Clinical immunogenicity outcomes from GENEr8-1, a phase 3 study of valoctocogene roxaparvovec, an AAV5-vectored gene therapy for hemophilia A.

Gene transfer therapies utilizing adeno-associated virus (AAV) vectors involve a complex drug design with multiple components that may impact immunogenicity. Valoctocogene roxaparvovec is an AAV serotype 5 (AAV5)-vectored gene therapy for the treatment of hemophilia A that encodes a B-domain-deleted human factor VIII (FVIII) protein controlled by a hepatocyte-selective promoter. Following previous results from the first-in-human phase 1/2 clinical trial, we assessed AAV5-capsid- and transgene-derived FVIII-specific immune responses with 2 years of follow-up data from GENEr8-1, a phase 3, single-arm, open-label study in 134 adult men with severe hemophilia A. No FVIII inhibitors were detected following administration of valoctocogene roxaparvovec. Immune responses were predominantly directed toward the AAV5 capsid, with all participants developing durable anti-AAV5 antibodies. Cellular immune responses specific for the AAV5 capsid were detected in most participants by interferon-γ enzyme-linked immunosorbent spot assay 2 weeks following dose administration and declined or reverted to negative over the first 52 weeks. These responses were weakly correlated with alanine aminotransferase elevations and showed no association with changes in FVIII activity. FVIII-specific cellular immune responses were less frequent and more sporadic compared with those specific for AAV5 and showed no association with safety or efficacy parameters.

Confirmatory detection of neutralizing antibodies to AAV gene therapy using a cell-based transduction inhibition assay.

Successful treatment with adeno-associated virus (AAV)-based gene therapies can be limited by pre-existing anti-AAV antibodies. Cell-based transduction inhibition (TI) assays are useful to characterize the neutralizing potential of anti-AAV antibodies in patient samples. While these assays are commonly used, they are not specific for neutralizing antibodies (NAbs) against AAV, also detecting non-antibody-based factors that inhibit AAV transduction in vitro but may not substantially decrease efficacy in vivo. This paper describes the development and bioanalytical validation of a confirmatory assay to improve the specificity of detecting anti-AAV5 NAbs in cell-based TI assays. Samples that screen positive for transduction inhibitors are subsequently depleted of all classes of immunoglobulins using agarose resins conjugated with protein A, G, and L (AGL), which restores AAV5 transduction for NAb-containing samples. Unconjugated agarose resin serves as a mock control for non-specific depletion effects and facilitates normalization of the transduction efficiencies between an AGL- and mock-treated sample; the normalized value is termed the AGL/mock ratio. During validation, a confirmatory cut point for the AGL/mock ratio was derived; sensitivity, precision, selectivity, and matrix interference were also assessed. This confirmatory TI assay facilitates a characterization of humoral immunity to AAV gene therapy by reliably distinguishing NAbs from non-antibody-based neutralizing factors.

Validation of an IFN-gamma ELISpot assay to measure cellular immune responses against viral antigens in non-human primates.

Adeno-Associated Virus (AAV)-based gene therapy vectors are in development for many inherited human disorders. In nonclinical studies, cellular immune responses mediated by cytotoxic T cells may target vector-transduced cells, which could impact safety and efficacy. Here, we describe the bioanalytical validation of an interferon-gamma (IFN-γ)-based Enzyme-Linked Immunospot (ELISpot) assay for measuring T cell responses against viral antigens in cynomolgus monkeys. Since ELISpots performed with antigen-derived peptides offer a universal assay format, method performance characteristics were validated using widely available peripheral blood mononuclear cells (PBMCs) responsive to cytomegalovirus peptides. The limit of detection and confirmatory cut point were established using statistical methods; precision, specificity, and linearity were confirmed. Monkey PBMCs from an AAV5 gene therapy study were then analyzed, using peptide pools spanning the vector capsid and transgene product. AAV5-specific T cell responses were detected only in 2 of 18 monkeys at Day 28, but not at Day 13 and 56 after vector administration, with no correlation to liver enzyme elevations or transgene expression levels. No transgene product-specific T cell responses occurred. In conclusion, while viral peptide-specific IFN-γ ELISpots can be successfully validated for monkey PBMCs, monitoring peripheral T cell responses in non-clinical AAV5 gene therapy studies was of limited value to interpret safety or efficacy.

Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer.

Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method.

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.

Pharmacokinetic, pharmacodynamic, and immunogenic rationale for optimal dosing of pegvaliase, a PEGylated bacterial enzyme, in adult patients with phenylketonuria.

Phenylketonuria (PKU), a deficiency in the activity of the enzyme phenylalanine hydroxylase, leads to toxic levels of phenylalanine (Phe) in the blood and brain. Pegvaliase (recombinant Anabaena variabilis phenylalanine ammonia lyase conjugated with polyethylene glycol) is approved to manage PKU in patients aged greater than or equal to 18 years in the United States and in patients aged greater than or equal to 16 years in the European Union. Pharmacokinetic, pharmacodynamic, and immunogenicity results from five open-label pegvaliase trials were assessed. Studies with induction/titration/maintenance (I/T/M) dosing regimens demonstrated pharmacokinetic stabilization and sustained efficacy associated with maintenance doses (20, 40, or 60 mg/day). Immune-mediated pegvaliase clearance was high during induction/titration phases when the early immune response was peaking. The combination of low drug dosage and high drug clearance led to low drug exposure and minimal decreases in blood Phe levels during induction/titration. Higher drug exposure and substantial reductions in blood Phe levels were observed later in treatment as drug clearance was reduced due to the maturation of the immune response, which allowed for increased dosing to target levels. The incidence of hypersensitivity reactions was temporally associated with the peaking of the early antidrug immune response and decreased with time as immune response matured after the first 6 months of treatment. These results support an I/T/M dosing regimen and suggest a strategy for administration of other nonhuman biologics to achieve efficacy and improve tolerability.

Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

Activity of transgene-produced B-domain-deleted factor VIII in human plasma following AAV5 gene therapy.

Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain-deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

Depletion of interfering IgG and IgM is critical to determine the role of IgE in pegvaliase-associated hypersensitivity.

Pegvaliase is an enzyme substitution therapy developed to lower blood phenylalanine (Phe) in adults with phenylketonuria (PKU). In phase 3 clinical studies, pegvaliase substantially reduced mean blood Phe in adult subjects with PKU. The most common type of adverse event observed in the pegvaliase clinical program was hypersensitivity adverse events (HAEs), which included occurrences of arthralgia, rash, and pruritis. The most clinically relevant HAEs were acute systemic hypersensitivity reactions consistent with anaphylaxis observed in 4.6% of phase 3 patients. HAEs were more commonly observed around the time of high circulating immune complex (CIC) levels and complement activation, and the majority of subjects that experienced acute systemic hypersensitivity events were able to continue treatment, which is atypical for a classical IgE-mediated anaphylactic event, but common for type III hypersensitivity reactions. To investigate the alternative mechanism of type III hypersensitivity, serum samples collected shortly after hypersensitivity events (in phase 2 and 3 studies) were tested for anti-pegvaliase IgE using custom radioallergosorbent test and/or ImmunoCAP® (ThermoFisher Scientific, Waltham, MA) assay methods. All subjects with acute systemic hypersensitivity that were tested for anti-pegvaliase IgE at or near the time of event with one or both assays tested negative for IgE. As presented here, an investigation using selected study samples with high anti-drug antibody (ADA) titers demonstrated that presence of IgM and/or IgG ADA can interfere with measurement of a human anti-pegvaliase IgE surrogate positive control. A depletion method was therefore developed using protein A- and G-conjugated Sepharose to remove interfering IgG and IgM in serum samples to low levels (<45 mg/dL) before IgE testing. A final 2× concentration step brought the IgE concentration in the depleted sample to approximately the same level of the starting serum. Phase 3 study samples with sufficient volume remaining that previously tested negative for anti-pegvaliase IgE were re-tested after depletion of IgG and IgM. All samples again tested negative, confirming the original test results. Taken together, the clinical presentation, temporal association of HAEs with CIC levels and complement activation, and lack of anti-pegvaliase IgE suggest pegvaliase-associated acute systemic hypersensitivity events were not IgE-mediated. Furthermore, we describe a universal method that is broadly applicable to enzyme therapies for detection of low concentrations of drug-specific IgE in the presence of high titer anti-drug antibodies of different isotypes.

