The engineered CD80 variant fusion therapeutic davoceticept combines checkpoint antagonism with conditional CD28 costimulation for anti-tumor immunity.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia.

With the demonstration of improved survival of some acute myeloid leukemia (AML) patients with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), CD33 has been validated as a target for antigen-specific immunotherapy. Since previous studies identified a CD33 splice variant missing exon 2 (CD33∆E2) and, consequently, the immune-dominant membrane-distal V-set domain, we investigated the expression and functional characteristics of CD33 transcript variants in AML. In primary AML specimens, we not only found full-length CD33 (CD33FL) and CD33∆E2 but also corresponding variants containing an alternate exon 7 predicted to encode a CD33 protein lacking most of the intracellular domain (CD33E7a and, not previously described, CD33∆E2,E7a) in almost all cases. In acute leukemia cell sublines engineered to express individual CD33 splice variants, all splice variants had endocytic properties. CD33FL and CD33E7a mediated similar degrees of GO cytotoxicity, whereas CD33∆E2 and CD33∆E2,E7a could not serve as target for GO. Co-expression of CD33∆E2 did not interfere with CD33FL endocytosis and did not impact CD33FL-mediated GO cytotoxicity. Together, our findings document a greater-than-previously thought complexity of CD33 expression in human AML. They identify CD33 variants that lack exon 2 and are not recognized by current CD33-directed therapeutics as potential target for future unconjugated or conjugated antibodies.

Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia.

PURPOSE: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. EXPERIMENTAL DESIGN: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. RESULTS: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice. CONCLUSIONS: Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR.

High expression of myocyte enhancer factor 2C (MEF2C) is associated with adverse-risk features and poor outcome in pediatric acute myeloid leukemia: a report from the Children's Oncology Group.

BACKGROUND: Recent studies have identified myocyte enhancer factor 2C (MEF2C) as cooperating oncogene in acute myeloid leukemia (AML) and suggested a contribution to the aggressive nature of at least some subtypes of AML, raising the possibility that MEF2C could serve as marker of poor-risk AML and, therefore, have prognostic significance. METHODS: To test this hypothesis, we retrospectively quantified MEF2C expression in pretreatment bone marrow specimens in participants of the AAML0531 trial by reverse-transcriptase polymerase chain reaction and correlated expression levels with disease characteristics and clinical outcome. RESULTS: In all 751 available patient specimens, MEF2C messenger RNA (mRNA) was detectable and varied >3000-fold relative to ß-glucuronidase. Patients with the highest relative MEF2C expression (4th quartile) less likely achieved a complete remission after one course of chemotherapy than the other patients (67 vs. 78 %, P = 0.005). They also had an inferior overall survival (P = 0.014; at 5 years 55 ± 8 vs. 67 ± 4 %), inferior event-free survival (P < 0.001; at 5 years 38 ± 7 vs. 54 ± 4 %), and higher relapse risk than patients within the lower 3 quartiles of MEF2C expression (P < 0.001; at 5 years 53 ± 9 vs. 35 ± 5 %). These differences were accounted for by lower prevalence of cytogenetically/molecularly defined low-risk disease (16 vs. 46 %, P < 0.001) and higher prevalence of standard-risk disease (68 vs. 42 %, P < 0.001) in patients with high MEF2C expression, suggesting that MEF2C cooperates with additional pathogenic abnormalities. CONCLUSIONS: High MEF2C expression identifies a subset of AML patients with adverse-risk disease features and poor outcome. With confirmation that high MEF2C mRNA expression leads to overexpression of MEF2C protein, these findings provide the rationale for therapeutic targeting of MEF2C transcriptional activation in AML.

The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003). AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

Multimerin-1 (MMRN1) as Novel Adverse Marker in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group.

PURPOSE: Exploratory gene expression array analyses suggested multimerin-1 (MMRN1) to be a predictive biomarker in acute myelogenous leukemia (AML). Following up on these studies, we evaluated the role of MMRN1 expression as outcome predictor in two recent Children's Oncology Group trials. EXPERIMENTAL DESIGN: We retrospectively quantified MMRN1 expression in 183 participants of AAML03P1 and 750 participants of AAML0531 by reverse-transcriptase PCR and correlated expression levels with disease characteristics and clinical outcome. RESULTS: In AAML03P1, the highest quartile of MMRN1 expression (expression ≥0.5 relative to ß-glucuronidase; n = 45) was associated with inferior event-free survival (EFS; P < 0.002) and higher relapse risk (P < 0.004). In AAML0531, in which we quantified MMRN1 mRNA for validation, patients with relative MMRN1 expression ≥0.5 (n = 160) less likely achieved remission (67% vs. 77%, P = 0.006), and more frequently had minimal residual disease (43% vs. 24%, P = 0.001) after one induction course. They had inferior overall survival (OS; 44% ± 9% vs. 69% ± 4% at 5 years; P < 0.001) and EFS (32% ± 8% vs. 54% ± 4% at 5 years; P < 0.001) and higher relapse risk (57% ± 10% vs. 35% ± 5% at 5 years; P < 0.001). These differences were partly attributable to the fact that patients with high MMRN1 expression less likely had cytogenetic/molecular low-risk disease (P < 0.001) than those with low MMRN1 expression. Nevertheless, after multivariable adjustment, high MMRN1 expression remained statistically significantly associated with shorter OS (HR, 1.57; 95% confidence interval, 1.17-2.12; P = 0.003) and EFS (HR, 1.34; 1.04-1.73; P = 0.025), and higher relapse risk (HR, 1.40; 1.01-1.94; P = 0.044). CONCLUSIONS: Together, our studies identify MMRN1 expression as a novel biomarker that may refine AML risk stratification.

