Toxicity In Nonhuman Primates Research Articles

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Abstract 5313: CB307: A dual targeting costimulatory Humabody® VH therapeutic for treating PSMA-positive tumors

Abstract INTRODUCTION: CD137 is a T and NK cell costimulatory receptor involved in consolidating immunological responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumour toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumour cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL PROCEDURES: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody® VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumour microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse anti-tumour pharmacology studies. Non-clinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with anti-tumour activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 immunohistochemistry (IHC) assay demonstrated a higher prevalence of CD137-postive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in non-human primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T cell activation thereby alleviating toxicological concerns against unrestricted CD137 agonism. Citation Format: Sophie Archer, Phillip M. Brailey, Minjung Song, Phillip D. Bartlett, Ines Figueiredo, Bora Gurel, Christina Guo, Verena Brucklacher-Waldert, H. Lorraine Thompson, Jude Akinwale, Samantha E. Boyle, Christine Rossant, Neil R. Birkett, Julia Pizzey, Mark Maginn, James Legg, Richard Williams, Colette M. Johnston, Philip Bland-Ward, Johann S. de Bono, Andrew J. Pierce. CB307: A dual targeting costimulatory Humabody® VH therapeutic for treating PSMA-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5313.

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.

Safety and immunogenicity of the anti-cocaine vaccine UFMG-VAC-V4N2 in a non-human primate model.

A promising strategy for cocaine addiction treatment is the anti-drug vaccine. These vaccines induce the production of anticocaine antibodies, capable of linking to cocaine, and decrease the passage of cocaine throughout the blood-brain barrier, decreasing drug activity in the brain. Our research group developed a new vaccine candidate, the UFMG-V4N2, to treat cocaine use disorders (CUD) using an innovative carrier based on calixarenes. This study assessed the safety and immunogenicity of the anti-cocaine vaccine UFMG-VAC-V4N2 in a non-human primate toxicity study using single and multiple vaccine doses. The UFMG-VAC-V4N2 yielded only mild effects in the injection site and did not influence the general health, feeding behavior, or hematological, renal, hepatic, or metabolic parameters in the vaccinated marmosets. The anti-cocaine vaccine UFMG-VAC-V4N2 presented a favorable safety profile and induced the expected immune response in a non-human primate model of Callithrix penicillata. This preclinical UFMG-VAC-V4N2 study responds to the criteria required by international regulatory agencies contributing to future anticocaine clinical trials of this anti-cocaine vaccine.

A CD79b Targeting ADC with Superior Anti-Tumor Activity and Safety Resulting in Significantly Improved Therapeutic Index (TI): Safe and Efficacious CD79b ADC

The Araris' site-specific and 1-step enzymatic linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC (ARC-02) with this technology showing a higher therapeutic index (TI) compared to the approved ADC polatuzumab-vedotin (Polivy®) in different preclinical models. Therefore, ARC-02 is considered a promising next generation treatment alternative for patients suffering from Non-Hodgkin lymphoma (NHL). Using native polatuzumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours the highly homogeneous and pure ARC-02 development candidate with a well-defined drug-to-antibody ratio (DAR) of 1.9 and with high purity (monomer content > 98%). ARC-02 is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In-vitro ARC-02 demonstrated potent cytotoxicity in four different NHL cell lines tested so far with similar potency (EC50 values) compared to the approved Polivy® ADC despite of the lower toxin load of ARC-02 (DAR of 1.9) compared to Polivy® (DAR of 3.5). ARC-02 clearly differentiates from Polivy® by extraordinary stability in mouse, cynomolgus monkey and human sera exemplified by the absence of payload deconjugation or linker cleavage under physiological conditions (at 37°C) and long-term incubation (>2 weeks). Exceptional stability could also be confirmed in vivo in pharmacokinetic studies in mice, rats and cynomolgus monkeys demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab parental antibody without premature toxin deconjugation in the circulation resulting in improved pharmacokinetics of ARC-02 compared to Polivy® (half-life in mice 10 days for ARC-02 vs 5 days for Polivy®). Despite the excellent stability of ARC-02 in vitro and in vivo, the MMAE toxin is still efficiently released from ARC-02 in lysosomal human Cathepsin B cleavage or human liver-lysosome release assays which is providing the foundation for the observed robust anti-tumor activity in vivo: ARC-02 was compared head-to-head to Polivy® in two different CD79b-expressing established tumor models in vivo (Granta-519 and Ramos). Single dose treatment of ARC-02 at very low payload doses induced effective and long-lasting anti-tumor responses with complete tumor eradication achieved at about half of the Polivy® payload dose in both NHL established tumor models. Based on these in vivo efficacy experiments, the minimal effective dose (MED) of ARC-02 that is still inducing eradication of established tumors is determined to be as low as 10 μg/kg based on the MMAE payload dose or 1 mg/kg based on the total ADC dose. Finally, the highest non-severely toxic dose (HNSTD) of ARC-02 in repeat dose toxicology studies was determined to be ≥30 mg/kg and ≥24 mg/kg in rats and cynomolgus monkeys, respectively, which is substantially higher than the HNSTD reported for Polivy® (i.e. 10 mg/kg in the rat and 3 mg/kg in the cynomolgus monkey as described in the Polivy® Biologic License Application). Therefore, the tolerability in rodents and non-human primates is at least 3 and 8 times higher when compared to historical safety data of Polivy®. Using the HNSTD derived from repeat dose non-human primate toxicity study (24 mg/kg) and the MED calculated from single dose rodent efficacy studies (1 mg/kg) an impressive TI of 24 can be calculated for ARC-02 which is substantially higher than the TI of Polivy®. These encouraging results obtained so far indicate that ARC-02 a) has very favorable biophysical properties, b) shows a consistent and highly defined DAR, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) has improved tolerability in rodents and non-human primates and f) shows an impressive improvement in TI and ultimately warrant further development of ARC-02.

Abstract 4189: Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates

Abstract Pro-inflammatory cytokines have been approved as a cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years. However, widespread use of cytokine therapy in the clinic is limited by its short half-life in circulation and the associated toxicities that emerge as a result of high systemic exposure. To overcome these limitations, we developed a clinically translatable cytokine delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These cells are able to produce a wide range of natural cytokines (IL2, IL7, IL10, or IL12) and allow for controlled and predictable dosing in vivo. In vivo administration of cytokine factories created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. Local, or tumor adjacent, administration of pro-inflammatory (IL-2-based) cytokine factories caused reduction of tumor burden by 70% after only 1 week of treatment when delivered as a monotherapy to mice with malignant pleural mesothelioma (MPM). Importantly, when administered in combination with local anti-PD1 injections, these cytokine factories lead to eradication of these highly aggressive tumors in 7/7 treated mice. To validate the translatability of this platform, we evaluated the safety profile in non-human primates. Significantly, this platform produced local and systemic T cell biomarker profiles that predict efficacy without renal, liver, or general toxicity in non-human primates. Our findings demonstrate the safety and efficacy of cytokine factories in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans. Citation Format: Amanda Nash, Samira Aghlara-Fotovat, Andrea Hernandez, Bertha Castillo, Alexander Lu, Aarthi Pugazenthi, Peter Rios, Sofia Ghani, Ira Joshi, Douglas Isa, Chunyu Xu, Rahul Sheth, Weiyi Peng, Jose Oberholzer, Amir Jazaeri, Hee-Jin Jang, Bryan Burt, Hyun-Sung Lee, Ravi Ghanta, Omid Veiseh. Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4189.

DMD - TREATMENT: EP.146 scAAV.U7snRNA-mediated therapy: prolonged dystrophin expression and muscle function correction in adult Dup2 mice

Exon 2 duplications of the DMD gene, encoding the dystrophin protein, account for around 6-11% of all duplication mutations associated with X-linked Duchenne muscular dystrophy (DMD). As part of the preclinical development of a U7snRNA vector currently in a clinical trial (ClinicalTrials.gov NCT04240314), we have previously evaluated the therapeutic efficacy, absence of off-target splicing effects in AAV9.U7snRNA-mediated skipping of exon 2 in a murine Dmd model, and lack of toxicity in non-human primates. Here we report that 3-month-old Dup2 mice systemically injected with scAAV9.U7.ACCA vector, containing four copies of U7snRNA targeted to the exon 2 splice acceptor and splice donor sites, showed efficient exon 2 skipping, long-term dystrophin expression, and skeletal muscle function correction 18-months post vector administration. The RT-PCR data showed that a single vector injection (3E13 vg/kg) resulted in significant exon 2 skipping in tibialis anterior (TA), diaphragm (Dia) and heart tissues, showing an average of 46%, 32% and 73% total therapeutic transcripts, respectively. To determine the degree of functional rescue, in situ and in vitro physiology studies on TA and Dia muscles were performed. Both Dia and TA from 21-month-old control Dup2 mice exhibited a functional deficit with a significant reduction in specific force output (45-61%) compared with Bl6 mice. The significant force drop was also observed in those mice compared with Bl6 following a rigorous fatigue protocol. The single vector infusion resulted in a dramatic improvement in specific force output up to 64-76% in Dia and TA, and better protection of the TA muscle (up to 73%) from repeated fatigue. Overall, our results confirm that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in straight muscles from Dup2 mice and functional recovery of TA and Dia muscles 18-month post vector administration.

Lack of Toxicity in Nonhuman Primates Receiving Clinically Relevant Doses of an AAV9.U7snRNA Vector Designed to Induce DMD Exon 2 Skipping.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an adeno-associated virus (AAV)-encapsidated viral vector containing four copies of the noncoding U7 small nuclear RNA (U7snRNA), each targeted to either the splice donor or the splice acceptor sites of DMD exon 2. We have previously shown that delivery of this vector (scAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length dystrophin from wild-type DMD mRNA, as well as an internal ribosome entry site (IRES)-driven isoform translated only in the absence of exon 2 (deletion exon 2 [Del2] mRNA). Here we present the data from a rigorous dose escalation toxicity study in nonhuman primates, encompassing two doses (3 × 1013 and 8 × 1013 vg/kg) and two time points (3 and 6 months postinjection). No evidence for significant toxicity was seen by biochemical, histopathologic, or clinical measures, providing evidence for safety that led to initiation of a first-in-human clinical trial.

Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

LATE BREAKING NEWS ORAL PRESENTATION: LBO 3 Expression of apparent full-length dystrophin in skeletal muscle in a first-in-human gene therapy trial using the scAAV9.U7-ACCA vector

Exon duplications that cause Duchenne muscular dystrophy (DMD) are promising candidates for exon skipping therapies because skipping a single exon copy should result in wild-type transcript and full-length dystrophin expression. We developed a therapeutic exon skipping viral vector (scAAV9.U7-ACCA) comprising four copies of a modified U7snRNA containing antisense sequences targeting the splice donor (2 copies) and splice acceptor (2 copies) of the DMD exon 2, the most commonly duplicated exon. Following dose finding studies in Dup2 mice and after demonstrating lack of toxicity in non-human primates, we initiated a first-in-human clinical trial (NCT04240314).

P0500RESULTS OF A PHASE 1 TRIAL TO INVESTIGATE THE SAFETY, TOLERABILITY, PHARMACOKINETICS, AND PHARMACODYNAMICS OF BION-1301 IN HEALTHY VOLUNTEERS

Abstract Background and Aims IgA nephropathy (IgAN), the leading cause of primary glomerulonephritis, is an autoimmune disease with no approved treatments. Slow progression to end-stage-renal disease occurs in up to 50% of patients with IgAN over approximately 20-25 years, resulting in the need for dialysis or kidney transplant. A critical step in IgAN pathogenesis is the production of galactose-deficient IgA1 (Gd-IgA1) leading to the generation of anti-Gd-IgA autoantibodies and the formation of immune complexes that result in kidney inflammation and damage. Patients with IgAN have elevated levels of A proliferation-inducing ligand (APRIL), which may contribute to IgAN pathogenesis by regulating differentiation and proliferation of B cells. In a study of patients with IgAN, those with high plasma APRIL levels had higher levels of Gd-IgA1 and proteinuria and lower estimated glomerular filtration rates compared to those with lower plasma APRIL levels. BION-1301 is a first-in-class humanized antagonistic antibody targeting APRIL. BION-1301 reduced serum levels of IgA, IgM, and IgG with no drug-related toxicity in nonhuman primates and was well-tolerated with no dose-limiting toxicities in a Phase 1/2 first-in-human clinical study in patients with late-stage multiple myeloma. The Phase 1 trial described here will characterize the safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of BION-1301 in healthy volunteers (HV) and patients with IgAN. Methods The protocol for this study (NCT03945318) is comprised of 3 parts, of which only Parts 1 and 2 are complete. Parts 1 and 2 assess BION-1301 in HV, and Part 3 will assess BION-1301 in patients with IgAN. Parts 1 and 2 are double-blind, randomized, placebo-controlled single and multiple ascending dose designs, respectively. Part 3 will assess a multiple dose regimen in IgAN patients. Part 1 enrolled 36 HV in 5 dose cohorts, randomized in a 3:1 ratio to receive a single dose of BION-1301 or placebo delivered by IV infusion. Part 2 enrolled 27 HV in 3 dose cohorts, randomized in a 2:1 ratio to receive 3 doses of BION-1301 or placebo delivered by IV infusion once every two weeks over a one month period. Dose escalation decisions were made by a Safety Review Team based on safety, tolerability, and PK-related stopping criteria. Results To-date, BION-1301 continues to be well tolerated. Its biological activity in the study is characterized by free APRIL, IgA, IgG, and IgM levels; unblinded safety, PK, and PD results for Parts 1 and 2 in Healthy Volunteers will be presented. Conclusion BION-1301, a humanized anti-APRIL antibody, may provide a novel avenue for the potential treatment of IgAN that offers potential advantages compared to currently used antihypertensive or immunosuppressive therapies.

A phase I study of the novel immunotoxin, MT-5111, in subjects (subj) with HER-2 positive tumors: Interim results.

e15567 Background: Engineered toxin bodies (ETBs), composed of a Shiga-like Toxin A subunit genetically fused to antibody-like binding domains, can force receptor internalization, self-route through intracellular compartments to the cytosol, and induce potent cell-kill via the enzymatic and permanent inactivation of ribosomes. MT-5111, a de-immunized 55 kD ETB targeting HER2 in solid tumors, binds to an epitope distinct from and non-competitive with trastuzumab and pertuzumab. Methods: The primary objective is to determine the maximum tolerated dose (MTD) of MT-5111 monotherapy in adults with advanced HER2+ solid tumors. Secondary objectives are pharmacokinetics, efficacy, and immunogenicity. Part 1 will identify the MTD via MT-5111 dose escalation (0.5, 1.0, 2.0, 3.0, 4.5, 6.75, 10 µg/kg/dose) according to a modified 3+3 design. Part 2 will further evaluate the safety of MT-5111 at the recommended phase 2 dose (RP2D) in 3 separate cohorts of subjs with a HER2 immunohistochemistry score ≥2 in a metastatic lesion of primary breast, gastroesophageal, or other cancers. All subjs receive MT-5111 as a 30-minute IV infusion on Days 1, 8, and 15 of each 21-day treatment (tx) cycle (C) until disease progression, unacceptable toxicity, death, or withdrawn consent. Details can be found on clinicaltrials.gov (NCT04029922). Results: The first cohort (0.5 µg/kg/dose) enrolled 4 subjs (metastatic breast cancer, n = 2; metastatic cholangiocarcinoma, n = 2). Three subjs were female and the mean age was 69 years (median 65, range 64-78). Subjs received a mean of 5 prior lines of therapy (median 4.5, range 3-8). Three subjs completed C1 of tx without dose-limiting toxicities; 1 subj was inevaluable. Two subjs had progressive disease in C2. A total of 23 AEs occurred in 4 subjs; all were grade (G) 1-2 except one G3 event of hypertension in a subj with a history of hypertension. There were 2 tx-related AEs (G1 chills; G2 aspartate aminotransferase increased in the setting of progressive liver metastases). There was 1 serious, non-tx-related AE (G2 dyspnea) that occurred in the inevaluable subj. No cardiac AEs were noted, nor clinically significant changes in cardiac biomarkers, an important safety parameter given non-human primate toxicity. Conclusions: MT-5111 appears to be well tolerated at the lowest dose with no apparent cardiotoxicity to date. Drug concentrations are expected to be below the level required for in vitro tumor cell killing. Safety and efficacy data from subsequent dose escalation cohorts are expected by May 2020. Clinical trial information: NCT04029922.

