An Optimized Full-Length FLT3/CD3 Bispecific Antibody Demonstrates Potent Anti-leukemia Activity and Reversible Hematological Toxicity.

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.

The IRF4 Gene Regulatory Module Functions as a Read-Write Integrator to Dynamically Coordinate T Helper Cell Fate.

Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses toward antibody production via Tfh cells or inflammation by Teff cells. Tfh-Teff cell fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6-Blimp-1 counter-antagonism. We found that the TCR-signal-induced transcription factor Irf4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised Irf4 amounts and promoted Teff cell fates at the expense of Tfh ones. Importantly, orthogonal induction of Irf4 expression redirected Tfh cell fate trajectories toward those of Teff. Mechanistically, we linked greater Irf4 abundance with its recruitment toward low-affinity binding sites within Teff cell cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the "reader" of TCR signal strength, and in turn, concentration-dependent activity of Irf4 "writes" T helper fate choice.

The transcription factor lymphoid enhancer factor 1 controls invariant natural killer T cell expansion and Th2-type effector differentiation.

Invariant natural killer T cells (iNKT cells) are innate-like T cells that rapidly produce cytokines that impact antimicrobial immune responses, asthma, and autoimmunity. These cells acquire multiple effector fates during their thymic development that parallel those of CD4(+) T helper cells. The number of Th2-type effector iNKT cells is variable in different strains of mice, and their number impacts CD8 T, dendritic, and B cell function. Here we demonstrate a unique function for the transcription factor lymphoid enhancer factor 1 (LEF1) in the postselection expansion of iNKT cells through a direct induction of the CD127 component of the receptor for interleukin-7 (IL-7) and the transcription factor c-myc. LEF1 also directly augments expression of the effector fate-specifying transcription factor GATA3, thus promoting the development of Th2-like effector iNKT cells that produce IL-4, including those that also produce interferon-γ. Our data reveal LEF1 as a central regulator of iNKT cell number and Th2-type effector differentiation.

Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway.

The chemokine receptor CCR9 controls the immigration of multipotent hematopoietic progenitor cells into the thymus to sustain T cell development. Postimmigration, thymocytes downregulate CCR9 and migrate toward the subcapsular zone where they recombine their TCR ß-chain and γ-chain gene loci. CCR9 is subsequently upregulated and participates in the localization of thymocytes during their selection for self-tolerant receptor specificities. Although the dynamic regulation of CCR9 is essential for early T cell development, the mechanisms controlling CCR9 expression have not been determined. In this article, we show that key regulators of T cell development, Notch1 and the E protein transcription factors E2A and HEB, coordinately control the expression of Ccr9. E2A and HEB bind at two putative enhancers upstream of Ccr9 and positively regulate CCR9 expression at multiple stages of T cell development. In contrast, the canonical Notch signaling pathway prevents the recruitment of p300 to the putative Ccr9 enhancers, resulting in decreased acetylation of histone H3 and a failure to recruit RNA polymerase II to the Ccr9 promoter. Although Notch signaling modestly modulates the binding of E proteins to one of the two Ccr9 enhancers, we found that Notch signaling represses Ccr9 in T cell lymphoma lines in which Ccr9 transcription is independent of E protein function. Our data support the hypothesis that activation of Notch1 has a dominant-negative effect on Ccr9 transcription and that Notch1 and E proteins control the dynamic expression of Ccr9 during T cell development.

Bcl-6 directly represses the gene program of the glycolysis pathway.

Despite the increasing knowledge of the molecular events that induce the glycolysis pathway in effector T cells, very little is known about the transcriptional mechanisms that dampen the glycolysis program in quiescent cell populations such as memory T cells. Here we found that the transcription factor Bcl-6 directly repressed genes encoding molecules involved in the glycolysis pathway, including Slc2a1, Slc2a3, Pkm and Hk2, in type 1 helper T cells (TH1 cells) exposed to low concentrations of interleukin 2 (IL-2). Thus, Bcl-6 had a role opposing the IL-2-sensitive glycolytic transcriptional program that the transcription factors c-Myc and HIF-1α promote in effector T cells. Additionally, the TH1 lineage-specifying factor T-bet functionally antagonized the Bcl-6-dependent repression of genes encoding molecules in the glycolysis pathway, which links the molecular balance of these two factors to regulation of the metabolic gene program.

Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

Targeting the Notch1 and mTOR pathways in a mouse T-ALL model.

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with gamma-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/- leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.

Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.

Activating Notch1 mutations in mouse models of T-ALL.

Recent studies have demonstrated that most patients with T-cell acute lymphocytic leukemia (T-ALL) have activating mutations in NOTCH1. We sought to determine whether these mutations are also acquired in mouse models of T-ALL. We sequenced the heterodimerization domain and the PEST domain of Notch1 in our mouse model of TAL1-induced leukemia and found that 74% of the tumors harbor activating mutations in Notch1. Cell lines derived from these tumors undergo G(0)/G(1) arrest and apoptosis when treated with a gamma-secretase inhibitor. In addition, we found activating Notch1 mutations in 31% of thymic lymphomas that occur in mice deficient for various combinations of the H2AX, Tp53, and Rag2 genes. Thus, Notch1 mutations are often acquired as a part of the molecular pathogenesis of T-ALLs that develop in mice with known predisposing genetic alterations.