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Figure S1: PRS-343 specificity for 4-1BB within TNF receptor superfamily. Seven recombinant human TNF receptor superfamily proteins were purchased from Sino Biological (4-1BB: 10041-H08H, RANK: 16078-H08H, GITR: 13643-H08H, Ox40: 10481-H08H) or R&D Systems (CD30: 6126-CD, TNF-RII: 1089-R2, TNF-RI: 636-R1) and used for determination of PRS-343 selectivity for 4-1BB. Proteins were coated to an ELISA plate, PRS-343 was added in a dilution series and detected via HRP-labeled anti-human IgG Fc antibody. Within the set of tested TNF receptor family proteins, PRS-343 binds exclusively to 4-1BB. Figure S2: Characterization of multiple bispecific formats of a 4-1BB targeting Anticalin protein recombinantly fused to an anti-HER2 antibody (A). ELISA based binding properties of all formats were compared to parental building blocks (C) while the ability to activate T-cells (IL2 induction) was assessed in a co-culture assay. Figure S3: Cytokine release assay with PRS-343. PBMC were isolated from the blood of twelve healthy donors and incubated for 72 hours with PRS-343 either air dried, in soluble form, or wet coated. Four concentrations of PRS-343 in a volume of 50 µl were tested in each setting as indicated in the figure. The anti-CD3 monoclonal antibody OKT3 at three different concentrations served as the positive control, and an IgG4 isotype antibody was the negative control. Supernatant levels of ten cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN-γ and TNF-α) were analyzed. The figure shows the average response for the ten donors that displayed a significant response to OKT3, and for a selection of the most relevant cytokines. Figure S4: Dose-dependent 4-1BB activation of a 4-1BB over-expressing Jurkat Nf-kB reporter cell line induced by PRS-343 with ON preincubation of the drug in the presence of NCI-N87 (HER2 high), MKN45 (HER2 low) and HepG2 (HER2 null) cell lines, or without tumor cells. Briefly, cancer cells were seeded onto tissue culture plates with PRS-343 (at 10, 1, 0.1 and 0,01 nM) and incubated ON. All plates were then washed twice with PBS. 4-1BB over-expressing Jurkat Nf-kB reporter (at 3:1 ratio) were added to each well. Following a 6 hours incubation, Bio-Glow luciferase reagent was added to each well and luminescence was measured. Figure S5: Fold increase changes of a panel of cytokines induced by human T-cells co-stimulated by PRS-343 in the presence of SKBR-3 (HER2 high) or MCF-7 (HER2 low). Figure S6: h-4-1BB expression in T-cells during co-culture assay. Using a similar set up as described in M&M, purified Pan T-cells (from healthy donors) and SK-BR3 high Her2 expressing cells were co-incubated in the presence of coated anti-CD3 antibody. After 24, 48 and 72 hours of co-incubation, Pan T-cells were collected and 4-1BB expression on CD3 positive cells was assessed by flow cytometry. (A) shows the % of 4-1BB positive T-cells for two donors and (B) shows histogram of h-4-1BB expression on CD3 positive T-cells from two donors (red 24 hours; blue 48 hours; brown 72 hours). Table S1: Relative HER2 cell surface expression on a panel of cell lines. Expression was experimentally determined using a specific anti-HER2-antibody binding capacity (sABC [HER2]) and quantitative indirect immunofluorescence in flow cytometry (QIFIKIT). HER2 surface levels are also provided relative to the level on SKBR3 cells which were chosen as a reference cell line.

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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

T2 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 16kD protein abundant in human periciliary fluid, has a robust drug-like structure well-suited to protein engineering, but has never been used to make an inhaled "Anticalin" protein therapeutic.To re-engineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile.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. As 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 hIL-4Ra, hIL-4, and hIL-13 at their correct syntenic murine loci to model clinical dosing.Inhaled PRS-060 strongly suppressed acute allergic inflammation indices in triple humanized mice with a duration of action longer than its bulk clearance suggesting it may act locally in the lung.Lcn1 can be re-engineered into the Anticalin antagonist PRS-060, exemplifying a new class of inhaled topical, long-acting therapeutic with potential to treat T2 endotype asthma.

