Selective support of engineered T cells using a cis-targeted interleukin-2 enhances anti-tumor activity and obviates the need for lymphodepletion

Selective support of engineered T cells using a cis-targeted interleukin-2 enhances anti-tumor activity and obviates the need for lymphodepletion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Selective support of engineered T cells using a cis-targeted interleukin-2 enhances anti-tumor activity and obviates the need for lymphodepletion Saar Gill, Sara Sleiman, Nathan Mathewson, Tyler Hansen, Paul Bessette, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5962170/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Adoptively transferred T cells require cytokine stimulation, which is achieved using lymphodepleting chemotherapy with or without administration of exogenous interleukin 2 (IL-2). Lymphodepleting chemotherapy (LDC) is associated with cytopenias and attendant complications, while high dose IL-2 causes severe infusion toxicity and can stimulate undesirable cell populations. To address these challenges, we developed cis-targeted IL-2 fusion molecules which are comprised of an IL-2 mutein with attenuated binding to IL-2Rα and IL-2Rβ linked to an antibody that targets a cell-surface molecule expressed specifically on engineered T cells. Using T cells from healthy donors as well as from lymphoma and melanoma patients, we selectively stimulated CAR-T cells or engineered TILs and enhanced their anti-tumor function in multiple tumor models. Finally, we eliminated the need for LDC by combining CAR-T cells with cis-targeted IL-2, leading to B cell aplasia in non-human primates. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Immunology/Cytokines/Interleukins Biological sciences/Immunology/Translational immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficacy in the treatment of B cell and plasma cell malignancies. Complete remission (CR) rates for B-ALL receiving CD19-targeted CAR T cell therapy exceed 80%, while NHL and MM patients achieve CR rates between 30% and 60% ( 1 – 7 ). The response rate to CAR-T therapy is highly correlated with the in vivo expansion and persistence of CAR-T cells ( 1 , 5 – 7 , 8 – 10 ). Therefore, strategies that can improve the durability and function of CAR-T cells have the potential to improve patient outcomes. One strategy for enhancing persistence involves providing cytokine support to the CAR-T cells. Currently, this is accomplished through lymphodepletion, which stimulates the production of homeostatic cytokines and transiently removes cellular “sinks” that compete with the adoptively transferred cells ( 4 , 11 – 14 ). However, lymphodepletion is associated with a significant incidence and severity of cytopenias and their attendant complications, such as susceptibility to infection ( 15 ). Therefore, an approach that could enhance CAR-T cell activity while minimizing the need for lymphodepleting chemotherapy would likely be of general interest and appeal. The cytokine interleukin (IL)-2 is crucial for the activation and expansion of CD4 and CD8 T cells. However, a demonstrable pharmacologic effect requires the administration of high doses of IL-2, as given in tumor-infiltrating lymphocyte (TIL) therapy, where it is accompanied by dose-limiting cardiovascular and respiratory toxicities. In addition, IL-2 promotes the proliferation of regulatory T (Treg) cells, which may restrain its anti-tumor effect ( 16 – 21 ). Thus, rhIL-2 therapy for cancer is limited by toxicity and by stimulation of undesirable immunosuppressive cell populations, which has limited its broader utility in combination with T-cell therapies. The intermediate-affinity IL-2 receptor complex is a heterodimer of the β chain (CD122) and the common γ chain (CD132), and is expressed broadly, including on resting effector T cells and natural killer cells. The high-affinity IL-2 receptor complex is a heterotrimer of the α chain (CD25) along with CD122 and CD132 and is expressed constitutively on Treg cells and innate lymphoid cells, and transiently on activated conventional T cells (Tconv) ( 16 , 17 , 20 – 22 ). Previous efforts to selectively activate effector T cells without stimulating Tregs led to the development of engineered IL-2 variants with attenuated affinity for IL-2Rα ( 19 , 23 – 25 ) (“not-α” IL-2 variants) or with enhanced affinity for IL2Rβ/γ (IL-2 ‘superkines’) ( 16 , 19 , 26 – 28 ). However, these variants are not completely selective for effector and memory T cells, since natural killer (NK) cells, innate lymphoid cells and endothelial cells also express IL-2 receptors and may contribute to toxicities of these variants in patients. Indeed, NK cells were found to mediate toxicities of the not-α IL-2 variants in mice ( 29 , 30 ), and clinical data with engineered IL-2 variants suggest that substantial improvements in IL-2’s therapeutic index could not be achieved in patients when signaling is delivered broadly to IL2Rβ/γ + cells ( 17 , 31 – 33 ) We set out to overcome the limitations of wild-type (WT) IL-2 and previously engineered IL-2 molecules in enhancing the potential of cell therapies by developing an approach that selectively delivers IL-2 stimulation to engineered T cells, without activating endogenous cells. We recently showed that a CD8 + T cell-selective IL-2 molecule, consisting of an attenuated IL-2 mutein linked to an antibody targeting the CD8b molecule, can enhance anti-tumor and anti-viral immunity while improving tolerability compared to “not-α” IL-2 ( 29 , 34 , 35 ). Building on this concept of cell type-selective targeting (cis-targeting) ( 29 , 36 ), here we engineered a cis-targeted IL-2 that is comprised of an IL-2 variant that does not bind to IL2Rα (CD25) and has reduced affinity for IL2Rβ (CD122), linked to a targeting antibody against a surface molecule specifically expressed on engineered cells. We show that targeting IL-2 to a truncated, non-signaling EGFR tag (EGFRt) expressed in CAR T cells can selectively and safely stimulate engineered human and non-human primate CAR-T cells resulting in substantially improved anti-tumor efficacy in mouse models, and prolonged B cell aplasia in non-human primates in the absence of lymphodepleting chemotherapy. Collectively, these data provide proof-of-concept enabling IL-2’s utility in combination with cell therapy and promise to enhance the efficacy of T cell therapies while avoiding the life-threating toxicities associated with wild-type IL-2 and lymphodepletion. RESULTS Cis-targeted IL-2 fusion molecules deliver selective IL-2 stimulation to CAR-T cells in vitro To develop an IL-2 molecule that can signal selectively on CD19 CAR-T cells, we fused a CAR-T cell-targeting antibody to an attenuated IL-2 mutein with no IL-2R⍺ binding and reduced IL-2Rβ binding (Fig. 1a). Attenuation of IL-2 affinity to both IL-2R⍺ and IL-2Rβ was required to avoid activation of endogenous Tregs and NK cells that express high levels of IL-2R⍺ and IL-2Rβ, respectively ( 29 ). Indeed, IL-2 mutein (IL2m1) fused to a control antibody did not activate conventional T cells in human PBMCs at concentrations below 100nM and regulatory T cells and NK cells below 10nM (Extended Data Fig. 1a, Supplementary Table 1). IL-2 muteins were targeted to CAR-T cells either in an idiotype-specific manner, using an antibody against the FMC63 clone of the CAR constructs employed in commercial CART19 products; or in a reporter-specific manner, using an antibody based on panitumumab directed against a non-signaling truncated EGFR (EGFRt) tag that is co-expressed with the CAR as used in