Association of immune response with efficacy and safety outcomes in adults with phenylketonuria administered pegvaliase in phase 3 clinical trials.

BACKGROUND: This study assessed the immunogenicity of pegvaliase (recombinant Anabaena variabilis phenylalanine [Phe] ammonia lyase [PAL] conjugated with polyethylene glycol [PEG]) treatment in adults with phenylketonuria (PKU) and its impact on safety and efficacy. METHODS: Immunogenicity was assessed during induction, upward titration, and maintenance dosing regimens in adults with PKU (n = 261). Total antidrug antibodies (ADA), neutralizing antibodies, immunoglobulin (Ig) M and IgG antibodies against PAL and PEG, IgG and IgM circulating immune complex (CIC) levels, complement components 3 and 4 (C3/C4), plasma Phe, and safety were assessed at baseline and throughout the study. Pegvaliase-specific IgE levels were measured in patients after hypersensitivity adverse events (HAE). FINDINGS: All patients developed ADA against PAL, peaking by 6 months and then stabilizing. Most developed transient antibody responses against PEG, peaking by 3 months, then returning to baseline by 9 months. Binding of ADA to pegvaliase led to CIC formation and complement activation, which were highest during early treatment. Blood Phe decreased over time as CIC levels and complement activation declined and pegvaliase dosage increased. HAEs were most frequent during early treatment and declined over time. No patient with acute systemic hypersensitivity events tested positive for pegvaliase-specific IgE near the time of the event. Laboratory evidence was consistent with immune complex-mediated type III hypersensitivity. No evidence of pegvaliase-associated IC-mediated end organ damage was noted. INTERPRETATION: Despite a universal ADA response post-pegvaliase administration, adult patients with PKU achieved substantial and sustained blood Phe reductions with a manageable safety profile. FUND: BioMarin Pharmaceutical Inc.

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug's pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples. In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-naïve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Antibodies that neutralize cellular uptake of elosulfase alfa are not associated with reduced efficacy or pharmacodynamic effect in individuals with Morquio A syndrome.

Many enzyme replacement therapies (ERTs) for lysosomal storage disorders use the cell-surface cation-independent mannose-6 phosphate receptor (CI-M6PR) to deliver ERTs to the lysosome. However, neutralizing antibodies (NAb) may interfere with this process. We previously reported that most individuals with Morquio A who received elosulfase alfa in the phase 3 MOR-004 trial tested positive for NAbs capable of interfering with binding to CI-M6PR ectodomain in an ELISA-based assay. However, no correlation was detected between NAb occurrence and clinical efficacy or pharmacodynamics. To quantify and better characterize the impact of NAbs, we developed a functional cell-based flow cytometry assay with a titer step that detects antibodies capable of interfering with elosulfase alfa uptake. Serum samples collected during the MOR-004 trial were tested and titers were determined. Consistent with earlier findings on NAb positivity, no correlations were observed between NAb titers and the clinical outcomes of elosulfase alfa-treated individuals with Morquio A.

Leveraging image cytometry for the development of clinically feasible biomarkers: evaluation of activated caspase-3 in fine needle aspirate biopsies.

Quantitation of activated caspases in xenograft models by laser scanning cytometry has demonstrated mechanism-specific biological activity of Anti-Trail Receptor immunoglobulin therapies in situ. These preclinical data confirmed that caspase activation is an early event that precedes tumor regression. To apply this platform for clinical monitoring of caspase activation using fine needle aspirate (FNA) biopsies, additional assay feasibility and validation experiments need be addressed. Furthermore, important instrument parameters should be considered including the maintenance and operation of the cytometer in a controlled state to ensure aspects like data traceability, reliability, and integrity. In the present chapter we describe a method to evaluate caspase activation in Colo205 cells and fine needle aspirate tumors by slide-based, laser scanning cytometry. This approach can be applied to cell cultures, preclinical and clinical fine needle aspirate material.

Measurement of conatumumab-induced apoptotic activity in tumors by fine needle aspirate sampling.

