Modulation of CD47-SIRPα innate immune checkpoint axis with Fc-function detuned anti-CD47 therapeutic antibody.

Cluster of differentiation 47 (CD47) is a transmembrane protein ubiquitously expressed on human cells but overexpressed on many different tumor cells. The interaction of CD47 with signal-regulatory protein alpha (SIRPα) triggers a "don't eat me" signal to the macrophage, inhibiting phagocytosis. Thus, overexpression of CD47 enables tumor cells to escape from immune surveillance via the blockade of phagocytic mechanisms. We report here the development and characterization of CC-90002, a humanized anti-CD47 antibody. CC-90002 is unique among previously reported anti-CD47 bivalent antibodies that it does not promote hemagglutination while maintaining high-affinity binding to CD47 and inhibition of the CD47-SIRPα interaction. Studies in a panel of hematological cancer cell lines showed concentration-dependent CC-90002-mediated phagocytosis in acute lymphoblastic leukemia, acute myeloid leukemia (AML), lenalidomide-resistant multiple myeloma (MM) cell lines and AML cells from patients. In vivo studies with MM cell line-derived xenograft models established in immunodeficient mice demonstrated significant dose-dependent antitumor activity of CC-90002. Treatment with CC-90002 significantly prolonged survival in an HL-60-disseminated AML model. Mechanistic studies confirmed the binding of CC-90002 to tumor cells and concomitant recruitment of F4-80 positive macrophages into the tumor and an increase in expression of select chemokines and cytokines of murine origin. Furthermore, the role of macrophages in the CC-90002-mediated antitumor activity was demonstrated by transient depletion of macrophages with liposome-clodronate treatment. In non-human primates, CC-90002 displayed acceptable pharmacokinetic properties and a favorable toxicity profile. These data demonstrate the potential activity of CC-90002 across hematological malignancies and provided basis for clinical studies CC-90002-ST-001 (NCT02367196) and CC-90002-AML-001 (NCT02641002).

Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

INBRX-120, a CD8α-targeted detuned IL-2 that selectively expands and activates tumoricidal effector cells for safe and durable in vivo responses.

BACKGROUND: As a major driver of lymphocyte proliferation and activation interleukin 2 (IL-2) is a crucial mediator for antitumor responses. Despite promising activity in a subset of patients, wider therapeutic utility of IL-2 (aldesleukin) has been hampered by severe dose-limiting toxicities, the expansion of immunosuppressive regulatory T cells and a poor pharmacokinetic (PK) profile. Recent engineering efforts, including non-α IL-2 variants, have lowered the toxicity profile, but have yet to induce meaningful antitumor activity in a wider patient population. METHODS: We engineered INBRX-120, a CD8α-targeted Cisleukin™ molecule consisting of an affinity tuned IL-2 (IL2-x) connected to two high affinity CD8α-specific single domain antibodies via an effector-silenced Fc domain. To show that this large affinity differential enables directed IL-2 cis-signaling exclusively on CD8α-expressing tumoricidal effector cell populations, INBRX-120 effects on target cell expansion, activation and antitumor activity were tested in vitro. In vivo antitumor efficacy was evaluated in syngeneic mouse models alone or in combination with programmed cell death protein-1 (PD-1) blockade. Preclinical safety, as well as pharmacodynamic (PD) and PK profiling was carried out in non-human primates. RESULTS: INBRX-120 effectively expanded and enhanced the cytotoxic capacity of CD8 T cells and natural killer cells towards tumor cells without affecting regulatory T cells in vitro and in vivo. In syngeneic mouse models, INBRX-120 surrogate showed safe, potent, and durable antitumor efficacy alone and in combination with PD-1 blockade. In non-human primates, INBRX-120 expanded and activated CD8α-expressing effector cells, showed a favorable PK profile, and was well tolerated up to a dose of 1 mg/kg. CONCLUSIONS: Through its unique cis-signaling activity on CD8α-expressing effector cells, INBRX-120 overcomes the major limitations of IL-2-based therapy and effectively harnesses IL-2's potent intrinsic antitumor activity. This novel therapeutic strategy promises safer clinical activity that could induce meaningful antitumor efficacy in a wider set of patients with various cancer indications.

Notochord-derived BMP antagonists inhibit endothelial cell generation and network formation.

