Elarekibep (PRS-060/AZD1402), a new class of inhaled Anticalin medicine targeting IL-4Ra for type 2 endotype asthma.

BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.

SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity.

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Tumor-Localized Costimulatory T-Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343.

PURPOSE: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation. EXPERIMENTAL DESIGN: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. RESULTS: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. CONCLUSIONS: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule.See related commentary by Su et al., p. 5732.

Therapeutic antibodies to human L1CAM: functional characterization and application in a mouse model for ovarian carcinoma.

Recent work has identified L1CAM (CD171) as a novel marker for human carcinoma progression. Functionally, L1CAM promotes tumor cell invasion and motility, augments tumor growth in nude mice, and facilitates experimental tumor metastasis. These functional features qualify L1 as an interesting target molecule for tumor therapy. Here, we generated a series of novel monoclonal antibodies (mAb) to the L1CAM ectodomain that were characterized by biochemical and functional means. All novel mAbs reacted specifically with L1CAM and not with the closely related molecule CHL1, whereas antibodies to the COOH terminal part of L1CAM (mAb2C2, mAb745H7, pcytL1) showed cross-reactivity. Among the novel mAbs, L1-9.3 was selected and its therapeutic potential was analyzed in various isotype variants in a model of SKOV3ip cells growing i.p. in CD1 nude mice. Only therapy with the IgG2a variant efficiently prolonged survival and reduced tumor burden. This was accompanied by an increased infiltration of F4/80-positive monocytic cells. Clodronate pretreatment of tumor-bearing animals led to the depletion of monocytes and abolished the therapeutic effect of L1-9.3/IgG2a. Expression profiling of tumor-derived mRNA revealed that L1-9.3/IgG2a therapy induced altered expression of cellular genes associated with apoptosis and tumor growth. Our results establish that anti-L1 mAb therapy acts via immunologic and nonimmunologic effector mechanism to block tumor growth. The novel antibodies to L1CAM could become helpful tools for the therapy of L1-positive human carcinomas.

Efficacy of oncolytic herpesvirus NV1020 can be enhanced by combination with chemotherapeutics in colon carcinoma cells.

NV1020, an oncolytic herpes simplex virus type 1, can destroy colon cancer cells by selectively replicating within these cells, while sparing normal cells. NV1020 is currently under investigation in a clinical phase I/II trial as an agent for the treatment of colon cancer liver metastases, in combination with conventional chemotherapeutic agents such as 5-fluorouracil (5-FU), SN38 (the active metabolite of irinotecan), and oxaliplatin. To study the synergy of NV1020 and chemotherapy, cytotoxicity and viral replication were evaluated in vitro by treating various human and murine colon carcinoma cell lines, using a colorimetric viability assay, a clonogenic assay, and a plaque-forming assay. In vivo experiments, using a subcutaneous syngeneic CT-26 tumor model in BALB/c mice, were performed to determine the efficacy of combination therapy. In vitro studies showed that the efficacy of NV1020 on human colon carcinoma cell lines HT-29, WiDr, and HCT-116 was additively or synergistically enhanced in combination with 5-FU, SN38, or oxaliplatin. The sequence of application was not important and effects were still apparent after a 21-day incubation period. Three intra-tumoral treatments with NV1020 (1 x 10(7) plaque-forming units), followed by three subcutaneous treatments with 5-FU (50 mg/kg), resulted in substantially higher inhibition of tumor growth and prolongation of survival compared with monotherapies (NV1020/5-FU vs. NV1020, p = 0.027). On WiDr cells, reduced replication of NV1020, in combination with 5-FU, indicated that additive and synergistic effects of combination therapy must be independent from viral replication. These results suggest that NV1020, in combination with chemotherapy, is a promising therapy for treating patients with metastatic colorectal cancer of the liver. We hypothesize that infection of cells with NV1020 sensitizes the infected cells for the cytotoxic effect of the chemotherapeutics.