Conatumumab is a monoclonal antibody specific for death receptor 5 (DR5) that activates caspases leading to DNA fragmentation and tumor-cell death. Like other Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) receptor therapies, conatumumab is currently being evaluated in clinical trials across a variety of tumor types. However, molecular evidence of on-target drug activity in tumors is often an elusive goal for clinical investigation. Here we evaluated a translational approach using a relevant biopsy method, fine needle aspirates (FNAs), to study the on-target pharmacodynamics of conatumumab pre-clinically. As detected by laser scanning cytometry, drug-induced caspase-3 activation in FNA biopsies of Colo205 xenografts correlated well with activated caspase-3 in conventional section-based samples. Furthermore, in tumor-bearing mice, surrogate assays of serum caspase-3/7 activity and serum drug exposure correlated with in situ caspase-3 activation. We found that one advantage of FNA sampling over other sampling techniques was the ability to measure caspase activity on a per cell basis using DNA content information. To adapt the utility of FNAs for measuring pharmacodynamic markers in humans, detection of activated caspase-3 was multiplexed with EpCAM to characterize mock and clinical FNAs from colorectal and nonsmall cell lung cancer patients. These data suggest that FNA sampling is a practical method to cytometrically evaluate tumors for pharmacological impact in a clinical setting.

Conatumumab, a fully human agonist antibody to death receptor 5, induces apoptosis via caspase activation in multiple tumor types.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG(1)) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122) and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the antitumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

Antagonists of CD117 (cKit) signaling inhibit mast cell accumulation in healing skin wounds.

Mast cells (MCs) have important functional roles in leukocyte recruitment, pain, and wound healing, and increased tissue resident MC function has been associated with several fibrotic diseases. Consequently, the study of MCs in situ can be a direct approach to studying the pharmacodynamic impact of MC-directed therapeutics in tissues. Here we describe an automated laser scanning cytometry assay that was used to characterize the kinetics of MC accumulation in healing skin wounds and to study the effect of inhibiting CD117 (cKit) signaling. The number of tryptase-positive MCs approximately doubled 14 days after cutaneous injury in nonhuman primates. Treatment of animals with anti-CD117 or imatinib mesylate (Gleevec) reduced MC accumulation at the edge of healing wounds in mice and nonhuman primates, respectively. In translating this MC assay to become a biomarker for human studies, no differences in dermal MC numbers were evident between genders, ages or body mass index from 20 healthy donors. These data suggest that skin is a practical and useful tissue for tracking pharmacodynamic effects of MC-directed therapies.

Signaling and protein associations of a cell permeable CD40 complex in B cells.

Signaling through the CD40 receptor activates diverse molecular pathways in a variety of immune cell types. To study CD40 signaling complexes in B cells, we produced soluble CD40 cytoplasmic domain multimers that translocate across cell membranes and engage intracellular CD40 signaling pathways. As visualized by fluorescence microscopy, rapid transduction of recombinant Antennapedia-isoleucine zipper (Izip)-CD40 cytoplasmic domain fusion protein (Antp-CD40) occurred in both the DND39 B cell line and human tonsillar B cells. Upon cellular entry, Antp-CD40 activated NF-kappaB-dependent transcription, induced proteolytic processing of p100 to the p52/NF-kappaB2 subunit, and increased expression of CD80 and CD54 on the surface of B cells. Antp-CD40 transduction of B cells did not, however, activate detectable levels of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase and did not up-regulate CD95 expression. Analysis of Antp-CD40 complexes recovered from transduced B cells revealed that Antp-CD40 associated with endogenous TRAF3 and Ku proteins. Multimerization of Antp-CD40, or extensive clustering of transmembrane CD40, diminished the disruptive effect of the T254A mutation in the TRAF2/3 binding site of the CD40 cytoplasmic domain. Taken together, these results indicate that Antp-CD40 mimics some of the natural CD40 signaling pathways in B cells by assembling partially functional signaling intermediates that do not require plasma membrane localization. We present a novel approach for delivering pre-activated, soluble receptor cytoplasmic domains into cells and recovering intact signaling complexes for molecular analysis.

Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo.

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors.