Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use. METHODS: In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3. RESULTS: Where the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non-tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo. CONCLUSIONS: By combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.

Antibody-mediated neutralization of autocrine Gas6 inhibits the growth of pancreatic ductal adenocarcinoma tumors in vivo.

Gas6 and its receptors Axl, Mer and Tyro-3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor-promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co-expressed and their co-expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6-induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl-Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6-induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100-1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6-induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.

Preclinical Evaluation of AZD6422, an Armored Chimeric Antigen Receptor T Cell Targeting CLDN18.2 in Gastric, Pancreatic, and Esophageal Cancer.

PURPOSE: CLDN18.2 is a surface membrane protein crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers. Thus, CLDN18.2 is suited for exploration as a clinical target for chimeric antigen receptor T-cell (CAR-T) therapy in these indications. Although CAR-T therapies show promise, a challenge faced in their development for solid tumors is the immunosuppressive tumor microenvironment, often characterized by the presence of immune and stromal cells secreting high levels of transforming growth factor beta (TGF-ß). Addition of TGF-ß armoring can potentially expand CAR-T activity in solid tumors. We report on the preclinical development of a CLDN18.2-targeting CAR-T showing effectiveness in CLDN18.2-positive gastric, esophageal, and pancreatic tumor models. EXPERIMENTAL DESIGN: The lead lentivirus product contains a unique single-chain variable fragment, CD28 and CD3z costimulatory and signaling domains, and dominant negative TGF-ß receptor armoring, enhancing targeting and safety and counteracting suppression. We developed a shortened cell manufacturing process to enhance the potency of the final product, AZD6422. RESULTS: AZD6422 exhibited significant antitumor activity and tolerability in multiple patient-derived tumor xenograft models with various CLDN18.2 and TGF-ß levels, as determined by immunohistochemistry. Efficacy of armored CAR-Ts in tumor models with elevated TGF-ß was increased in vitro and in vivo. In vitro restimulation assays established greater persistence and cytolytic function of AZD6422 compared with a traditionally manufactured CAR-T. CONCLUSIONS: AZD6422 was safe and efficacious in patient-derived, CLDN18.2-positive murine models of gastrointestinal cancers. Our data support further clinical development of AZD6422 for patients with these cancers.

DIALing-up the preclinical characterization of gene-modified adoptive cellular immunotherapies.

The preclinical characterization of gene modified adoptive cellular immunotherapy candidates for clinical development often requires the use of mouse models. Gene-modified lymphocytes (GML) incorporating chimeric antigen receptors (CAR) and T-cell receptors (TCR) into immune effector cells require in vivo characterization of biological activity, mechanism of action, and preclinical safety. Typically, this characterization involves the assessment of dose-dependent, on-target, on-tumor activity in severely immunocompromised mice. While suitable for the purpose of evaluating T cell-expressed transgene function in a living host, this approach falls short in translating cellular therapy efficacy, safety, and persistence from preclinical models to humans. To comprehensively characterize cell therapy products in mice, we have developed a framework called "DIAL". This framework aims to enable an end-to-end understanding of genetically engineered cellular immunotherapies in vivo, from infusion to tumor clearance and long-term immunosurveillance. The acronym DIAL stands for Distribution, Infiltration, Accumulation, and Longevity, compartmentalizing the systemic attributes of gene-modified cellular therapy and providing a platform for optimization with the ultimate goal of improving therapeutic efficacy. This review will discuss both existent and emerging examples of DIAL characterization in mouse models, as well as opportunities for future development and optimization.

Antitumor activity of AZD0754, a dnTGFßRII-armored, STEAP2-targeted CAR-T cell therapy, in prostate cancer.

Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-ß type II receptor, bolstering its activity in the TGF-ß-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-ß-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

Efficacy of ganitumab (AMG 479), alone and in combination with rapamycin, in Ewing's and osteogenic sarcoma models.

Ewing's and osteogenic sarcoma are two of the leading causes of cancer deaths in children and adolescents. Recent data suggest that sarcomas may depend on the insulin-like growth factor type 1 (IGF-1) receptor (IGF1R) and/or the insulin receptor (INSR) to drive tumor growth, survival, and resistance to mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We evaluated the therapeutic value of ganitumab (AMG 479; C(6472)H(10028)N(1728)O(2020)S(42)), an anti-IGF1R, fully human monoclonal antibody, alone and in combination with rapamycin (mTORC1 inhibitor) in Ewing's (SK-ES-1 and A673) and osteogenic (SJSA-1) sarcoma models. IGF1R was activated by IGF-1 but not by insulin in each sarcoma model. INSR was also activated by IGF-1 in the SJSA-1 and SK-ES-1 models, but not in the A673 model where insulin was the preferred INSR ligand. Ganitumab significantly inhibited the growth of SJSA-1 and SK-ES-1 xenografts; inhibition was associated with decreased IGF1R and Akt phosphorylation, reduced total IGF1R and bromodeoxyuridine detection, and increased caspase-3 expression. Ganitumab inhibited rapamycin-induced IGF1R, Akt, and glycogen synthase kinase-3ß hyperphosphorylation in each sarcoma model. However, ganitumab in combination with rapamycin also resulted in a marked increase in INSR expression and activity in the SJSA-1 and A673 models. The in vivo efficacy of ganitumab in the two ganitumab-sensitive models (SJSA-1 and SK-ES-1) was significantly enhanced in combination with rapamycin. Our results support studying ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas and suggest that INSR signaling is an important mechanism of resistance to IGF1R blockade.