lisocabtagene ciloleucel ( 37 ). Whereas WT IL-2 stimulated all cells expressing its receptor, cis-targeted IL-2 fusion molecules targeting the CAR (CAR-IL2m1) or EGFRt (panit-IL2m1) mediated selective IL-2 signaling due to their preferential stimulation of cells that express both the IL-2R and the targeting antigen (CAR or EGFRt) (Fig. 1b-d). Using STAT5 phosphorylation as a readout, stimulation of CAR-T cells with the cis-targeted IL-2 muteins led to > 100-fold increase in selectivity compared with WT IL-2, suggesting that IL-2 fusion binding to a cell surface antigen in cis compensated for its low affinity binding to IL-2R (Fig. 1d; Extended Data Fig. 1b). Consistent with selective IL-2 signaling in vitro, CAR-IL2m1 and panit-IL2m1 induced robust and selective in vivo expansion of CAR-T cells in immunodeficient NOD-SCIDγc-/- (NSG) mice (Fig. 1e). To demonstrate the potential of CAR-T cells stimulated with IL-2 mutein to mediate an anti-tumor effect in vivo, we engrafted NSG mice with the B-ALL cell line NALM6 and down-titrated the CART19 dose. We found that the addition of CAR-IL2m1 could rescue sub-therapeutic doses of CART-19 (Supplementary Fig. 1). Anti-idiotype CAR cis-targeting induces antigen-independent cytokine release and toxicity Chimeric antigen receptors contain intracellular CD3z and costimulatory signaling domains that become activated upon binding to tumor antigen ( 38 – 40 ). Given that anti-idiotype CAR targeting of IL-2 brings in proximity two signaling receptors (CAR and IL-2R) which could induce their potential cross talk ( 41 ), we were concerned that CAR-IL2m1 could activate CAR-T cells in the absence of CD19 antigen. Indeed, CAR-IL2m1 stimulation of human CART19 cells induced the production of interferon-γ (IFN-γ), tumor necrosis factor (TNF) and IL-5 in an antigen-independent manner at levels comparable to that induced by anti-CD3 and anti-CD28 stimulation (Extended Data Fig. 1c). In contrast, Panit-IL2m1 targeting the non-signaling truncated EGFR tag induced significantly lower cytokine secretion and this was further attenuated when the Panit-targeting antibody was fused with IL2m2, an IL-2 mutein with 5-10-fold lower affinity than IL2m1 (Extended Data Fig. 1a; Supplementary Table 1). We administered a single dose of CAR-IL2m1 or panit-IL2m2 one day after CART-19 to compare their ability to safely enhance CAR-T cell function in vivo (Fig. 1f). Although both molecules prolonged the survival of tumor-bearing mice compared to mice that received control CAR-T cells, Panit-IL2m2 showed better tumor control than CAR-IL2m1 and induced median survival of 78 days vs 57 days, respectively (p = 0.028) (Fig. 1g-h). In addition, transient body weight loss indicative of toxicity was observed in mice receiving CAR-IL2m1, suggesting that targeting of IL-2 to CAR has a narrower therapeutic window than targeting to a non-signaling tag such as EGFRt (Fig. 1i). Thus, by selecting the optimal combination of targeting antigen (EGFRt instead of CAR) and of IL-2 mutein affinity (IL2m2 instead of IL2m1), we developed a cis-targeted IL-2 that can substantially and safely enhance the anti-tumor activity of CAR-T cells. Development of IL-2 fusion molecule that is selective for truncated EGFR over EGFR The targeting antibody in panit-IL2m2 is based on panitumumab and binds the full-length EGFR as well as the truncated EGFR that is used as an extracellular tag in CAR-T cells. To prevent the binding of cis-targeted IL-2 to cells that express full-length EGFR (such as epithelial cells), which could have unintended off-target toxicity, we developed an antibody that is selective for EGFRt over EGFR. The EGFRt-selective antibody Ab43 recognizes only the truncated EGFR expressed in CAR-T cells and does not bind to HE293 cells that express full length EGFR (Fig. 2a-b). When fused to IL2m2, this EGFRt-selective molecule, referred to as EGFRt-IL2, stimulated CAR-T cells with equivalent potency to panit-IL2m2 in vitro (Fig. 2c) and in vivo (Fig. 2d). Furthermore, CAR-T cells stimulated with the EGFRt-IL2 one day after infusion were able to selectively re-expand following a prolonged rest of 2 months in NSG mice when stimulated with a second dose of EGFRt-IL2 (Supplementary Fig. 2). To confirm that EGFRt-IL2 can bind and stimulate patient-derived CAR-T cells, we obtained residual cells saved from a lisocabtagene ciloleucel product, a commercial CART19 co-expressing EGFRt (Supplementary Fig. 3a). While exposure to WT IL-2 used at a high concentration of 10nM mediated superior overall T cell expansion (likely due to the fact that WT IL-2 induces IL2Rα and becomes more potent by binding to the trimeric high affinity receptor), only stimulation with EGFRt-IL2 led to a selective expansion of CAR + T cells in the CART19 product (Supplementary Fig. 3b-d). EGFRt-IL2 mediates strong CD19 CART expansion and anti-leukemia activity without toxicity We next sought to define the in vivo anti-tumor activity of EGFRt-IL2 when administered early (day 1) or delayed (day 7) after CD19 CAR-T cells. NSG mice bearing NALM6 leukemia cells were treated with a “stress dose” of CART19, followed 1 or 7 days later by a single dose of EGFRt-IL2 (Fig. 2e). EGFRt-IL2 treatment on day 1 induced strong anti-tumor activity with 5/5 complete tumor regressions and no toxicity as measured by weight loss (Fig. 2f; Extended Data Fig. 2a,b). EGFRt-IL2 strongly and preferentially expanded CAR-T cells reaching a peak of over 2,400-fold expansion by day 13 in mice that received both CAR-T cells and EGFRt-IL2 compared to mice that received only CAR-T cells (Fig. 2g-i). Early EGFRt-IL2 treatment resulted in improved survival exceeding 100 days (Fig. 2j). While delayed treatment with EGFRt-IL2 did potentiate CART19 anti-leukemia activity, the magnitude of this effect was less than with treatment on day 1 (Fig. 2f, j). Delayed administration of the cytokine was associated with lower peak expansion of CAR-T cells (Fig. 2g-i). EGFRt-IL2-treated mice that had cleared tumors were re-challenged with NALM6 140 days post CAR-T infusion and showed moderate tumor control compared to naïve mice suggesting remaining CAR-T activity even 140 days after initial infusion (Extended Data Fig. 2c,d). These results suggest that early (pre-emptive) administration of EGFRt-IL2 may be preferable to delayed administration, at least in the setting of a fast-growing leukemia xenograft. EGFRt-IL2 enhanced CAR-T cell expansion and survival of mice using a sub-optimal dose of CAR19 T cells manufactured from a lymphoma patient PBMCs The above experiments were largely performed using CART19 made from healthy donor T cells. To assess whether EGFRt-IL2 could potentiate the activity of CAR-T cells manufactured from a cancer patient, CART19 cells were prepared from cryopreserved PBMCs that had been collected from a patient with B cell lymphoma (Supplementary Fig. 4a-b). NALM6-bearing NSG mice were treated with 0.25x10 6 CAR-T cells followed by EGFRt-IL2 1 day later (Fig. 3a). Mice treated with EGFRt-IL2 experienced improved tumor control, attributed to marked expansion of patient-derived CAR-T cells (Fig. 3b-d). To test whether repeated administration of EGFRt-IL2 could provide ongoing CAR-T cell expansion and further enhance their anti-tumor activity, we treated one group with 3 doses of EGFRt-IL2, on day 1, day 21 and day 49 post-CAR-T cell infusion (every 3 to 4 weeks) and found EGFRt-IL2 dependent re-expansion in majority of these mice (Fig. 3d), with a trend toward delayed tumor growth and prolonged survival. (Fig. 3b, c). Among the mice treated with EGFRt-IL2, a