High expression of suppressor of cytokine signaling-2 predicts poor outcome in pediatric acute myeloid leukemia: a report from the Children's Oncology Group.

Deregulated cytokine signaling is a characteristic feature of acute myeloid leukemia (AML), and expression signatures of cytokines and chemokines have been identified as a significant prognostic factor in this disease. Given this aberrant signaling, we hypothesized that expression of suppressor of cytokine signaling-2 (SOCS2), a negative regulator of cytokine signaling, might be altered in AML and could provide predictive information. Among 188 participants of the Children's Oncology Group AAML03P1 trial, SOCS2 mRNA levels varied > 6000-fold. Higher (> median) SOCS2 expression was associated with inferior overall (60 ± 10% vs. 75 ± 9%, p = 0.026) and event-free (44 ± 10% vs. 59 ± 10%, p = 0.031) survival. However, these differences were accounted for by higher prevalence of high-risk and lower prevalence of low-risk disease among patients with higher SOCS2 expression, limiting the clinical utility of SOCS2 as a predictive marker. It remains untested whether high SOCS2 expression identifies a subset of leukemias with deregulated cytokine signaling that could be amenable to therapeutic intervention.

Cellular determinants for preclinical activity of a novel CD33/CD3 bispecific T-cell engager (BiTE) antibody, AMG 330, against human AML.

CD33 is a valid target for acute myeloid leukemia (AML) but has proven challenging for antibody-drug conjugates. Herein, we investigated the cellular determinants for the activity of the novel CD33/CD3-directed bispecific T-cell engager antibody, AMG 330. In the presence of T cells, AMG 330 was highly active against human AML cell lines and primary AML cells in a dose- and effector to target cell ratio-dependent manner. Using cell lines engineered to express wild-type CD33 at increased levels, we found a quantitative relationship between AMG 330 cytotoxicity and CD33 expression; in contrast, AMG 330 cytotoxicity was neither affected by common CD33 single nucleotide polymorphisms nor expression of the adenosine triphosphate-binding cassette (ABC) transporter proteins, P-glycoprotein or breast cancer resistance protein. Unlike bivalent CD33 antibodies, AMG 330 did not reduce surface CD33 expression. The epigenetic modifier drugs, panobinostat and azacitidine, increased CD33 expression in some cell lines and augmented AMG 330-induced cytotoxicity. These findings demonstrate that AMG 330 has potent CD33-dependent cytolytic activity in vitro, which can be further enhanced with other clinically available therapeutics. As it neither modulates CD33 expression nor is affected by ABC transporter activity, AMG 330 is highly promising for clinical exploration as it may overcome some limitations of previous CD33-targeted agents.

Significance of expression of ITGA5 and its splice variants in acute myeloid leukemia: a report from the Children's Oncology Group.

Acute myeloid leukemia (AML) encompasses a heterogeneous group of diseases, and novel biomarkers for risk refinement and stratification are needed to optimize patient care. To identify novel risk factors, we performed transcriptome sequencing on 68 diagnostic AML samples and identified 2 transcript variants (-E2 and -E2/3) of the α-subunit (ITGA5) of the very late antigen-5 integrin. We then quantified expression of ITGA5 and these splice variants in specimens from participants of the AAML03P1 trial. We found no association between ITGA5 expression and clinical outcome. In contrast, patients with the highest relative expression (Q4) of the -E2/3 ITGA5 splice variant less likely had low-risk disease than Q1-3 patients (21% vs. 38%, P = 0.027). Q4 patients had worse response to chemotherapy with a higher proportion having persistent minimal residual disease (50% vs. 23%, P = 0.003) and inferior overall survival (at 5 years: 48% vs. 67%, P = 0.015); the latter association was limited to low-risk patients (Q4 vs. Q1-3: 56% vs. 85%, P = 0.043) and was not seen in standard-risk (51% vs. 60%, P = 0.340) or high-risk (33% vs. 38%, P = 0.952) patients. Our exploratory studies indicate that transcriptome sequencing is useful for biomarker discovery, as exemplified by the identification of ITGA5 -E2/3 splice variant as potential novel adverse prognostic marker for low-risk AML that, if confirmed, could serve to further risk-stratify this patient subset.

AKT signaling as a novel factor associated with in vitro resistance of human AML to gemtuzumab ozogamicin.

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.