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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, CD56 bright CD16 - and CD56 dim CD16 + , 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.

BackgroundSOT201 and its murine surrogate mSOT201 are novel cis-acting immunocytokines consisting of a humanized/murinized/, Fc-silenced anti-programmed cell death protein 1 (PD-1) monoclonal antibody (mAb) fused to an attenuated human interleukin (IL)-15 and the IL-15Rα sushi+ domain. Murine mPD1-IL2v is a conjugate of a murinized, Fc silenced anti-PD-1 mAb bearing human IL-2 with abolished IL-2Rα binding. These immunocytokines spatiotemporally reinvigorate PD-1+ CD8+ tumor-infiltrating lymphocytes (TILs) via cis-activation and concomitantly activate the innate immunity via IL-2/15Rβγ signaling.MethodsHuman peripheral blood mononuclear cell and cell lines were used to evaluate cis/trans activity of SOT201. Anti-PD-1 mAb responsive (MC38, CT26) and resistant (B16F10, CT26 STK11 KO) mouse tumor models were used to determine the anticancer efficacy, and the underlying immune cell activity was analyzed via single-cell RNA sequencing and flow cytometry. The expansion of tumor antigen-specific CD8+ T cells by mSOT201 or mPD1-IL2v and memory CD8+ T-cell generation in vivo was determined by flow cytometry.ResultsSOT201 delivers attenuated IL-15 to PD-1+ T cells via cis-presentation, reinvigorates exhausted human T cells and induces higher interferon-γ production than pembrolizumab in vitro. mSOT201 administered as a single dose exhibits strong antitumor efficacy with several complete responses in all tested mouse tumor models. While mPD1-IL2v activates CD8+ T cells with a 50-fold higher potency than mSOT201 in vitro, mSOT201 more effectively reactivates effector exhausted CD8+ T cells (Tex), which demonstrate higher cytotoxicity, lower exhaustion and lower immune checkpoint transcriptional signatures in comparison to mPD1-IL2v in MC38 tumors in vivo. This can be correlated with a higher rate of complete responses in the MC38 tumor model following mSOT201 treatment when compared with mPD1-IL2v. mSOT201 increased the relative number of tumor antigen-specific CD8+ T cells, and unlike mPD1-IL2v stimulated greater expansion of adoptively transferred ovalbumin-primed CD8+ T cells simultaneously limiting the peripheral CD8+ T-cell sink, leading to the development of memory CD8+ T cells in vivo.ConclusionsSOT201 represents a promising therapeutic candidate that preferentially targets PD-1+ TILs, delivering balanced cytokine activity for reviving CD8+ Tex cells in tumors. SOT201 is currently being evaluated in the Phase I clinical study VICTORIA-01 (NCT06163391) in patients with advanced metastatic cancer.

