Bispecific T-cell engagers (Bi-TCEs) have revolutionized the treatment and management of both hematological and solid tumor indications with opportunities to become best-in-class therapeutics for cancer. However, defining the dose and dosing regimen for the first-in-human (FIH) studies of Bi-TCEs can be challenging, as a high starting dose can expose subjects to serious toxicity while a low starting dose based on traditional minimal anticipated biological effect level (MABEL) approach could lead to lengthy dose escalations that exposes seriously ill patients to sub-therapeutic dosing. Leveraging our in-depth and broad clinical development experience across three generations of Bi-TCEs across both liquid and solid tumor indications, we developed an innovative modified MABEL approach for starting dose selection that integrates knowledge based on the target biology, indication, toxicology, in vitro, in vivo pharmacological evaluations, and translational pharmacokinetic/pharmacodynamic (PK/PD) modeling, together with anticipated safety profile. Compared to the traditional MABEL approach in which high effector to target (E:T) cell ratios are typically used, our innovative approach utilized an optimized E:T cell ratio that better reflects the tumor microenvironment. This modified MABEL approach was successfully applied to FIH dose selection for a half-life extended (HLE) Bi-TCE for gastric cancer. This modified MABEL approach enabled a 10-fold higher starting dose that was deemed safe and well tolerated and saved at least two dose-escalation cohorts before reaching the projected efficacious dose. This approach was successfully accepted by global regulatory agencies and can be applied for Bi-TCEs across both hematological and solid tumor indications for accelerating the clinical development for Bi-TCEs.

Includes supplemental methods, tables and figures.

Abstract MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo. In vitro, the MUC12 HLE BiTE molecule mediated T-cell–redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 μg/kg with a step dose to 1,000 μg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.

Immune checkpoints are promising targets in cancer therapy. Recently, poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2) have been identified as novel immune checkpoints. In this investigation we show that acute myeloid leukemia (AML) cell lines and AML patient samples highly express the T-cell immunoreceptor with Ig and ITIM domains (TIGIT) ligands PVR and PVRL2. Using two independent patient cohorts, we could demonstrate that high PVR and PVRL2 expression correlates with poor outcome in AML. We show for the first time that antibody blockade of PVR or PVRL2 on AML cell lines or primary AML cells or TIGIT blockade on immune cells increases the anti-leukemic effects mediated by PBMCs or purified CD3+ cells in vitro. The cytolytic activity of the BiTE® antibody construct AMG 330 against leukemic cells could be further enhanced by blockade of the TIGIT-PVR/PVRL2 axis. This increased immune reactivity is paralleled by augmented secretion of Granzyme B by immune cells. Employing CRISPR/Cas9-mediated knockout of PVR and PVRL2 in MV4-11 cells, the cytotoxic effects of antibody blockade could be recapitulated in vitro. In NSG mice reconstituted with human T cells and transplanted with either MV4-11 PVR/PVRL2 knockout or wildtype cells, prolonged survival was observed for the knockout cells. This survival benefit could be further extended by treating the mice with AMG 330. Therefore, targeting the TIGIT-PVR/PVRL2 axis with blocking antibodies might represent a promising future therapeutic option in AML.

Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14+ monocytic MDSCs in newly diagnosed AML that co-express CD33 but lack HLA-DR (HLA-DRlo). AML-blasts induce HLA-DRlo cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33+ MDSCs. In fact, T-cells eliminate IDO+CD33+ MDSCs in the presence of AMG 330. Depletion of total CD14+ cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33+ MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy.

Acute Myeloid Leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 25%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. In this study we sought to investigate whether the CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with documented pre-clinical activity against AML blasts can also target CD33+ AML-MDSCs by redirecting and activating T-cells and thereby enhance AMG 330 mediated anti-leukemic activity.