AAV9-Mediated Expression of SMN Restricted to Neurons Does Not Rescue the Spinal Muscular Atrophy Phenotype in Mice

Spinal muscular atrophy (SMA) is a neuromuscular disease mainly caused by mutations or deletions in the survival of motor neuron 1 (SMN1) gene and characterized by the degeneration of motor neurons and progressive muscle weakness. A viable therapeutic approach for SMA patients is a gene replacement strategy that restores functional SMN expression using adeno-associated virus serotype 9 (AAV9) vectors. Currently, systemic or intra-cerebrospinal fluid (CSF) delivery of AAV9-SMN is being explored in clinical trials. In this study, we show that the postnatal delivery of an AAV9 that expresses SMN under the control of the neuron-specific promoter synapsin selectively targets neurons without inducing re-expression in the peripheral organs of SMA mice. However, this approach is less efficient in restoring the survival and neuromuscular functions of SMA mice than the systemic or intra-CSF delivery of an AAV9 in which SMN is placed under the control of a ubiquitous promoter. This study suggests that further efforts are needed to understand the extent to which SMN is required in neurons and peripheral organs for a successful therapeutic effect.

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

Histopathologic Characterization of Mifepristone-induced Ovarian Toxicity in Cynomolgus Monkeys.

Mifepristone, which is an orally active synthetic steroid with antiprogesterone activity, is known as an ovarian toxicant. Because the available data regarding the histopathologic characteristics of ovarian toxicity in nonhuman primates are limited, the present study was undertaken in order to investigate detailed histopathologic changes accompanying mifepristone-induced ovarian toxicity and its relationship to changes in menstrual cycle and circulating sex steroid hormone. Twenty mg/kg of mifepristone was orally administered daily to 4 cynomolgus monkeys for 2 months. Mifepristone inhibited the cyclic increases in circulating estradiol-17β and progesterone levels with associated absence of menstruation. Histopathologically, the ovary in the treated animals showed follicular phase without changes in the percentage of atretic antral follicles, and reduced endometrial thickness was noted in the uterus. These changes indicated that a certain degree of antral follicle development had been retained in spite of the menstrual cycle having been arrested in mifepristone-treated animals. Our investigation suggested that it is important to perform detailed histopathologic examination of reproductive organs with precise knowledge of the characteristics of each menstrual stage to detect ovarian toxicity in nonhuman primates. Monitoring menstrual signs and circulating sex steroid hormone levels provides additional evidence for the investigation of the mechanism of ovarian toxicity.

Severe Toxicity in Nonhuman Primates and Piglets Following High-Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN.

Neurotropic adeno-associated virus (AAV) serotypes such as AAV9 have been demonstrated to transduce spinal alpha motor neurons when administered intravenously (i.v.) at high doses. This observation led to the recent successful application of i.v. AAV9 delivery to treat infants with spinal muscular atrophy, an inherited deficiency of the survival of motor neuron (SMN) protein characterized by selective death of lower motor neurons. To evaluate the efficiency of motor neuron transduction with an AAV9 variant (AAVhu68) using this approach, three juvenile nonhuman primates (NHPs; aged 14 months) and three piglets (aged 7-30 days) were treated with an i.v. injection of an AAVhu68 vector carrying a human SMN transgene at a dose similar to that employed in the spinal muscular atrophy clinical trial. Administration of 2 × 1014 genome copies per kilogram of body weight resulted in widespread transduction of spinal motor neurons in both species. However, severe toxicity occurred in both NHPs and piglets. All three NHPs exhibited marked transaminase elevations. In two NHPs, the transaminase elevations resolved without clinical sequelae, while one NHP developed acute liver failure and shock and was euthanized 4 days after vector injection. Degeneration of dorsal root ganglia sensory neurons was also observed, although NHPs exhibited no clinically apparent sensory deficits. There was no correlation between clinical findings and T-cell responses to the vector capsid or transgene product in NHPs. Piglets demonstrated no evidence of hepatic toxicity, but within 14 days of vector injection, all three animals exhibited proprioceptive deficits and ataxia, which profoundly impaired ambulation and necessitated euthanasia. These clinical findings correlated with more severe dorsal root ganglia sensory neuron lesions than those observed in NHPs. The liver and sensory neuron findings appear to be a direct consequence of AAV transduction independent of an immune response to the capsid or transgene product. The present results and those of another recent study utilizing a different AAV9 variant and transgene indicate that systemic and sensory neuron toxicity may be general properties of i.v. delivery of AAV vectors at high doses, irrespective of the capsid serotype or transgene. Preclinical and clinical studies involving high systemic doses of AAV vectors should include careful monitoring for similar toxicities.