pre-defined group was sacrificed on day 14 to assess CAR-T cell engraftment and tumor burden in the spleen. We found a significantly higher number of CAR-T cells in mice treated with EGFRt-IL2 compared to the CART-only group (Fig. 3e). In addition, CAR-T cells from mice treated with EGFRt-IL2 displayed lower levels of PD1 and LAG3 exhaustion markers as compared to CAR-T cells that received PBS alone (Fig. 3f). To gain further insight regarding the impact of EGFRt-IL2 treatment in vivo, CD45 + cells from CART19 + PBS and CART19 + EGFRt-IL2 treatment groups were harvested from the bone marrows on day 14 for single cell transcriptomics. Clustering of all CAR-T cells identified 2 different populations for mice treated with CART19 only vs CAR19 with the EGFRt-IL2 (Fig. 3g). Compared with CART19 alone, CART19 exposed to EGFRt-IL2 had higher expression of some genes associated with activation (ZNF683, CD40LG, CD52, TIMP1, ANXA1), adhesion (ITGA1, ITGB1), the immune synapse (TAGLN2), and metabolism (ALOX5AP); and lower expression of transcription factors associated with exhaustion (ID3, TOX2, IKZF3), inhibitory molecules (CRTAM, CD38, CD200, TNIP3, LAG3, IL10) (Fig. 3h). T cell subset analysis showed more proliferating CD8 + T cells in the control group (Fig. 3i) which may be related to a higher tumor burden than in EGFRt-IL2 group at the time of analysis (47% vs 13% respectively). In addition, slightly higher effector memory CD8 + T cells were observed in EGFRt-IL2 group compared to PBS group (35% vs 25% respectively). These findings show that EGFRt-IL2 enhances the anti-tumor activity of patient-derived CAR-T cells in a xenograft model by promoting expansion, reducing exhaustion, and improving tumor control. Transcriptomic and pathway analyses revealed increased activation and reduced exhaustion markers in CAR-T cells treated with EGFRt-IL2. These results suggest that EGFRt-IL2 can sustain CAR-T cell function and improve therapeutic outcomes. EGFRt-IL2 converts B7H3 CART cells into a curative therapy in a solid tumor model To evaluate the potential of EGFRt-IL2 in the context of CAR-T cell therapy for solid tumors, we utilized a human A375 melanoma NSG xenograft model that expresses the B7H3 tumor antigen. A375 melanoma-bearing mice were treated with 4x10 6 CAR-T cells co-expressing an anti-B7H3 CAR along with the EGFRt tag with or without EGFRt-IL2 given either weekly or biweekly (Fig. 4a). WT IL-2 in combination with anti-B7H3 CAR-T cells was also tested. In this model, 4x10 6 CAR-T cells alone resulted in only a minor tumor growth delay, whereas complete tumor rejection occurred in all mice treated with either weekly or biweekly administration of EGFRt-IL2 (Fig. 4b-c, Extended Data Fig. 3a), with no apparent toxicity (Fig. 4d). In contrast, although the WT IL-2 combination induced a similar level of tumor regression as EGFRt-IL2 initially (Fig. 4b), significant toxicity developed in mice treated with WT IL-2 within 20 days, resulting in greater than 20% weight loss in all mice and treatment-related mortality (Fig. 4c-d). We further characterized the effects of EGFRt-IL2 on anti-B7H3 CAR-T cells by isolating peripheral blood and tumors from some mice that received PBS or weekly doses of EGFRt-IL2 at day 18 post initial CAR-T dose (Fig. 4a). In the blood, EGFRt-IL2 treatment selectively expanded CAR + T cells by approximately 80-fold over PBS control, resulting in the enrichment of CAR + T cells to over 90% compared to 20% in the inbound CAR-T product (Fig. 4e). Within the tumor, CAR + T cells from both PBS and EGFRt-IL2 groups expanded compared to CAR- T cells and were enriched to over 90% in both groups. However, EGFRt-IL2 treatment resulted in over 86-fold more CAR + T cells in the tumor compared to PBS controls (Fig. 4f). Furthermore, close to 60% of CD8 + CAR + T cells in the tumor from PBS-treated mice were of the exhausted phenotype, as defined by co-expression of PD1, LAG3, and TIM3, compared to only 25% in EGFRt-IL2-treated mice (Fig. 4g, Extended Data Fig. 3b-c)). Similar pattern was observed with CD4 + CAR + T cells of which 30% co-expressed the 3 inhibitory markers in EGFRt-treated mice compared to 45% in PBS-treated mice (Fig. 4g). This likely explains the lack of meaningful anti-tumor activity observed in the CAR-T alone treatment group. Overall, the results presented here suggest that EGFRt-IL2 selectively and strongly expanded CAR + T cells and reduced the expression of inhibitory/exhaustion markers, dramatically enhancing CAR-T cells' anti-tumor activity in a solid tumor setting. Next, we sought to selectively stimulate tumor-infiltrating lymphocytes (TILs) expressing EGFRt using our EGFRt-IL2 molecule. To this end, we generated EGFRt + TILs from an excisional biopsy of a patient with metastatic melanoma (Extended Data Fig. 4a). These cells were added to an autologous organoid model ( 42 ) derived from the same patient’s melanoma tissue. Flow cytometry analysis revealed a pronounced selective expansion of EGFRt + TILs by day 7 post-stimulation with EGFRt-IL2, compared to WT-IL2 (Extended Data Fig. 4b,d), while the anti-tumor effect in this short-term assay was similar between TILs stimulated with WT-IL2 and EGFRt-IL2 (Extended Data Fig. 4c). EGFRt-IL2 allows autologous anti-CD20 CAR to mediate B cell aplasia without lymphodepleting chemotherapy in non-human primates. CART19 do not cross-react with non-human primate (NHP) B cells. We therefore generated autologous anti-CD20 CAR-T cells from 2 adult rhesus macaques (RM) that recognize rhesus CD20 and co-express EGFRt (Supplementary Fig. 5a-c). We also selected a new EGFRt-IL2 molecule for rhesus macaque CAR-T cell experiments (Ab43-IL2m3) since Ab43-IL2m2 bound and stimulated rhesus CART cells poorly due to the binding specificity difference of the IL-2 mutein toward rhesus and human IL-2 receptors (Supplementary Fig. 6). On day 0, both animals were injected with 2.5 x 10 6 CAR-T cells/kg. RM#1 received 0.3 mg/kg of EGFRt-IL2 IV 30 minutes post CAR-T cell infusion while RM#2 received CAR-T cells alone (Fig. 5a, b). B cell counts began to fall on day 3 in RM#1 and remained undetectable in the blood until day 35 post infusion (Fig. 5c, Supplementary Fig. 7). In addition, bone marrow flow cytometry showed B cell aplasia in RM#1 through day 35. (Fig. 5d). This was accompanied by a transient increase in CD3 + T cells (Fig. 5e) of which approximately 14% expressed the CAR on day 8 (Extended Data Fig. 5a). Notably, on day 7 RM#1 developed hyperthermia, cutaneous erythema of face and neck, and was found to have biochemical features (marked renal azotemia and elevations of transaminases) consistent with dehydration induced by cytokine release syndrome (CRS) (Fig. 5f), while hematologic parameters were relatively stable (Fig. 5g). The animal was therefore treated on day 7 with intravenous fluids and dexamethasone (3mg/kg), and on day 8 with the IL-6 receptor antagonist tocilizumab (8mg/kg) and a second dose of dexamethasone (2mg/kg). These interventions resulted in prompt clinical and laboratory improvement (Fig. 5f,g). These clinical findings were associated with marked increase in MIP1 \(\:\alpha\:\) and modest elevation in IFN- \(\:\gamma\:\) and IL-6, which could correspond to CRS (Extended Data Fig. 5b). At the first indication of B cell recovery, RM#1 was re-dosed with EGFRt-IL2 on day 48 without additional CART cells. This time there were no clinical or lab abnormalities, no reduction in B cells and no increases in T cells (Fig. 5c, e-g). Retrospective analysis of animal serum showed the development of anti-CAR antibodies