Bispecific antibodies which bind a tumor antigen and the CD3 heterodimer of the T cell receptor to form a synthetic immunological synapse represent a class of T cell engagers that has been clinically validated with the approvals of blinatumomab (Blinctyo®) and mosunetuzumab (Lunsumio®), targeting CD19 and CD20 respectively, for the treatment of B cell acute lymphoblastic leukemia and follicular lymphoma. Despite being a potent class of therapeutics, not all patients respond to therapy. Recent studies into state of blinatumomab-treated T cells highlight exhaustion and the lack of co-stimulation as potential factors in resistance. In solid tumors, the immunosuppressive tumor microenvironment is also a major factor while cytokine release is a safety consideration for all bispecifics that bind CD3 with strong affinity. To address the challenges of CD3 bispecifics, we have developed the EVOLVE™ platform, a trispecific antibody that consists of tumor targeting and attenuated CD3 binding affinity coupled with a CD2 agonist. We show that CD2 costimulation is superior to other pathways in its ability to sustain T cell activation and expansion and cytokine production over repeated cycles of stimulation. By integrating a CD2 agonist with an attenuated CD3 affinity bispecific, we can restore cytolytic potential without concomitant increase in cytokine release as compared to a strong CD3 affinity bispecific. The EVOLVE platform is also capable of inducing tumor cell killing by in vitro exhausted T cells whereas the bispecific did not. In terms of safety, the EVOLVE platform does not induce peripheral T cell activation and cytokine release in PBMCs in the absence of target as compared to CD3 and CD28 agonist antibodies. The superiority of the EVOLVE platform compared to a bispecific was also demonstrated in vivo where we observed 8/8 complete responses in EVOLVE-treated animals whereas the strong CD3 affinity bispecific showed no difference compared to isotype control using a CORL105 lung cancer xenograft model. We believe these experiments recapitulate the continual clinical challenges of using strong CD3 affinity bispecifics while demonstrating the advantages of the EVOLVE platform. Finally, we demonstrate the modular nature of the EVOLVE platform across diverse tumor antigens including B7H4, PSMA, CD20, and a novel squamous tumor antigen ULBP2. Together our data highlight the broad applications of the EVOLVE platform to improve T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mohosin Sarkar, Eric M. Tam, Guixian Jin, Shu Shien Chin, Abudukadier Abulizi, Oksana A. Sergeeva, Zengzu Lai, Evelyn Teran, Hayden Karp, Danielle Klaskin, Nana Adjoa Pels, Xingyue An, Jennifer Ziegler, Changqing Yuan, Maria Hackettt, Sonali Dhindwal, Amber Fearnley, Julio Rodriguez, Louis Matis, Jay S. Fine, Jeremy S. Myers. EVOLVETM: A novel T cell engager platform with integrated CD2 costimulation engineered for the treatment of immune suppressive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2971.

T cell exhaustion is a hallmark of tumor-infiltrating T cells and a major therapeutic challenge for immunotherapy which is not sufficiently understood. To identify T cell exhaustion promoting genes which may be targeted by new therapies, we combined highly efficient CRISPR/Cas9 gene knockouts in primary human T cells with functional assessment of effector function under exhaustion-inducing chronic T cell activation conditions. We conducted an initial CRISPR/Cas9 T cell exhaustion screen focused on previously identified genes reported as enhancers of anti-tumor T cell response in mouse tumor models, published exhaustion genes, and genes associated with CD8 T cell activation. Based on these results, we constructed gene networks from these screen hits and performed a secondary CRISPR-Cas9-based screen. We found that eight genes, when efficiently knocked out, limited T cell exhaustion, as measured by increased proliferation under chronic activation, reduced expression of T cell exhaustion markers, and improved tumor cell killing in CD3-bispecific antibody-mediated tumor/T cell co-culture assays. Prioritized T cell exhaustion gene hits were also evaluated in assays assessing anti-CD19 CAR-T cell function. The CRISPR-mediated ablation of five T cell exhaustion gene hits resulted in significantly more effective CD19-positive human tumor cell killing, compared to the non-targeting control CAR-T cells. These five genes included a transcriptional regulator, a member of a CD28-independent costimulatory pathway, and a series of ligand/receptor pairs that have been previously associated with T cell exhaustion and dysfunction. Interestingly, knockdown of combinations of the top three T cell exhaustion gene hits resulted in CAR-T cells that were more effective in tumor killing than any of the single gene knockouts, suggesting a non-redundant role for each gene, most likely comprising independent pathways. Furthermore, enhanced tumor cell killing was observed when each T cell exhaustion gene hit was ablated in combination with PDCD1 (PD-1) in double knockout CAR-T cells, suggesting that the T cell exhaustion pathways we identified were complementary to the PD1-PDL1 immune checkpoint pathway in regulating effector T cell function. By using a multidimensional, functional CRISPR-Cas9-based T cell exhaustion screen, we have uncovered genes that are involved in T cell exhaustion. These gene knockouts can be universally applied individually or in combination to create CAR-T cells with more durable effector activity, with the potential to achieve superior patient outcomes in a variety of tumor indications. Citation Format: Oksana Sergeeva, Amber Fearnley, Shu Shien Chin, Xingyue An, Nana Adjoa Pels, Kristen Metzler, Kyla Joinville, Lizet Aquino Calderon, Abudukadier Abulizi, Hayden Karp, Julio Rodriguez, Louis Matis, Jay Fine, Jeremy Myers. Enhanced CRISPR-Cas9 edited CAR-T cells through simultaneous inactivation of multiple T cell exhaustion pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4094.