The last stages of T-lineage-restriction occur in the thymus. Therefore, bone marrow derived thymus-seeding progenitors (TSPs) must continuous migrate and colonize the thymus in order to sustain T-cell production. However, the identity and lineage potentials of TSPs remains elusive, reflecting the inability to visualize and characterize TSPs prior to their seeding of the adult vascularized thymus. Contrarily, the first embryonic TSPs enter a non-vascularized thymus-rudiment, allowing the direct imaging and to establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before entering and interacting with the thymic micro-environment. Previously, hematopoietic stem cells have been suggested to seed the embryonic thymus, whereas other studies were more compatible with initiation of embryonic thymopoiesis by more lineage-restricted progenitors. Using different reporter mouse lines, whole-mount imaging, clonal assays and fate mapping analysis we have showed that the first hematopoietic progenitors migrating to the E11.25 thymic rudiment uniformly express Rag1 prior to their thymus entry and possess combined lymphoid and myeloid (GM), but no erythroid and megakaryocytic potentials at the single cell level. These studies establish the exact lineage commitment step at which the T-lymphocyte lineage restriction process must migrate to the thymus. Moreover, global RNA sequencing of embryonic thymopoiesis-initiating progenitors established their unique molecular signature and insights into pathways critical for the thymus-seeding and T lineage restriction processes. Moreover, Studies on Rbpj-deficient embryos unequivocally disproved claims that canonical Notch-signaling is involved in pre-thymic T-lineage-restriction and in the initial colonization of the embryonic thymus.

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.

Using single-cell transcriptome profiling of pre-granulocyte-macrophage progenitor cells, Nerlov and colleagues identify an early hematopoietic lineage bifurcation that segregates mast cells, eosinophils, megakaryocytes and erythroid cells from monocytes, neutrophils and lymphocytes. According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin−Sca-1+c-Kit+CD34+Flt3hi) and common myeloid progenitors (CMPs) (Lin−Sca-1+c-Kit+CD34+CD41hi) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre–granulocyte-macrophage progenitor (pre-GM) (Lin−Sca-1−c-Kit+CD41−FcγRII/III−CD150−CD105−). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.

1 All articles published in this journal are protected by copyright. 2014 – POST-TRANSCRIPTIONAL REGULATION OF AML PROPAGATION BY ONCOGENIC RNA BINDING PROTEINS Takahiro Ito, Ayuna Hattori 1 1 University of Georgia Acute myelogenous leukemia is characterized by uncontrolled propagation of abnormal myeloid cells with a limited capacity to differentiate into mature cells. Recent studies have shown that cell fate determinants can modulate differentiation potential during stem and progenitor cell division, although their regulatory roles in hematologic malignancies remain poorly understood. In this study we focus on the RNA binding protein MSI2, which is known to play a role in cell fate determination in metazoans. MSI2 is functionally essential for the development of AML or blast crisis CML, and high expression is associated with unfavorable clinical outcomes in AML and ALL. Because MSI2 encodes an RNA binding protein, we hypothesized that MSI2 regulates expression of genes required for aggressive expansion of immature myeloid cells. Through bioinformatics analysis, we identified FLT3 tyrosine kinase as a MSI2 target transcript. RNA immunoprecipitation assays revealed that MSI2 physically interacts with FLT3 mRNA in leukemia cells. Loss of MSI2 functionally impaired clonogenic growth of FLT3-positive AML cells and led to a reduction of FLT3 protein expression without affecting FLT3 mRNA levels. Taken together these data suggest that MSI2 posttranscriptionally regulates FLT3 expression via RNA binding to drive leukemia propagation.