BackgroundNanrilkefusp alfa (SOT101, RLI-15) is a high affinity 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. Nanrilkefusp alfa induces proliferation and activation of CD8+ T cells, memory CD8+ T cells, NK cells, γ∂ T cells and NKT cells but not T regs.MethodsBlood and tumor samples from patients with advanced/metastatic solid tumors participating in a Phase clinical I study (NCT04234113) were analyzed by flow cytometry, immunohistochemistry and NanoString analyses for immune cells activation and tumor infiltration induced by Nanrilkefusp alfa monotherapy or in combination with pembrolizumab.ResultsNanrilkefusp alfa monotherapy or combined with pembrolizumab markedly increased proliferation of CD8+ T cells, memory CD8+ T cells, NK cells and NKT cells, the absolute NK cell, CD8+ and memory CD8+ T cell counts, as well as IFN-γ levels without concomitantly increasing Tregs in peripheral blood. Whereas strong proliferation of NK cells was detected already at the lowest dose level of 0.25 µg/kg, proliferation of CD8+ T cells, memory CD8+ T cells and NKT cells was dose-dependent, reaching maximal activity at 12 µg/kg. High NK-cell proliferation was maintained over repeated cycles of the treatment, while NKT and CD8+ T cell proliferation peaked in cycle 1 and then declined slightly. In tumor tissues, nanrilkefusp alfa increased the density of NK cells, CD3+, CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), the CD8+/Treg ratio and the densities of proliferating CD8+ and CD4+ TILs, while Tregs in the tumor remained low. Consistent with the increased number of TILs, nanrilkefusp alfa increased the expression of gene sets related to innate and adaptive immune responses, including NK cell function, Th1 activation, regulation of the immune response, chemokines, and γ∂ T cells. Pharmacodynamic responses were the most pronounced in patients showing a clinical benefit as determined by stable disease or partial response.ConclusionsNanrilkefusp alfa boosts both the innate and adaptive immune system and induces proinflammatory changes in the microenvironment of multiple tumor types as single-agent and in combination with pembrolizumab. An extended evaluation of nanrilkefusp alfa in combination with pembrolizumab or cetuximab is currently ongoing in phase 2 clinical trials in patients with selected advanced solid tumors (NCT05256381, NCT05619172).Ethics ApprovalThe study obtained ethics approval and participants gave informed consent before taking part. Comite de Protection des Personnes, CPP Ouest 6 – CPP 1157 ME1; CEIm Hospital Universitari Vall d´Hebron, ID-RTF096; Western Institutional Review Board, 20192896; U.T.MD Anderson Cancer Center Institutional Review Board, 20192896; Multicenter ethics committee University Hospital Brno, 82/20MEK;Ethics committee Masaryk Memorial Cancer Institute, R 22/20.

BackgroundSOT201 is a novel cis-acting immunocytokine consisting of a humanized, Fc-silenced monoclonal antibody against PD-1 fused to a covalent RLI-15 complex of a human attenuated IL-15 mutein linked to the high-affinity binding site of the IL-15Rα, the sushi+ domain. The activity of SOT201 is based on spatiotemporal reinvigoration of PD-1+ CD8+tumor infiltrating lymphocytes (TILs) via cis activation and concomitant activation of innate immunity by IL-15-mediated signaling via the IL-2/IL-15βγ receptor.MethodsHuman PBMC, wt or PD-1-transfected Kit225 or Raji cells and in vitro exhausted human T cells were used to evaluate cis/trans activity of SOT201. Mouse surrogate SOT201-induced expansion and activation of ovalbumin-primed adoptively transferred OT-I CD8+ T cells in vivo was detected by flow cytometry. PD-1 responsive (MC38, CT26) and resistant mouse models (B16F10, CT26 STK11 ko) were used to determine the anti-tumor efficacy. The pharmacodynamics and pharmacokinetics of SOT201 were evaluated in cynomolgus monkeys.ResultsSOT201 delivers attenuated RLI-15 mutein to PD1+ TILs via cis presentation, stimulates in vitro exhausted T cells and expands antigen-specific PD-1+ CD8+ T cells in vivo. SOT201 treatment showed strong anti-tumor efficacy in PD-1 responsive and resistant tumor models in vivo and was shown to be superior to mouse PD-1-IL-2Rβγ agonist. Studies in cynomolgus monkeys showed that decreased affinity of the novel IL-15 mutein in SOT201 for reduced IL-15Rβγ binding is well optimized to ensure favorable pharmacokinetic properties while potently stimulating PD-1+ CD8+ T cells and NK cells.ConclusionsThis data confirms SOT201 to be a promising therapeutic candidate molecule directed preferentially to the PD-1+ T cell tumor microenvironment. SOT201 is currently being prepared for evaluation in a Phase I clinical study in metastatic advanced cancer patients as well as for PD-1 resistant/refractory patients.