In BriefMost studies assessing decision-making under uncertainty exploit events with probabilities that are typically above 10%. Here, to study decision-making in radical uncertainty conditions, Degoulet, Willem, Baunez, Luchini and Pintus provide a novel modelling and experimental approach that aims at measuring the extent to which rats are sensitive - and how they respond - to events that are both very rare (probability below 1%) and very extreme in their consequences, in a novel four-armed bandit task. Gains (sugar pellets) and losses (time-out punishments) are such that large - but rare - outcomes materialize or not depending on the option chosen. The results show that all rats diversify their choices mainly across two options, out of four, and that most rats feature sensitivity to rare and extreme events despite their infrequent occurrence. They do so by combining more often options with extreme gains (Jackpots) and/or avoidance of extreme losses (Black Swans). Notably, most rats’ choices feature one-sided sensitivity in favor of trying more often to avoid extreme losses than to seek extreme gains - that is, they feature the Black Swan Avoidance.Highlights-Novel modelling and experimental design are proposed to measure the extent to which whether rats are sensitive - and how they respond - to radical uncertainty materialized by events that are both very rare and extreme in their consequences, in a novel four-armed bandit task;-Most rats exhibit moderate to high sensitivity to rare and extreme events;-Most rats opt more often to avoid rare and extreme losses than to seek rare and extreme gains: they feature the Black Swan avoidance.

CD3-bispecifics are antibody-based therapies that can simultaneously bind to a tumor cell surface antigen and T cells to establish a synapse between the tumor and T cell and activate T cell to induce specific killing of the tumor cell. CD3-bispecifics have demonstrated clinical success in B cell acute lymphoblastic leukemia and follicular lymphoma with approvals of that blinatumomab and mosunetuzumab that target B cell lineage antigens CD19 and CD20, respectively. However, clinical progress in deploying CD3-bispecifics for positive patient outcomes in solid tumors has been slow, due to tumor microenvironmental factors such as induction of T cell exhaustion, as well as the potential of CD3-bispecifics to mediate T cell anergy and dysfunction in the absence of adequate co-stimulation. Here we describe the development and preclinical validation of the EVOLVE platform, a tumor-targeted biologic that induces the formation of a synthetic synapse that simultaneously activates the T cell receptor complex and the CD2 receptor to optimize CD8 T cell effector phenotype and improve tumor cell killing ex vivo and in vivo, compared to matched CD3-bispecifics. We demonstrate that CD2 co-stimulation is superior to other forms of T cell co-stimulation in its ability to promote cytolytic co-stimulation, T cell cytokine production and T cell expansion. Furthermore, CD2 receptor expression is markedly elevated in tumor infiltrating lymphocytes across a broad set of tumor types, relative to the CD28 and 4-1BB costimulatory receptors. EVOLVE-mediated T cell activation is conditionally dependent on tumor antigen binding and can be tuned to promote optimal co-stimulation without increasing cytokine release relative to matched CD3-bispecifics. We also demonstrate the modular nature of the EVOLVE platform across diverse solid tumor antigens including B7H4 (VTCN1), and a novel squamous tumor antigen ULBP2. Our data highlight the broad applications of the EVOLVE platform to improve CD8 T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mandy Shu Shien Chin, Mohosin Sarkar, Eric Tam, Abudukadier Abulizi, Guixian Jin, Xingyue An, Evelyn Teran, Danielle M Klaskin, Nana Adjoa Pels, Maria Hackett, Oksana A Sergeeva, Hayden Karp, Julio Rodriguez, Sonali Dhindwal, Changqing Yuan, Zengzu Lai, Jennifer Zeiger, Amber Fearnley, Louis Matis, Jay Fine, Jeremy S Myers. EVOLVE: A novel costimulatory T cell engager platform engineered for the treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B44.