starting from day 28, coinciding with B cell recovery (Fig. 5h). Notably, antibodies directed against the EGFRt-IL2 molecule did not develop until 62 (14 days post re-dosing) (Supplementary Fig. 8a). In contrast to RM#1, RM#2 received autologous CART-20 without EGFRt-IL2. This animal did not show any evidence of B cell reduction, and no clinical or laboratory changes up to 14 days post CAR-T infusion (Fig. 5i,j; Extended Data Fig. 5c,d). The animal then received EGFRt-IL2 on day 14 post CART-20, again with no meaningful changes in B cell count (Fig. 5i,j), despite mild clinical changes including mild facial edema, photophobia and mild tremors that were treated with diphenhydramine and levetiracetam on day 14, and 1 dose of dexamethasone (3mg/kg). In RM#2, anti-CAR antibodies were detected from day 12 onwards and may have contributed to the lack of CAR-T cell expansion and activity from day 7 onward (Fig. 5k). Anti EGFRt-IL2 antibodies were not detected through day 166 (Supplementary Fig. 8b). These results suggest that administration of a single dose of EGFRt-IL2 along with autologous CAR-T cells can obviate the need for lymphodepletion and achieve B cell aplasia at the dose provided. ADA against CAR-T cells, which contain non-rhesus sequences, may have limited the potential for the repeat dose of EGFRt-IL2 to induce CAR-T re-expansion in this setting as re-dosing was performed well after the initiation of the ADA response against CAR-Ts. DISCUSSION Expansion and persistence are considered crucial to the immediate and durable anti-tumor effects of adoptively transferred cells such as CAR-T cells and TILs ( 1 – 4 , 43 ). The IL-2 cytokine family plays a key role in mediating T cell survival, proliferation, and memory formation ( 44 ). While current CAR-T cell therapy is not accompanied by exogenous cytokine administration, it is thought that lymphodepleting chemotherapy works, in part, by stimulating the production of homeostatic cytokines and by transiently depleting cell populations that can compete with the adoptively transferred T cells for these cytokines. IL-2 was the first cytokine used in cancer immunotherapy, with early clinical trials showing its effectiveness in stimulating “lymphokine activated killer cells” against solid tumors. However, the cytokine has a short half-life and the high doses required in TIL therapy are dose-limiting ( 45 ). In addition, the pleiotropic nature of IL-2 cytokine may hamper the immune response due to the activation of regulatory T cells and toxicities, both of which were linked to IL-2 binding to IL-2R \(\:\alpha\:\) ( 16 , 17 , 20 , 22 ). Consequently, efforts have been made to engineer IL-2 molecules with a bias towards IL-2R \(\:\beta\:\gamma\:\) , thus avoiding both immune suppression and toxicity and opening possibilities of combinations with T cell therapies ( 16 , 19 , 24 , 26 – 28 , 46 ). However, many of the recently engineered IL-2 variants have been tested in the clinic and have shown limited success, including preferential expansion of NK cells over T cells in patients. Therefore, there is a need to further improve IL-2 therapeutics, including in the setting of adoptive T cell therapies. We have recently shown that a protein engineering approach referred to as cis-targeting can be used to engineer a more selective IL-2 to use as an immunotherapy for cancer and viral infections ( 29 , 34 , 35 ). We previously developed AB248, a CD8-targeted IL-2, by utilizing an IL-2 mutein with attenuated affinity to IL-2R \(\:\alpha\:\) and IL-2R \(\:\beta\:\) fused to a targeting antibody recognizing CD8b, a molecule that is specific for cytotoxic CD8 + T cells with limited to no expression on NK cells. AB248 recapitulated the effect of IL-2 on CD8 + T cells, induced their selective expansion in mice and primates, and resulted in superior anti-tumor and anti-viral activity and enhanced tolerability when compared to an IL-2Rβγ agonist. Efficacy in mouse models was associated with the emergence of a tumor-infiltrating effector population and the rescue of dysfunctional or exhausted T cells in ex vivo cultured human tumor fragments. AB248 is currently in clinical development for the treatment of cancer alone and in combination with anti-PD1 (NCT05653882). Here we used a similar cis-targeting technology to demonstrate that IL-2 can be specifically directed to engineered T cells, in order to limit any off-target effects that may arise from long-term stimulation of endogenous T cells. We directed engineered IL-2 molecules to CAR-T cells by linking them to antibodies directed against the CAR idiotype or against the EGFRt extracellular tag. The molecules tested here showed high selectivity for engineered T cells, using pSTAT5 assays, proliferation, cytokine production, and tumor cell killing in vitro; as well as in vivo proliferation and persistence, durable tumor responses and prolonged survival in xenografted mice, including those treated with CAR-T cells made from the blood of a lymphoma patient. The IL-2 molecule directed specifically to the CAR led to antigen-independent cytokine release, likely due to induced proximity of the two signaling receptors, CAR and IL-2R, and this translated to toxicity in mice. We therefore prioritized anti-tag IL-2 molecules, ultimately generating a first-in-class antibody selective for the EGFRt tag. This construct was able to stimulate CAR-T cells from a patient treated with lisocabtagene (a commercial CART19 product that contains an EGFRt tag), and does not cross-react with the wild type EGFR expressed on epithelial cells, which should translate into a further improvement in therapeutic index in patients. We then showed in a second tumor model that the anti-EGFRt-IL2 molecule can enhance the activity of anti-B7H3 CART cells in a melanoma xenograft model, while reducing toxicity compared with wild-type (un-engineered) IL-2. Our data suggest that a single cis-targeted IL-2 mutein could be used to partner with different adoptive cell therapies, as long as these contain a specific extracellular tag. Lifileucel is an autologous TIL therapy that recently obtained FDA approval for melanoma, and that must be combined with high-dose IL-2. While TIL products are usually not genetically engineered, we demonstrated here that TILs can be transduced to express a tag and that administration of the EGFRt-IL2 molecule can further enhance their function, and perhaps this approach could be tested in future to improve TIL function without exposing the patient to the toxicity of high-dose IL-2. Finally, we reasoned that potent IL-2 stimulation could obviate the need for lymphodepleting chemotherapy. In rhesus macaques we found that EGFRt-IL2 could indeed mediate B cell aplasia. As expected, in the absence of chemotherapy there was no hematopoietic toxicity. Notably, in this model we found that B cell depletion was profound yet transient, lasting approximately 6 weeks. We note that this is similar to the B cell depletion that occurs in patients recently treated with LD chemotherapy and CART19 cells for autoimmune diseases ( 47 ). B cell recovery was accompanied by the development of ADA, initially anti-CAR antibodies, and later, antibodies against EGFRt-IL2. The early occurrence of anti-CAR antibodies may be attributable to the human and murine components of the CAR construct. Future studies may evaluate earlier redosing of EGFRt-IL2 prior to the development of ADA which may result in even longer B cell aplasia. Whether such ADA will occur in humans is unknown. Biweekly dosing of EGFRt-IL2 alongside CAR-T cells may be a feasible approach