Embryonic blood vessel formation is initially mediated through the sequential differentiation, migration, and assembly of endothelial cells (ECs). While many molecular signals that promote vascular development have been identified, little is known about suppressors of this process. In higher vertebrates, including birds and mammals, the vascular network forms throughout the embryonic disk with the exception of a region along the midline. We have previously shown that the notochord is responsible for the generation and maintenance of the avascular midline and that BMP antagonists expressed by this embryonic tissue, including Noggin and Chordin, can mimic this inhibitory role. Here we report that the notochord suppresses the generation of ECs from the mesoderm both in vivo and in vitro. We also report that the notochord diminishes the ability of mature ECs to organize into a primitive plexus. Furthermore, Noggin mimics notochord-based inhibition by preventing mesodermal EC generation and mature EC network formation. These findings suggest that the mesoderm surrounding the midline is competent to give rise to ECs and to form blood vessels, but that notochord derived-BMP antagonists suppress EC differentiation and maturation processes leading to inhibition of midline vessel formation.

Profiling constitutive proteolytic events in vivo.

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.

Recruitment of the proneural gene scute to the Drosophila sex-determination pathway.

In flies, scute (sc) works with its paralogs in the achaete-scute-complex (ASC) to direct neuronal development. However, in the family Drosophilidae, sc also acquired a role in the primary event of sex determination, X chromosome counting, by becoming an X chromosome signal element (XSE)-an evolutionary step shown here to have occurred after sc diverged from its closest paralog, achaete (ac). Two temperature-sensitive alleles, sc(sisB2) and sc(sisB3), which disrupt only sex determination, were recovered in a powerful F1 genetic selection and used to investigate how sc was recruited to the sex-determination pathway. sc(sisB2) revealed 3' nontranscribed regulatory sequences likely to be involved. The sc(sisB2) lesion abolished XSE activity when combined with mutations engineered in a sequence upstream of all XSEs. In contrast, changes in Sc protein sequence seem not to have been important for recruitment. The observation that the other new allele, sc(sisB3), eliminates the C-terminal half of Sc without affecting neurogenesis and that sc(sisB1), the most XSE-specific allele previously available, is a nonsense mutant, would seem to suggest the opposite, but we show that housefly Sc can substitute for fruit fly Sc in sex determination, despite lacking Drosophilidae-specific conserved residues in its C-terminal half. Lack of synergistic lethality among mutations in sc, twist, and dorsal argue against a proposed role for sc in mesoderm formation that had seemed potentially relevant to sex-pathway recruitment. The screen that yielded new sc alleles also generated autosomal duplications that argue against the textbook view that fruit fly sex signal evolution recruited a set of autosomal signal elements comparable to the XSEs.

Streptolysin O Rapidly Impairs Neutrophil Oxidative Burst and Antibacterial Responses to Group A Streptococcus.

Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance.

N-terminomics: a high-content screen for protease substrates and their cleavage sites.

Proteases play vital roles in many cellular processes and signaling cascades through specific limited cleavage of their targets. It is important to identify what proteins are substrates of proteases and where their cleavage sites are so as to reveal the molecular mechanisms and specificity of signaling. We have developed a method to achieve this goal using a strategy that chemically tags the substrate's alpha amine generated by proteolysis, enriches for tagged peptides, and identifies them using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Peptide MS/MS data are searched against a database to reveal what proteins are cleaved, whereby peptide N-termini demarcate sites of protease cleavage.

Streptolysin O promotes group A Streptococcus immune evasion by accelerated macrophage apoptosis.

Group A Streptococcus (GAS) is a leading human bacterial pathogen capable of producing invasive infections even in previously healthy individuals. As frontline components of host innate defense, macrophages play a key role in control and clearance of GAS infections. We find GAS induces rapid, dose-dependent apoptosis of primary and cultured macrophages and neutrophils. The cell death pathway involves apoptotic caspases, is partly dependent on caspase-1, and requires GAS internalization by the phagocyte. Analysis of GAS virulence factor mutants, heterologous expression, and purified toxin studies identified the pore-forming cytolysin streptolysin O (SLO) as necessary and sufficient for the apoptosis-inducing phenotype. SLO-deficient GAS mutants induced less macrophage apoptosis in vitro and in vivo, allowed macrophage cytokine secretion, and were less virulent in a murine systemic infection model. Ultrastructural evidence of mitochondrial membrane remodeling, coupled with loss of mitochondrial depolarization and cytochrome c release, suggests a direct attack of the toxin initiates the intrinsic apoptosis pathway. A general caspase inhibitor blocked SLO-induced apoptosis and enhanced macrophage killing of GAS. We conclude that accelerated, caspase-dependent macrophage apoptosis induced by the pore-forming cytolysin SLO contributes to GAS immune evasion and virulence.

Structural and kinetic determinants of protease substrates.

Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in alpha-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.

Identification of proteolytic cleavage sites by quantitative proteomics.

The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes) from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10 cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.