BackgroundSO-C101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain. SO-C101 effectively stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cells which translates to anti-tumor efficacy in mouse.MethodsIn this study, we investigated different administration schedules of SO-C101 in cynomolgus monkeys to assess its pharmacodynamics and pharmacokinetics properties using intravenous (IV) and subcutaneous (SC) routes of administration.ResultsSubcutaneous administration of SO-C101 was more effective than IV administration in terms of activating thee target immune cells which was correlated to the differences in SO-C101 exposure. Repeated administration of SO-C101 over two weeks promoted an increase of absolute lymphocyte counts and of the circulating NK and CD8+ T cell numbers. Moreover, two administrations on consecutive days were optimal and comparable to four daily administrations. We further determined an optimal schedule for a repetitive SO-C101 SC administration to achieve a cycle-dependent stimulation of NK and CD8+ T cells over the course of 10 weeks. These studies allowed to correlate the concentration to response relationship in vitro with the relationship between Cmax following SC administration and the resulting NK and CD8+ T cell activation levels in vivo. These data were used to determine the starting dose and subsequent dose escalation steps of SO-C101 in an ongoing Phase I clinical trial in patients with advanced solid tumors.ConclusionsSince the potency of SO-C101 to activate NK and CD8+ T cells in vitro is equivalent between human and cynomolgus monkeys, these studies informed the dose and schedule selection for the ongoing Phase I clinical study (NCT04234113).Ethics ApprovalPharmacodynamics and pharmacokinetics studies in cynomolgous monkeys were approved by Ethics Board of an appropriate contract research organizations (CROs)

Patients with gastric and pancreatic cancers, as well as other solid tumors including ovarian, lung, liver, and colon cancers, often lack effective therapeutic options. Claudin 18.2 (CLDN18.2) is a tumor-associated target that is predominantly expressed in gastric and pancreatic cancers but also found in several other tumor types. SOT102 is a novel antibody-drug conjugate directed against CLDN18.2, developed to provide a new therapeutic strategy for patients with CLDN18.2-positive tumors. SOT102, composed of a proprietary monoclonal antibody (mAb) conjugated to the cytotoxic payload PNU-159682, was evaluated for binding, internalization, and cytotoxic effects in vitro. The in vivo antitumor activity was assessed in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) mouse models, both as monotherapy and the latter in combination with anti-PD1 antibody therapy. SOT102 pharmacokinetics and tolerability were further investigated in cynomolgus monkeys following intravenous administration. SOT102 demonstrated selective binding to CLDN18.2, with no detectable cross-reactivity to CLDN18.1, and efficient internalization into CLDN18.2-expressing cell lines, resulting in potent cytotoxic effects against tumor organoids with half-maximal activity ranging from 0.2 nM to 19.4 nM. Antitumor activity against PDX-derived mouse models was observed at a minimum effective dose of 0.2 mg/kg, with enhanced efficacy when combined with anti-PD1 antibody treatment. SOT102 exposure in cynomolgus monkeys was dose-dependent at doses between 0.3 mg/kg and 1 mg/kg with a half-life of approximately 7 days. An acceptable tolerability profile was observed, and the therapeutic window was defined between the minimum effective dose in mice and the highest non-severe toxic dose (HNSTD) of 0.6 mg/kg in cynomolgus monkeys. SOT102 exhibited strong antitumor activity in preclinical models of CLDN18.2-positive cancers and demonstrated a favorable pharmacokinetic and safety profile in non-human primates. These data were used to support clinical evaluation of SOT102 as a potential treatment option for patients with CLDN18.2-expressing solid tumors.

BackgroundSO-C101 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. SO-C101 specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment.MethodsHuman NK cell proliferation, the expression of NK cell receptors and ADCC activity of human PBMC after stimulation with SO-C101 in vitro in combination with monoclonal antibodies were detected by flow cytometry. The anti-tumor efficacy of SO-C101 in combination with Daratumumab was assessed in a multiple myeloma SCID xenograft mouse model in vivo.ResultsIn this study, we show that SO-C101 induced proliferation and expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+. Furthermore, SO-C101 induced expression of the cytotoxic receptors NKp30 and NKG2D whereas no upregulation of the inhibitory receptors CD158a, CD158b and NKG2A was detected. Both NK cell subsets activated by SO-C101 exhibited cytotoxicity towards cancer cells in vitro. Using human PBMCs, we show that SO-C101 potentiated killing of tumor cells induced by several clinically approved therapeutic monoclonal antibodies such as Cetuximab, Daratumumab and Obinutuzumab in vitro. SO-C101 and Daratumumab monotherapy treatment inhibited tumor growth in vivo, however, their combination showed the strongest anti-tumor efficacy. Specifically, sequential administration of Daratumumab, followed by SO-C101 promoted complete tumor regression, compared to partial anti-tumor responses induced by the respective monotherapies.ConclusionsSO-C101 augments the anti-tumor activity of therapeutic antibodies by increasing NK cells mediated antibody-dependent cell cytotoxicity. These results support the evaluation of SO-C101 in combination with monoclonal therapeutic antibodies in clinical studies.Ethics ApprovalThe anti-tumor efficacy studies in mice were approved by the internal ethics board of the respective contract research organization (CRO).