in patients to maintain long term B cell and tumor cell depletion. There are other approaches to obviate the need for lymphodepletion. These include constitutive STAT5 activation, or administration of synthetic IL-2 or IL-2 family cytokines ( 11 , 48 , 49 ). The orthogonal IL-2/IL-2R system showed selective stimulation of engineered CAR T cells in murine models ( 46 , 49 , 50 ). However, orthogonal IL-2/IL-2R pairs require further genetic modification of the T cells, and therefore, could not be readily added to current standard of care CAR T therapeutics. In conclusion, our data show that combining the EGFRt-IL2 with CAR T cell therapy markedly enhances the anti-tumor efficacy of the CAR T cell therapy in two preclinical xenograft models models. We also showed that human melanoma TILs can be transduced with an extracellular tag and stimulated using EGFRt-IL2, and their anti-tumor function augmented against autologous melanoma organoids. EGFRt-IL2 eliminated the need for conditioning lymphodepletion chemotherapy prior to an autologous CART cell treatment in an NHP model. Together, these studies support cis-targeted IL-2 as a valuable addition to multiple aspects of therapeutic adoptive T cell therapy, which should be tested in appropriately designed clinical trials. MATERIALS AND METHODS Generation of interleukin (IL)-2 molecules Antibodies against the FMC63 and EGFRt were generated via hybridoma or phage display. The amplified cDNA fragments of heavy and light chain V-domains were inserted in frame into a human IgG1 construct with knob-into-hole modification in the IgG CH3 domains ( 51 ). IL-2 portions of the constructs were cloned in frame with the “hole” heavy chain using a flexible linker between the C-terminus of the IgG heavy chain and the N-terminus of IL-2. To abolish FcγR binding/effector function and prevent FcγR co-activation, the following mutations were introduced into the CH2 domain of each of the IgG heavy chains: L234A/L235A/G237A (EU numbering). Molecules were expressed in HEK293 cells and then purified using Protein A affinity chromatography, followed by ion-exchange chromatography and then size exclusion chromatography. SDS-PAGE analyzed the purity, integrity, and monomeric state of the fusion constructs. The protein concentration of purified IL-2 fusion constructs was determined by measuring the optical density at 280 nm. Human T-cell transduction Healthy donor T cells were obtained from the Human Immunology Core (HIC) at the University of Pennsylvania, or from the Stanford Blood Bank. Deidentified lymphoma patient PBMCs were obtained from the Stem Cell and Xenograft Core at the University of Pennsylvania. Cells were expanded with antiCD3/CD28 beads (ThermoFisher, Catalogue# 11132D) for up to 14 days (until cell size is less than 350fL) and transduced with concentrated lentivirus from HEK293 T cells transfected with anti-CD19-41BB- CD3ζ plasmid DNA beginning on day + 1 at MOI of 3. CAR-T cells utilized in mouse pharmacodynamic studies were generated using a construct generated with either an anti-CD19 [FMC63] scFv or anti-B7H3 “clone CD276.MG, ( 52 )” scFv fused to human CD8 transmembrane region that was upstream of 41BB costimulatory molecule and the CD3zeta signaling domain. This CAR cassette was encoded upstream of a self-cleaving P2A peptide that drove expression of truncated epidermal growth factor receptor (EGFRt), consisting of only the transmembrane region and extracellular domain 3 and domain 4 of EGFR, and cloned into a lentiviral vector under control of EF-1⍺ promoter. Cell Lines The NALM6 cell line was obtained from the ATCC and maintained in RPMI media supplemented with 10% fetal calf serum, penicillin, and streptomycin (R10). The cells were transduced with a luc2-EGFP lentiviral construct and sorted twice by positive-selection flow cytometry to > 99% purity. Cells were viably cryopreserved in 90% fetal calf serum and 10% dimethylsulfoxide until required for use. For all functional studies, NALM6 cells were thawed at least 12 hours before analysis and rested overnight at 1 × 10 6 per mL in R10. In vitro cytokine release assay Antigen-independent cytokine release assays were performed by incubating 5 x 104 thawed human CAR-T cells with the indicated stimulus in X-VIVO 15 media (Lonza) in a 96-well flat bottom plate (Corning) for 72 hours. Cell culture supernatants were collected and stored at -80°C until analysis was performed by MSD U-plex plates (Mesoscale Discovery) to quantify IFN𝛾, TNF⍺, IL-5, MIP1⍺, and IL-6 following the manufacturer’s instructions. Flow cytometry Anti-human antibodies were purchased from BioLegend, and Thermofisher. Cells were first added to ACK lysing buffer, then they were stained with Fc block. They were then stained with our flow cytometry panel. Counting beads were used to quantify the concentration of cells from peripheral blood as per manufacture’s protocol (Invitrogen, CountBright Absolute Counting Beads). In all analyses, the population of interest was gated based on forward vs side scatter characteristics followed by singlet gating, and live cells were gated using Live Dead Aqua (Invitrogen). Analysis of pSTAT5 activity assays were performed using FlowJo v10.9. Briefly, CD3 + T cells were gated based on CD8 + high population, shown to consistently exhibit pSTAT5 activity in response to WT IL-2. Cells were further gated upon staining with anti-FMC63 idiotype antibody (Acro Biosystems) to determine the percent or gMFI of pSTAT5 activity in CAR + or non-CAR (CAR-) T cells. Human pSTAT5 Assay STAT5 phosphorylation was assessed on a mixed population of CD3 + T cells consisting of CAR-T cells (30–50% CAR+) and non-CAR T cells in the same well of a 96-well plate. Briefly, blood was treated for 25 minutes with indicated molecules at 37°C at a 2X final dilution in unsupplemented RPMI-1640 media. Surface staining antibodies were added and incubated at 4°C for 10 minutes and cells were washed twice with PBS containing 2% BSA. Pre-warmed Lyse/Fix (BD Biosciences) was added and incubated at 37°C for 10 minutes. Following fixation, cells were washed and resuspended in chilled BD Perm Buffer III (BD Biosciences) and incubated for 1 hour at -20°C. Cells were then washed and stained for remaining surface and intracellular markers in TFP Perm/Wash buffer (BD Biosciences) for 45 minutes at 4°C. After washing, cells were resuspended in FACS buffer and analyzed by flow cytometry as described above. In vivo murine models Male and female 8–12 week old NOD-SCID-IL2rγ−/− (NSG) mice were purchased from Jackson laboratories or bred in-house. All experiments were performed on protocols approved by the institutional animal care and use committees of the University of Pennsylvania. These studies were conducted in accordance with the Declaration of Helsinki. The experimental outline schema of the used xenograft models is available in Fig. 1. NALM6 cells (1x10 6 cells/ mouse) were injected in 200 µL of PBS into the tail veins of mice; tumor burden was assessed by live imaging of animals for bioluminescence. Mice were injected intraperitoneally (IP) with 150 µL of luciferin (15 mg/mL) and imaged starting on Day − 1 (6 days post NALM6 transplant) with a bioluminescence imager (Xenogen IVIS-200 Spectrum). Animals were imaged once weekly up to Day 110. Anti-CD19-redirected T cells (CART19), or untransduced (UTD) human T cells were injected in 200 µL of PBS at multiple doses ranging from 0.1 to 1x10 6 cells/mouse into the tail vein. Mice were monitored for signs of severe disease and euthanized when they met pre-specified endpoints according to IACUC