Background Anticalin® proteins are recombinantly engineered human proteins based on lipocalins. PRS-343 is a first-in-class bispecific antibody-Anticalin fusion protein targeting the oncogenic tumor antigen HER2 and the costimulatory immune receptor 4-1BB on T and other immune cells. Here, we report the results of a phase 1 single-agent dose escalation trial in patients with HER2+ solid tumors. Methods PRS-343 has been evaluated in sequential dose cohorts from 0.0005 to 8 mg/kg i.v. Doses were administered Q3W and the 8 mg/kg dose was also given Q2W. An accelerated titration design was utilized for the initial dose escalation followed by a modified 3+3 design and the option to back-fill cohorts. Dose-limiting toxicities (DLTs) were reported during the first cycle of each schedule. The primary study objectives include the safety profile and RP2D of PRS-343. Secondary objectives include ORR and DCR, PD biomarker response and PK profile. PD response was assessed in tumor biopsies (CD8+ T cell IHC) pre- and post- PRS-343 treatment. Results 51 patients (median age 61.2 years, 61% female, 82% caucasian, 57% with more than three lines of prior therapy) with a variety of solid tumor indications [gastric/GEJ (n=19); BC (n=12); gynecological cancer (n=6); CRC (n=5); BTC (n=4); UC (n=2); melanoma, pancreatic and salivary duct (n=1 each)] have been treated with PRS-343. Based on pharmacokinetic analyses and observed kinetics of the CD8+ T cell expansion post-treatment, the low end of the active dose range is considered 2.5 mg/kg. 19 patients treated at active dose levels before the data cut-off on 09-06-2019 were evaluable for response [DCR 58% (11% confirmed PR) as per RECIST 1.1]. At the active doses, we observed significant and pronounced post-treatment expansion of CD8+ T cells particularly in the tumor nests, consistent with the MoA of PRS-343, while there was no increase in the doses below 2.5 mg/kg. The post-treatment expansion of CD8+ T cells was more pronounced in patients with a confirmed PR or prolonged SD. PRS-343 was very well tolerated, with no SAEs reported. The most frequent TRAEs were fatigue (9%), chills (6%) and diarrhea (5%) of mild to moderate severity. None qualified as a DLT. Conclusions PRS-343 is the first molecule of its kind to demonstrate encouraging evidence of safety and clinical benefit with a correlative PD effect in a heavily pre-treated population. These initial data suggest that PRS-343, the first 4-1BB bispecific to enter clinical development, merits further investigation in clinical trials. Trial Registration NCT03330561

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

Background. 4-1BB (CD137) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome toxicity and efficacy limitations of current 4-1BB targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific based on Anticalin® technology. We have previously reported on the generation and characterization of PRS-343 with regard to preclinical proof-of-concept and basic drug-like properties.1 Here, we describe the preclinical dataset supporting initiation of a first-in-patient trial. Methods and Results. The pharmacology of PRS-343 was investigated by ex vivo assays based on mixed culture of human PBMC and tumor cell lines. We find that 4-1BB costimulated T cells prominently increase production of IL-2, GM-CSF, TNF-α and IFN-γ. Using a set of immortal cancer cell lines spanning a range of HER2 surface copy numbers, we identified a copy number threshold above which PRS-343 reliably elicited T cell costimulation with a high potency and an EC50 in the subnanomolar range. PRS-343 was well tolerated in a repeat-dose study in cynomolgus monkeys, with no overt toxicity and no significant drug-related toxicological findings. Pharmacokinetic assessment confirmed dose-proportional exposure of the animals during the course of the study. In a mouse model of human PBMC-induced xenograft-vs-host disease (xGvHD), PRS-343 did not show an acceleration of xGvHD development, in contrast to a 4-1BB targeting benchmark. Again utilizing ex vivo assays, we found no PRS-343 induced T cell costimulation in a panel of primary cells, showing that physiological levels of HER2 are insufficient for activation. In a cytokine release assay, proinflammatory cytokine induction by PRS-343 in the absence of a primary TCR stimulation was negligible. Conclusion. The ex vivo experiments described indicate that HER2 expression level is expected to be a reliable marker for patient stratification for PRS-343. The toxicology assessment of PRS-343 indicates that the benign toxicity profile of trastuzumab is retained in PRS-343 with regard to HER2 targeting, and that PRS-343 is expected to elicit its costimulatory effects strictly on T cells also receiving a primary TCR signal and strictly localized to HER2-positive tumors. This is in agreement with in vivo mouse model data showing PRS-343 leads to tumor-localized CD8+ T cell expansion,1 and supports the potential of PRS-343 as an efficacious yet well tolerable 4-1BB costimulating agent. The reported data is the basis for the trial design of a first in patient Phase 1 trial with PRS-343. 1Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Thomas Jaquin, Sven Berger, Manuela Dürr, Corinna Schlosser, Andrea Allersdorfer, Christine Rothe, Louis A. Matis, Shane A. Olwill. Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2017-3673

Despite intensive efforts, the general rules for γδ T cell recognition remain undefined. Here, we take advantage of the detailed knowledge of the molecular structure and biosynthetic pathways of major histocompatibility complex (MHC) molecules to analyze the recognition properties of the γδ T cell clones LBK5 (specific for the class II MHC, IE k ) and G8 (specific for the nonclassical class I MHC, TL 10 b ). We find that the activation of these clones requires neither class I nor class II antigen-processing and that peptides do not confer specificity. Epitope mapping also shows that the topology of γδ T cell receptor interaction with the MHC is distinct from that of αβ T cells. These results suggest that the molecular nature of γδ T cell recognition is fundamentally different than that of αβ T cells.