BackgroundCD137 is a potent costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The receptor, also known as 4-1BB, is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. While multiple lines of evidence show that CD137 is a highly promising therapeutic target, current approaches are not designed to achieve a tumor-target driven activation. Thus, these approaches may display a limited therapeutic window due to peripheral T cell and NK cell activation, leading to unwanted toxicity. To overcome this limitation, we generated a bispecific protein therapeutic binding to CD137 and to a differentially expressed tumor target, HER2.MethodsAnticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. The CD137-specific Anticalin protein was genetically fused to a trastuzumab variant at different positions, yielding four different constructs covering a range of distances between the binding sites of the T cell-target and the tumor cell target. To minimize Fcγ-receptor interaction of the resulting bispecific and concomitant potential toxicity towards CD137-positive cells, the backbone of trastuzumab was switched from IgG1 to an engineered IgG4 isotype.ResultsAll four bispecific constructs bound the targets CD137 and HER2 with a nearly identical affinity compared to the parental building blocks, and both targets could be simultaneously bound. Compared to non-engineered trastuzumab, binding to human receptors FcγRI and FcγRIII was significantly reduced, while binding to the neonatal Fc receptor (FcRn) was retained. All constructs were found to be fully stable in human and mouse plasma, and in mice displayed pharmacokinetics similar to trastuzumab. HER2-dependent agonistic engagement of CD137 was demonstrated in ex-vivo T cell activation assays utilizing HER2-positive human cell lines. The functional activity of the bispecific constructs was found to be dependent on their geometry.ConclusionWe report the first bispecific therapeutic protein that targets the potent costimulatory immunoreceptor CD137 in a tumor-target dependent manner, utilizing HER2 as the tumor target. Compared to currently existing CD137-targeting antibodies, this approach has the potential to provide a more localized activation of the immune system with reduced peripheral toxicity. Bispecific T cell engagers based on CD137 and HER2 may have utility in HER2-positive cancers where there is a significant unmet medical need.

We describe a detailed mapping of the class II major histocompatibility complex (MHC) binding site using site-directed mutagenesis in conjunction with high-resolution CD4 structural data. Residues on all lateral surfaces of domain 1 and the neighboring portions of domain 2 participate in contacting class II MHC. Thus, in addition to the C'C'' ridge that forms the human immunodeficiency virus type 1 gp120 binding site, apparent MHC contacts extend over the BED face of domain 1 and across the interdomain groove onto the FG loop of domain 2. Several models of the CD4/class II MHC interaction accounting for the extent of the CD4 surface involved are discussed, including the possibility that CD4 may contact more than one class II MHC molecule using different surfaces.

Using site-directed mutagenesis informed by high-resolution CD4 structural data, we have investigated the role of residues of the C'C'' ridge region of human CD4 on class II major histocompatibility complex (MHC) binding. This C'C'' ridge is homologous to the CDR2 loop of an immunoglobulin variable domain and is known to contain the binding site for human immunodeficiency virus (HIV) coat glycoprotein gp120. Here we report that this region is also involved in interaction with class II MHC. Exposed positively charged residues Lys-35, Lys-46, and Arg-59 and the exposed hydrophobic residue Phe-43 contribute significantly to class II MHC binding. Moreover, mutations in the buried residues Trp-62 and Ser-49, which support the top and bottom of the C'C'' ridge, respectively, disrupt class II MHC interaction. The HIV binding region appears to involve a restricted area of the larger class II MHC binding site on CD4. Strategies of drug design aimed at interrupting CD4-HIV interaction will need to consider the extensive overlap between class II MHC and HIV gp120 binding surfaces in this region of CD4.