protocol monitoring plan including losing > 20% body weight or hind limb paralysis. Mice were also monitored for any GvHD signs including hair loss and cachexia. Mice were injected with PBS, WT IL-2, or the indicated targeted-IL2 fusion molecules at the doses described in the figure legends and/or main text. Peripheral blood was collected via retroorbital bleed and stained for Live/dead (ThermoFisher), anti-mouse CD45, and anti-human CD45, CD3, CD8, CD4, PD-1, LAG3, TIM3, and either anti-FMC63 (Acro Biosystems) to detect the CD19 CAR-T cells or anti-G4S linker antibody to detect the B7H3 CAR-T cells. Excised tumors were digested using the gentleMACS Octo dissociator and stained with the above panel for quantification of T cells. Single-cell RNA-seq Analysis Raw single-cell RNA sequencing (scRNA-seq) data were processed using Cell Ranger (version 7.1.0, 10x Genomics). Transcript reads were aligned to the GRCh38 human reference genome, which was supplemented with the scFv domain sequence of the CAR19 transcript. Filtered gene expression matrices were further analyzed in R (version 4.3.1) using the Seurat package (version 5.1.0). To exclude low-quality cells, cells were filtered out if they met any of the following criteria: fewer than 20 expressed genes, abnormal total UMI counts ( 30,000), or elevated mitochondrial gene content (> 10%). Doublets were identified and removed using DoubletFinder (version 2.0.3). Batch correction and sample integration were performed using Seurat’s SCTransform workflow. Cell types were annotated prior to integration using the Azimuth package (version 0.5.0) with both the built-in peripheral blood mononuclear cells (PBMCs) reference dataset (version 1.0.0) and an external PBMC reference from the Tabula Rasa project, provided by the Chan Zuckerberg Foundation. Following annotation, all scRNA-seq samples were integrated into a single dataset. Dimensionality reduction was performed by calculating principal components (PCs) using the RunPCA function in Seurat. The first 10 PCs were used as input for Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction via the RunUMAP function, enabling visualization and clustering of cells in reduced dimensions. Differential Expression Analysis After integration, the dataset was subset to include only the cells expressing CD3E and scFvCAR19. Differential gene expression between experimental conditions was assessed using the FindMarkers function in Seurat with the Wilcoxon rank-sum test. Only genes expressed in more than 5% of cells in at least one condition were considered for analysis. Generation of EGFRt + TILs Tumor-infiltrating lymphocytes (TILs) are T cells isolated from melanoma tumor fragments obtained from the Tara Miller Melanoma Center at the University of Pennsylvania. Fresh melanoma tumor tissue was physically and enzymatically digested using a solution containing collagenase/hyaluronidase and DNase. The cells were then cultured in a medium containing IL-7 and IL-15. TILs were isolated using a CD45 + selection kit and further analyzed by flow cytometry for the expression of CD3, CD4, and CD8 markers. The isolated TILs were stimulated with anti-CD3/anti-CD28 beads for activation and expansion. On day 2 post-stimulation, the TILs were transduced with an EGFRt lentiviral vector and kept in culture with 50U/ml of rhIL2. On day 14, they were harvested and used fresh for the in vitro organoid experiment. Cytotoxicity Assay for NHP CART cells Effector Cells (CAR20 T cells) or untransduced (UTD) control T cells were thawed and rested 12–24 hours in R10 media (2 x 10 6 cells/mL; RPMI + 10% FBS, 100 U/mL penicillin/streptomycin, 1% GlutaMAX). Within 24 hours effector cells were counted and resuspended to appropriate concentrations to set up co-cultures with target cells at multiple E:T ratios. Target cells were luciferase-expressing Raji target cells maintained in culture, counted, and resuspended to a concentration of 1 x 10 6 cells/mL in R10. We tested several E:T ratios, the highest being 5:1 and the lowest 0.075:1 in for NHP RM#1 CAR20 T cells. One hundred microliters of target cells (1 x 105 cells) were added to all wells containing varying amounts of effector cells in 100 µL to achieve the specified E:T ratios, including a triplicate set of wells with target cells only. Wells were gently mixed and incubated at 37°C for 24 and 48 hours. At both time points, 2 µL of diluted Luciferin (1:10 dilution in PBS) was added to each well to determine luciferase levels. Bioluminescence was assessed on the Synergy H4 plate reader on auto-gain. Non-human primate studies The rhesus macaque (RM) study was reviewed and approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) committee. Animals were housed in state of the art AAALAC International accredited facilities. Animals were monitored twice daily (or more often) and kept in temperature and humidity controlled rooms. NHPs were fed twice daily with Lab diet 5038 (for NHPs) and provided daily fruits and vegetables. RM (n = 1 per group, both males) were dosed intravenously with CAR20-EGFRt T cells (2.5 x 10 6 cells/kg) on day 0. Cells were infused in 20-30mL of sterile PBS. RM#1 was also injected with EGFRt-IL2m on day 0. RM#2 was dosed intravenously with EGFRt-IL2 on day 14. Cells and muteins were dosed over 10–20 minutes and vitals were monitored. Peripheral blood samples were collected and PBMCs isolated by lysing RBCs from peripheral blood samples with ACK lysing buffer. PBMCs were analyzed via flow cytometry. Cell blood counts and serum chemistries were routinely monitored and submitted to the clinical pathology laboratory at Penn Vet. Absolute counts were determined by relating the percentage of each lymphocyte population as determined by flow cytometry to the absolute lymphocyte counts as determined by hematology. Detection of ADAs against CAR-T cells Serum ADAs against CAR-T cells were assessed by flow cytometry. Rhesus CAR-T cells or untransduced controls (7.5x10 4 each) were incubated with diluted serum samples (1:20, 1:100, 1:500) in 96-well plates for 30min at 4°C. Cells were washed, then incubated with a labeled ant-rhesus IgG for detection (20 min, at 4°C). Cells were also stained for viability and cell surface expression of CAR (using labeled panitumumab), CD45, CD3, CD4, and CD8. After a final wash, cells were analyzed on a flow cytometer. Detection of ADAs against EGFRt-IL2 An electrochemiluminescent homogeneous bridging assay on the MSD platform was used to detect EGFRt-IL2 specific ADA response. Briefly, MSD GOLD streptavidin 96-well plates were blocked with 200 µL/well of assay buffer (2% BSA in 1X PBS) for 1 to 3 hours at room temperature with shaking at 700 rpm. Test rhesus monkey serum samples, negative control (NC – drug-naïve pooled sera), and a positive control (PC – 100 ng/mL anti-IL2 specific mAb prepared in drug-naïve pooled sera) were diluted 5-fold in assay buffer. The 5-fold diluted samples were further diluted 4-fold into a master mix solution of 50 µL 0.25 µg/mL of biotinylated EGFRt-IL2, 50 µL 0.5 µg/mL ruthenylated EGFRt-IL2, and 50 µL of assay buffer (or 40 µg/mL unlabeled EGFRt-IL2 to assess assay specificity) for a final minimum required dilution of 1:20, and incubated at room temperature for 1 hour in the dark with shaking at 700 rpm. The MSD plates were washed three times with 300 µL/well of wash buffer (1X TBST). The samples and controls in master mix were transfer 50 µL/well to the blocked streptavidin plate and incubated at room temperature for 1 hour in the dark with shaking at 700 rpm. Subsequently, the plates were washed