Severe Toxicity in Nonhuman Primates and Piglets with Systemic High-Dose Administration of Adeno-Associated Virus Serotype 9–Like Vectors: Putting Patients First

Severe Toxicity in Nonhuman Primates and Piglets with Systemic High-Dose Administration of Adeno-Associated Virus Serotype 9–Like Vectors: Putting Patients First

RN927C, a Site-Specific Trop-2 Antibody-Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models.

Trop-2, also known as TACSTD2, EGP-1, GA733-1, and M1S1, is frequently expressed on a variety of human carcinomas, and its expression is often associated with poor prognosis of the diseases. However, it is also present on the epithelium of several normal tissues. A comprehensively designed Trop-2-targeting antibody-drug conjugate (ADC), balancing both efficacy and toxicity, is therefore necessary to achieve clinical utility. To this end, we developed a cleavable Trop-2 ADC (RN927C) using a site-specific transglutaminase-mediated conjugation method and a proprietary microtubule inhibitor (MTI) linker-payload, PF-06380101. Robust in vitro cytotoxicity of RN927C was observed on a panel of Trop-2-expressing tumor cell lines, with IC50 generally in the subnanomolar range. As expected for an MTI-containing ADC, RN927C readily induced mitotic arrest of treated cells in vitro and in vivo, followed by subsequent cell death. The in vivo efficacy of RN927C was tested in multiple cell line and patient-derived xenograft tumor models, including pancreatic, lung, ovarian, and triple-negative breast tumor types. Single-dose administration of RN927C at 0.75 to 3 mg/kg was generally sufficient to induce sustained regression of Trop-2-expressing tumors and showed superior efficacy over standard treatment with paclitaxel or gemcitabine. Administration of RN927C in nonhuman primate toxicity studies resulted in target-mediated effects in skin and oral mucosa, consistent with Trop-2 expression in these epithelial tissues with minimal, non-dose limiting off-target toxicities. On the basis of the combined efficacy and safety results, RN927C is postulated to have a favorable therapeutic index for treatment of solid tumors. Mol Cancer Ther; 15(11); 2698-708. ©2016 AACR.

SIRPα-Antibody Fusion Proteins Selectively Bind and Eliminate Dual Antigen-Expressing Tumor Cells

CD47 is highly expressed on a variety of tumor cells. The interaction of CD47 with signal regulatory protein alpha (SIRPα), a protein on phagocytic cells, transmits a "don't eat me" signal that negatively regulates phagocytosis. CD47-SIRPα antagonists enable phagocytosis by disrupting the inhibitory signal and can synergize with Fc-mediated pro-phagocytic signals for potent elimination of tumor cells. A potential limitation of therapeutic CD47-SIRPα antagonists is that expression of CD47 on normal cells may create sites of toxicity or an "antigen sink." To overcome these limitations and address selective tumor targeting, we developed SIRPabodies to improve the therapeutic benefits of CD47-SIRPα blockade specifically toward tumor. SIRPabodies were generated by grafting the wild-type SIRPα either to the N-terminus or to the C-terminus of the heavy chain of rituximab. Selective tumor binding was tested using CFSE-labeled human primary CLL cells in the presence of 20-fold excess of human RBCs. NSG mice were transplanted with Raji-luciferase cells and were assigned to controls versus SIRPabody treatment. Cynomolgus nonhuman primates were administered a single intravenous infusion of SIRPabody at 3, 10, or 30 mg/kg. SIRPabodies selectively bound to dual antigen-expressing tumor cells in the presence of a large antigen sink. SIRPabody reduced tumor burden and extended survival in mouse xenograft lymphoma models. SIRPabody caused no significant toxicity in nonhuman primates. These findings establish SIRPabodies as a promising approach to deliver the therapeutic benefit of CD47-SIRPα blockade specifically toward tumor cells. SIRPabodies may be applied to additional cancer types by grafting SIRPα onto other tumor-specific therapeutic antibodies. Clin Cancer Res; 22(20); 5109-19. ©2016 AACR.

Sensitivity of male reproductive endpoints in nonhuman primate toxicity studies: A statistical power analysis

Sensitivity of male reproductive endpoints in nonhuman primate toxicity studies: A statistical power analysis