Abstract Background: T-cell activity is regulated by immune checkpoints to maintain the sensitive balance of co-stimulatory and inhibitory immune signals. Therapeutic blockade of checkpoint molecules on tumor or T cells such as CTLA-4 or PD-L1 has shown clinical success in several tumor types including Hodgkin´s disease. Furthermore, Blinatumomab, a bispecific T-cell engager (BiTE antibody construct) directing cytotoxic T cells to CD19 positive leukemic cells has been approved for treatment of acute lymphoblastic leukemia. The CD33 specific BiTE antibody construct AMG 330 has been developed for the therapy of acute myeloid leukemia (AML) and will be evaluated in phase I studies shortly. In our current study, we investigated the therapeutic utility of blockade of the novel checkpoint proteins PVR (poliovirus receptor) and PVRL2 (poliovirus receptor-related 2) alone and in combination with AMG 330 in AML. Methods and results: Samples from 140 treatment naive patients with newly diagnosed AML (AMLSG 07-04, NCT00151242) were analyzed by RT-qPCR for expression of the immune checkpoint molecules PVR, PVRL2 and Galectin-9 (Gal-9). Expression was correlated with patient demographics (age, karyotype, FLT3 mutation status) and clinical survival data by multivariate cox regression. The majority of patients showed mRNA expression of PVR (94%), PVRL2 (95%) and Gal-9 (92%). In a multivariate stepwise cox regression for overall survival, an unfavorable karyotype, high PVR and high Gal-9 expression were identified as independent prognostic markers (p<0.001, HR: 2.10, CI 1.39-3.15 for the karyotype; p=0.001, HR: 1.64, CI 1.21-2.21 for PVR and p<0.001, HR: 0.67, CI 0.54-0.84 for Gal-9). Due to a high correlation between PVR and PVRL2 (Pearson's rho=0.827, p<0.001), PVRL2 was removed during the stepwise process. Nevertheless, if PVR was excluded from the multivariate cox regression, PVRL2 remained as significant term in the stepwise procedure in addition to the karyotype and Gal-9 (p=0.003, HR: 1.58, CI 1.17-2.13 for PVRL2). In a second, independent patient cohort containing microarray-based gene expression and clinical data of 291 AML patients (Verhaak et.al., Haematologica 2009;94) a high PVR and PVRL2 expression in contrast to expression of CD80, CD86 or PD-L1 was associated with poor overall survival (log-rank test p=0.003 and p=0.032, respectively). In in vitro killing assays the therapeutic effect of PVR and PVRL2 blockade was studied by FACS using 7-AAD staining. AML cell lines MV4-11, Kasumi-1 and Molm-13 were preincubated with blocking antibodies against PVR, PVRL2 or both and co-cultured for 24h with peripheral blood mononuclear cells (PBMCs) of healthy donors in the presence or absence of AMG 330. In the absence of AMG 330, the cell kill of MV4-11 increased from 12.6±4.7% (control) to 33.0±8.8% (PVR), to 40.4±10.4% (PVRL2) and to 56.0±12.0% (both PVR + PVRL2). In the presence of suboptimal concentration of AMG 330 (0.1 ng/ml) MV4-11 cell lysis was 29.4±9.0% (AMG 330 alone), 49.7±12.6% (AMG 330 + PVR), 57.9±11.3% (AMG 330 + PVRL2) and 70.0±9.8% (AMG 330 + PVR + PVRL2; n=4, p<0.05 for all comparisons). Comparable results were found for Kasumi-1 and Molm-13 with blockade of both checkpoint inhibitors being the most effective treatment, although additive effects of antibodies against PVR and PVRL2 could not be verified in all cases (data not shown). To confirm specificity of the approach and to exclude effects caused by antibody dependent cellular cytotoxicity (ADCC), PVR and PVRL2 double knockouts of the cell line MV4-11 were generated by CRISPR/Cas-9. Significantly increased killing was observed in PVR and PVRL2 double knockout cells compared to wild-type cells (40.5±8.1 % vs. 25.9±9.1; n=3, p<0.001). Further experiments using an irrelevant antibody against CD117 or Fcγ receptor blockade by purified IgG antibodies excluded ADCC confirming the functional relevance of PVR/PVRL2 blockade. Conclusion: The expression of immune checkpoint ligands PVR and PVRL2 confers a negative prognosis to AML patients possibly due to immune evasion. We could further show that the killing of AML cells by PBMCs could be augmented by blockade of these novel checkpoint inhibitors. Furthermore, addition of PVR and/or PVRL2 blocking antibodies to AMG 330 could enhance cytotoxicity. Therefore, blockade of PVR and PVRL2 represents a promising target for the treatment of AML. Disclosures Kischel: Amgen Research (Munich) GmbH: Employment. Stienen:Amgen Research (Munich) GmbH: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment. Lutteropp:Amgen Research (Munich) GmbH: Employment. Nagorsen:Amgen: Employment, Equity Ownership, Patents & Royalties: Inventor on blinatumomab-related patent.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.