Intratumoral IL12 mRNA Therapy Promotes TH1 Transformation of the Tumor Microenvironment.

PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.

Discovery and in Vivo Evaluation of Macrocyclic Mcl-1 Inhibitors Featuring an α-Hydroxy Phenylacetic Acid Pharmacophore or Bioisostere.

Overexpression of the antiapoptotic protein Mcl-1 provides a survival advantage to some cancer cells, making inhibition of this protein an attractive therapeutic target for the treatment of certain types of tumors. Herein, we report our efforts toward the identification of a novel series of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. This work led to the discovery of 1, a potent Mcl-1 inhibitor (IC50 = 19 nM in an OPM-2 cell viability assay) with good pharmacokinetic properties and excellent in vivo efficacy in an OPM-2 multiple myeloma xenograft model.

AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies.

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing.

For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.

IGF1R blockade with ganitumab results in systemic effects on the GH-IGF axis in mice.

Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding assays showed that ganitumab recognized murine IGF1R with sub-nanomolar affinity (KD=0.22 nM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon carcinoma cells and tumors. Addition of ganitumab to 5-fluorouracil resulted in enhanced inhibition of tumor growth in the CT26 model. Pharmacological intervention with ganitumab in naïve nude mice resulted in a number of physiological changes described previously in animals with targeted deletions of Igf1 and Igf1r, including inhibition of weight gain, reduced glucose tolerance and significant increase in serum levels of GH, IGF1 and IGFBP3. Flow cytometric analysis identified GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These findings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly regulates serum IGF1 levels through Gh signaling.

Ganitumab (AMG 479) inhibits IGF-II-dependent ovarian cancer growth and potentiates platinum-based chemotherapy.

PURPOSE: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer. EXPERIMENTAL DESIGN: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I-, IGF-II-, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo. RESULTS: In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho-IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I-, IGF-II-, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo. CONCLUSIONS: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho-IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947-58. ©2014 AACR.

Molecular mechanism of hepcidin-mediated ferroportin internalization requires ferroportin lysines, not tyrosines or JAK-STAT.

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. In vitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. In vivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys.

Interferon-alpha (IFN-alpha) is indicated for the treatment of certain viral infections including hepatitis B and C, and cancers such as melanoma. The short circulating half-life of unmodified IFN-alpha makes frequent dosing (daily or three times weekly) over an extended period (6-12 months or more) necessary. To improve the pharmacokinetics of IFN-alpha and decrease dosing frequency, IFN-alpha was fused to human serum albumin producing a new protein, Albuferon. In vitro comparisons of Albuferon and IFN-alpha showed similar antiviral and antiproliferative activities, although Albuferon was less potent on a molar basis than IFN-alpha. Pharmacokinetic and pharmacodynamic properties of the fusion protein were enhanced in monkeys. After a single intravenous injection (30 microg/kg,) clearance was 0.9 ml/h/kg, and the terminal half-life was 68 h. After 30 microg/kg subcutaneous injection, apparent clearance (clearance divided by bioavailability) was 1.4 ml/h/kg, the terminal half-life was 93 h, and bioavailability was 64%. The rate of clearance of Albuferon was approximately 140-fold slower, and the half-life 18-fold longer, than for IFN-alpha given by the subcutaneous route in other monkey studies. Sera from Albuferon-treated monkeys demonstrated dose-related antiviral activity for > or =8 days based on an in vitro bioassay, whereas antiviral activity from IFN-alpha-treated animals was only slightly elevated relative to vehicle on day 0. Significant increases in 2',5'-oligoadenylate synthetase mRNA relative to IFN-alpha- or vehicle-treated animals were maintained for > or =10 days after subcutaneous dosing. The improved pharmacokinetics of Albuferon are accompanied by an improved pharmacodynamic response suggesting that Albuferon may offer the benefits of less frequent dosing and a potentially improved efficacy profile compared with IFN-alpha.