoption of high-throughput technologies has led to a transformation in our ability to classify diffuse large B-cell lymphoma (DLBCL) into unique molecular subtypes. In parallel, the expansion of agents targeting key genetic and gene expression signatures has led to an unprecedented opportunity to personalize cancer therapies, paving the way for precision medicine. Areas covered: This review summarizes the key molecular subtypes of DLBCL and outlines the novel technology platforms in development to discriminate clinically relevant subtypes. Expert commentary: The application of emerging diagnostic tests into routine clinical practise is gaining momentum following the demonstration of subtype specific activity by novel agents. Co-ordinated efforts are required to ensure that these state of the art technologies provide reliable and clinically meaningful results accessible to the wider haematology community.

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.