The identification of a functionally distinct subset of haematopoietic stem cells (HSCs) that is primed for platelet-specific gene expression is described; the cells frequently have long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs, and may enable the design of therapies for enhancing platelet reconstitution.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the evolutionary older innate immune-system are formed early in development, while lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we prospectively identified a lympho-myeloid restricted embryonic progenitor as early as E11.5 that expresses IL7Rα and that sustains combined lymphoid and GM transcriptional lineage priming and lineage potentials at the single cell level but no MkE potential. Using Rag1-GFP knock-in mice, we tracked the establishment of this lympho-myeloid restricted progenitor back to as early as E9.5, preceding both hematopoietic colonization of the FL and the establishment of definitive HSCs. Moreover, through in vivo fate mapping we confirmed the inability of Rag1 expressing early embryonic progenitors to contribute to the MkE lineage, while unequivocally and robustly contributing to the myeloid innate as well as lymphoid adaptive immune systems of the mammalian embryo. These findings identify the developmentally earliest immune-restricted progenitor and establish the lympho-myeloid restriction step as a physiologically important lineage commitment step in embryonic mammalian hematopoiesis, preceding the emergence of definitive HSCs.

MicroRNAs (miRs) are involved in many aspects of normal and malignant hematopoiesis, including hematopoietic stem cell (HSC) self-renewal, proliferation, and terminal differentiation. However, a role for miRs in the generation of the earliest stages of lineage committed progenitors from HSCs has not been identified. Using Dicer inactivation, we show that the miR complex is not only essential for HSC maintenance but is specifically required for their erythroid programming and subsequent generation of committed erythroid progenitors. In bipotent pre-MegEs, loss of Dicer up-regulated transcription factors preferentially expressed in megakaryocyte progenitors (Gata2 and Zfpm1) and decreased expression of the erythroid-specific Klf1 transcription factor. These results show a specific requirement for Dicer in acquisition of erythroid lineage programming and potential in HSCs and their subsequent erythroid lineage differentiation, and in particular indicate a role for the miR complex in achieving proper balance of lineage-specific transcriptional regulators necessary for HSC multilineage potential to be maintained.

The stepwise commitment from hematopoietic stem cells in the bone marrow (BM) to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus.

Lymphoid-primed multipotent progenitors with down-regulated megakaryocyte-erythroid (MkE) potential are restricted to cells with high levels of cell-surface FLT3 expression, whereas HSCs and MkE progenitors lack detectable cell-surface FLT3. These findings are compatible with FLT3 cell-surface expression not being detectable in the fully multipotent stem/progenitor cell compartment in mice. If so, this process could be distinct from human hematopoiesis, in which FLT3 already is expressed in multipotent stem/progenitor cells. The expression pattern of Flt3 (mRNA) and FLT3 (protein) in multipotent progenitors is of considerable relevance for mouse models in which prognostically important Flt3 mutations are expressed under control of the endogenous mouse Flt3 promoter. Herein, we demonstrate that mouse Flt3 expression initiates in fully multipotent progenitors because in addition to lymphoid and granulocyte-monocyte progenitors, FLT3(-) Mk- and E-restricted downstream progenitors are also highly labeled when Flt3-Cre fate mapping is applied.

Adult mouse LSK cells unable to undergo autophagy contain fewer HSCs, accumulate mitochondria, and fail to reconstitute lethally irradiated mice.