The apoptosome activates caspase-9 by dimerization.

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.

The activin signaling pathway promotes differentiation of dI3 interneurons in the spinal neural tube.

The generation of the appropriate types and numbers of mature neurons during the development of the spinal cord requires the careful coordination of patterning, proliferation, and differentiation. In the dorsal neural tube, this coordination is achieved by the combined action of multiple ligands of both the Wnt and TGF-beta families, and their effectors, such as the bHLH proteins. TGF-beta signaling acting through the BMP receptors is necessary for the generation of several dorsal interneuron types. Other TGF-beta ligands expressed in the dorsal neural tube interact with the Activin receptors, which signal via a different set of SMAD proteins than BMPs. The effects of Activin signaling on the developing neural tube have not been described. Here we have activated the Activin signal transduction pathway in a cell-autonomous manner in the developing chick neural tube. We find that a constitutively active Activin receptor promotes differentiation throughout the neural tube. Although most differentiated cell populations are unaffected by Activin signaling, the number of dorsal interneuron 3 (dI3) cells is specifically increased. Our data suggest that Activin signaling may promote the formation of the dI3 precursor cells within a region circumscribed by BMP signaling and that this function is not dependent upon BMP signaling.

Tissue morphogenesis and vascular stability require the Frem2 protein, product of the mouse myelencephalic blebs gene.

Adhesive properties of cells undergoing morphogenetic rearrangements can be regulated either at the cellular level or by altering the environment in which rearrangements occur. Here, we describe the identification of a mutation (my(F11)) in the mouse extracellular matrix component Frem2, and provide evidence that suggests Frem2 expression creates an environment conducive to morphogenetic events. Loss of Frem2 function results in defects in developmental events associated with morphogenetic rearrangements of the vasculature and of tissues arising from all germ layers. The Frem2 transcript is restricted both spatially and temporally and appears in advance of cell rearrangement events. Thus, expression of Frem2 may dynamically alter the extracellular matrix to provide a substrate for cell migration and rearrangements during embryogenesis.

Analysis of mouse embryonic patterning and morphogenesis by forward genetics.

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

BMP signaling patterns the dorsal and intermediate neural tube via regulation of homeobox and helix-loop-helix transcription factors.

In the spinal neural tube, populations of neuronal precursors that express a unique combination of transcription factors give rise to specific classes of neurons at precise locations along the dorsoventral axis. Understanding the patterning mechanisms that generate restricted gene expression along the dorsoventral axis is therefore crucial to understanding the creation of diverse neural cell types. Bone morphogenetic proteins (BMPs) and other transforming growth factor beta (TGFbeta) proteins are expressed by the dorsal-most cells of the neural tube (the roofplate) and surrounding tissues, and evidence indicates that they play a role in assigning cell identity. We have manipulated the level of BMP signaling in the chicken neural tube to show that BMPs provide patterning information to both dorsal and intermediate cells. BMP regulation of the expression boundaries of the homeobox proteins Pax6, Dbx2 and Msx1 generates precursor populations with distinct developmental potentials. Within the resulting populations, thresholds of BMP act to set expression domain boundaries of developmental regulators of the homeobox and basic helix-loop-helix (bHLH) families, ultimately leading to the generation of a diversity of differentiated neural cell types. This evidence strongly suggests that BMPs are the key regulators of dorsal cell identity in the spinal neural tube.

Zic1 represses Math1 expression via interactions with the Math1 enhancer and modulation of Math1 autoregulation.

Math1 is a basic helix-loop-helix transcription factor expressed in progenitor cells that give rise to dorsal commissural interneurons in the spinal cord, granule cells of the cerebellum, and sensory cells in the inner ear and skin. Transcriptional regulation of this gene is tightly controlled both temporally and spatially during nervous system development. The signals that mediate this regulation are likely integrated at the Math1 enhancer, which is highly conserved among vertebrate species. We have identified the zinc-finger transcription factor Zic1 as a regulator of Math1 expression. Zic1 binds a novel conserved site within the Math1 enhancer, and represses both the expression of endogenous Cath1 (chicken homolog of Math1) and the activity of a Math1 enhancer driven lacZ reporter when expressed in chick neural tubes. Repression by Zic1 blocks the autoregulatory activity of Math1 itself. Although previous reports have shown that Zic1 and Math1 are both induced by BMP signaling, these genes appear to have opposing functions, as Math1 acts to promote neuronal differentiation in the chick neural tube and excess Zic1 appears to block differentiation. Zic1-mediated repression of Cath1 transcription may modulate the temporal switch between the progenitor state and differentiating dorsal cell types during neural tube development.