three times with 300 µL/well of wash buffer, 150 µl MSD GOLD Read Buffer A was added and the electrochemiluminescence signal was then measured on a MESO Quickplex SQ120 (MSD). Serum cytokine detection in NHP Rhesus monkey serum samples were collected at the indicated time points pre-injection and post-injection of EGFRt-IL2. IFNγ, TNFα, GM-CSF, IL-5, IL-6, and MIP1a were concurrently quantified using a U-PLEX 6-assay kit for non-human primates from MSD following the manufacturers protocol. Briefly, plates were labeled with 50 µL of 1x multiplex coating solution and incubated overnight at 4°C with shaking at 650 rpm. The plates were washed three times with 400 µl of manufacturer recommended PBS-T (PBS with 0.05% Tween-20) wash buffer, followed by the addition of 25 µL of assay buffer and either 25 µL of calibrator standards or test serum. Plates were incubated for 1 hour at room temperature with shaking at 700 rpm, then washed three times with 400 µL PBS-T per well. Manufacturer Read Buffer (2x) was added to each plate with 150 µL per well and the plate was analyzed with MSD instrument (Rockville, Maryland) using Methodical Mind software. Statistical analysis and reproducibility All statistical analysis was performed using GraphPad Prism, where comparisons between groups were performed using either unpaired t test or ordinary one-way ANOVA with Turkey’s multiple comparison test. Unless noted otherwise, data are reported as median ± SD. Survival data were analyzed using the log-rank (Mantel-Cox) test. Declarations Data availability All data associated with this study are present in the manuscript. Materials used in this study are available from the corresponding author upon reasonable request. Experimental Schemes Experimental schemes were generated using biorender.com. Competing interests: S.G. has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. S.G. is a scientific co-founder and holds equity in Interius Biotherapeutics and Carisma Therapeutics. S.G. is a scientific advisor to Carisma, Currus, Interius, Kite, NKILT, Mission Bio, and Vor Bio. J.F. holds patents and intellectual property in T-cell-based cancer immunotherapy with royalties, has received funding from Tmunity Therapeutics and Danaher Corporation, consults for Retro Biosciences, and serves on the scientific advisory boards of Cartography Bio, Shennon Biotechnologies Inc., CellFe Biotech, OverT Bio, Inc., and Tceleron Therapeutics, Inc. P.B., C.K., M.S., D.C.P., T.P., S.L., and K.D.M are employees of Asher Biotherapeutics. Y.A.Y. and I.D. are employees of and own stock in Asher Biotherapeutics. Author contributions: S.S., N.D.M, Y.A.Y, I.J. and S.I.G. conceptualized the project, analyzed the data and wrote the paper. S.S., T.H., N.D.M, F.S., O.S., R.D.S., A.B.B. and M.K. carried out the translational experiments. D.J. and J.F. performed antigen quantification on rhesus macaques peripheral blood samples. P.B., C.K. designed and generated the proteins. S.L.C. performed the testing of ADA. S.S., N.D.M., T.H., K.D.M, I.D., and M.S. visualized the data. S.I.G. supervised the project. J.S., H.T., and R.D.S handled the treatment and care of the rhesus macaques in the NHP model. Acknowledgments: We would like to acknowledge the human immunology core, the cell center service core, and the stem cell and xenograft core at the University of Pennsylvania. References Fraietta, J. A. et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 24, 563–571 (2018). Cappell, K. M. et al. Long-Term Follow-Up of Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy. JCO 38, 3805–3815 (2020). Grover, P. et al. Chimeric antigen receptor T-cell therapy in adults with B-cell acute lymphoblastic leukemia. Blood Advances 6, 1608–1618 (2022). Kochenderfer, J. N. et al. Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels. JCO 35, 1803–1813 (2017). Neelapu, S. S. et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med 377, 2531–2544 (2017). Schuster, S. J. et al. Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas. N Engl J Med 377, 2545–2554 (2017). Munshi, N. C. et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med 384, 705–716 (2021). Porter, D. L. et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med 7 , 303ra139 (2015). Mueller, K. T. et al. Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia. Blood 130 , 2317–2325 (2017). Melenhorst, J. J. et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 602 , 503–509 (2022). Tennant, M. D., New, C., Ferreira, L. M. R. & O’Neil, R. T. Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling. Molecular Therapy 31, 2591–2599 (2023). Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells | Journal of Experimental Medicine | Rockefeller University Press. https://rupress.org/jem/article/202/7/907/40182/Removal-of-homeostatic-cytokine-sinks-by. Klebanoff, C. A., Khong, H. T., Antony, P. A., Palmer, D. C. & Restifo, N. P. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol 26 , 111–117 (2005). North, R. J. Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells. J Exp Med 155 , 1063–1074 (1982). Rejeski, K. et al. Immune effector cell-associated hematotoxicity: EHA/EBMT consensus grading and best practice recommendations. Blood 142 , 865–877 (2023). Spolski, R., Li, P. & Leonard, W. J. Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat Rev Immunol 18, 648–659 (2018). Overwijk, W. W., Tagliaferri, M. A. & Zalevsky, J. Engineering IL-2 to Give New Life to T Cell Immunotherapy. Annu. Rev. Med. 72, 281–311 (2021). Ko, B., Takebe, N., Andrews, O., Makena, M. R. & Chen, A. P. Rethinking Oncologic Treatment Strategies with Interleukin-2. Cells 12, 1316 (2023). Raeber, M. E., Sahin, D. & Boyman, O. Interleukin-2–based therapies in cancer. Sci. Transl. Med. 14 , eabo5409 (2022). Briukhovetska, D. et al. Interleukins in cancer: from biology to therapy. Nat Rev Cancer 21 , 481–499 (2021). Krieg, C., Létourneau, S., Pantaleo, G. & Boyman, O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 107 , 11906–11911 (2010). Reinhardt, J. et al. Distinguishing activated T regulatory cell and T conventional cells by single‐cell technologies. Immunology 166, 121–137 (2022). Parisi, G. et al. Persistence of adoptively transferred T cells with a kinetically engineered IL-2 receptor agonist. Nat Commun 11, 660 (2020). Ptacin, J. L. et al. An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism. Nat Commun 12, 4785 (2021). Rouanne, M., Zitvogel, L. & Marabelle, A. Pegylated Engineered IL2 plus Anti-PD-1 Monoclonal Antibody: The Nectar Comes from the Combination. Cancer Discov 10 , 1097–1099 (2020). Levin, A. M. et al. Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533 (2012). Ren, J. et al. Interleukin-2 superkines by computational design. Proc. Natl. Acad. Sci. U.S.A. 119, e2117401119 (2022). Merchant, R. et al. Fine-tuned long-acting interleukin-2 superkine potentiates durable immune responses in mice and non-human primate. J Immunother Cancer 10 , e003155 (2022). Moynihan, K. D. et al. IL2 Targeted to CD8+ T Cells Promotes Robust Effector T-cell Responses and Potent Antitumor Immunity. Cancer Discovery 14 , 1206–1225 (2024). Sun, Z. et al. A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+ T-cell response and effective tumor control. Nat Commun 10, 3874 (2019). Italiano, A. et al. Clinical activity and safety of simlukafusp alfa, an engineered interleukin-2 variant targeted to fibroblast activation protein-α, combined with atezolizumab in patients with recurrent or metastatic cervical cancer. Journal of Clinical Oncology (2021) doi:10.1200/JCO.2021.39.15_suppl.5510. Janku, F. et al. Abstract LB041: THOR-707 (SAR444245), a novel not-alpha IL-2 as monotherapy and in combination with pembrolizumab in advanced/metastatic solid tumors: Interim results from HAMMER, an open-label, multicenter phase 1/2 Study. Cancer Research 81 , LB041 (2021). Boni, V. et al. ARTISTRY-1: Nemvaleukin alfa monotherapy and in combination with pembrolizumab in patients (pts) with advanced solid tumors. Journal of Clinical Oncology (2021) doi:10.1200/JCO.2021.39.15_suppl.2513. Andreata, F. et al. CD8 cis-targeted IL-2 drives potent antiviral activity against hepatitis B virus. Sci Transl Med 16, eadi1572 (2024). Kaptein, P. et al. CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool. Cancer Discov 14, 1226–1251 (2024). Taylor, N. D., Way, J. C., Silver, P. A. & Cironi, P. Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity. Protein Eng Des Sel 23, 251–260 (2010). Abramson, J. S. et al. Lisocabtagene maraleucel as second-line therapy for large B-cell lymphoma: primary analysis of the phase 3 TRANSFORM study. Blood 141, 1675–1684 (2023). Lindner, S. E., Johnson, S. M., Brown, C. E. & Wang, L. D. Chimeric antigen receptor signaling: Functional consequences and design implications. Science Advances 6 , eaaz3223 (2020). Guedan, S., Calderon, H., Posey, A. D. & Maus, M. V. Engineering and Design of Chimeric Antigen Receptors. Molecular Therapy Methods & Clinical Development 12 , 145–156 (2019). Rohrs, J. A., Zheng, D., Graham, N. A., Wang, P. & Finley, S. D. Computational Model of Chimeric Antigen Receptors Explains Site-Specific Phosphorylation Kinetics. Biophysical Journal 115 , 1116–1129 (2018). Fernandes, R. A. et al. Immune receptor inhibition through enforced phosphatase recruitment. Nature 586, 779–784 (2020). Ou, L. et al. Patient-derived melanoma organoid models facilitate the assessment of immunotherapies. EBioMedicine 92 , 104614 (2023). Jafarzadeh, L., Masoumi, E., Fallah-Mehrjardi, K., Mirzaei, H. R. & Hadjati, J. Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward. Front Immunol 11 , 702 (2020). Goff, S. L. et al. Randomized, Prospective Evaluation Comparing Intensity of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating Lymphocytes for Patients With Metastatic Melanoma. J Clin Oncol 34, 2389–2397 (2016). Lotze, M. T., Frana, L. W., Sharrow, S. O., Robb, R. J. & Rosenberg, S. A. In vivo administration of purified human interleukin 2. I. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2. The Journal of Immunology 134, 157–166 (1985). Aspuria, P.-J. et al. An orthogonal IL-2 and IL-2Rβ system drives persistence and activation of CAR T cells and clearance of bulky lymphoma. Sci. Transl. Med. 13, eabg7565 (2021). Mackensen, A. et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat Med 28 , 2124–2132 (2022). Avanzi, M. P. et al. Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System. Cell Rep 23, 2130–2141 (2018). Kalbasi, A. et al. Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature 607, 360–365 (2022). Zhang, Q. et al. A human orthogonal IL-2 and IL-2Rβ system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia. Sci. Transl. Med. 13, eabg6986 (2021). Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat Biotechnol 16, 677–681 (1998). Majzner, R. G. et al. CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clin Cancer Res 25 , 2560–2574 (2019). Additional Declarations Yes there is potential Competing Interest. S.G. has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. S.G. is a scientific co-founder and holds equity in Interius Biotherapeutics and Carisma Therapeutics. S.G. is a scientific advisor to Carisma, Currus, Interius, Kite, NKILT, Mission Bio, and Vor Bio. J.F. holds patents and intellectual property in T-cell-based cancer immunotherapy with royalties, has received funding from Tmunity Therapeutics and Danaher Corporation, consults for Retro Biosciences, and serves on the scientific advisory boards of Cartography Bio, Shennon Biotechnologies Inc., CellFe Biotech, OverT Bio, Inc., and Tceleron Therapeutics, Inc. P.B., C.K., M.S., D.C.P., T.P., S.L., and K.D.M are employees of Asher Biotherapeutics. Y.A.Y. and I.D. are employees of and own stock in Asher Biotherapeutics. Supplementary Files SupplementaryTableS1.docx Supplementary Table S1 SupplementaryTableS2.docx Supplementary Table S2 ExtendedFigure1.ai ExtendedFigure2.ai ExtendedFigure3.ai ExtendedFigure4.ai ExtendedFigure5.ai SupplementaryFig1.ai SupplementaryFig2.ai SupplementaryFig3.ai SupplementaryFig4.ai SupplementaryFig5.ai SupplementaryFig6.ai SupplementaryFig7.ai SupplementaryFig8.ai Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5962170","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":415996856,"identity":"6541b7a1-e485-4d48-ad92-05200ffafee3","order_by":0,"name":"Saar Gill","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-7946-7778","institution":"University of Pennsylvania","correspondingAuthor":true,"prefix":"","firstName":"Saar","middleName":"","lastName":"Gill","suffix":""},{"id":415996857,"identity":"ae484f02-4673-4c58-bf89-abc36b785bb7","order_by":1,"name":"Sara Sleiman","email":"","orcid":"https://orcid.org/0009-0006-1922-4663","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Sleiman","suffix":""},{"id":415996858,"identity":"79f47c5f-49b1-433c-b2b8-042f8e8d6647","order_by":2,"name":"Nathan Mathewson","email":"","orcid":"","institution":"Asher Biotherapeutics","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Mathewson","suffix":""},{"id":415996859,"identity":"5683068c-e13a-458c-9f33-8c4c699d689a","order_by":3,"name":"Tyler Hansen","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Tyler","middleName":"","lastName":"Hansen","suffix":""},{"id":415996860,"identity":"e84487c4-3ec6-41d3-851a-0c0e3d245f93","order_by":4,"name":"Paul Bessette","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Bessette","suffix":""},{"id":415996861,"identity":"d177d42b-d0ff-4d63-acf6-9a0e48d5b5e5","order_by":5,"name":"Chris Kimberlin","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Chris","middleName":"","lastName":"Kimberlin","suffix":""},{"id":415996862,"identity":"e07536aa-bc86-4491-b4e1-02bc5f54fc49","order_by":6,"name":"Feng Shen","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Shen","suffix":""},{"id":415996863,"identity":"6bde34da-7abf-4d23-b508-54a162c4bc35","order_by":7,"name":"Olga Shestova","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"","lastName":"Shestova","suffix":""},{"id":415996864,"identity":"50049958-adde-4884-8097-fef7d45f00a8","order_by":8,"name":"Maksim Shestov","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Maksim","middleName":"","lastName":"Shestov","suffix":""},{"id":415996865,"identity":"242e6516-7b12-494c-b3c8-e3f675f8ec1c","order_by":9,"name":"Miroslaw Kozlowski","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Miroslaw","middleName":"","lastName":"Kozlowski","suffix":""},{"id":415996866,"identity":"34bd131c-a199-4c99-8574-8ccfdec0ec8f","order_by":10,"name":"Danuta Jarocha","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Danuta","middleName":"","lastName":"Jarocha","suffix":""},{"id":415996867,"identity":"93e50249-b1ad-4282-a9d8-9337f5c41724","order_by":11,"name":"Joseph Fraietta","email":"","orcid":"https://orcid.org/0000-0001-7900-8993","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Fraietta","suffix":""},{"id":415996868,"identity":"c689f87f-7507-482e-ba29-5b84df52be05","order_by":12,"name":"Marco Ruella","email":"","orcid":"https://orcid.org/0000-0003-4301-5811","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Ruella","suffix":""},{"id":415996869,"identity":"d3c5375d-b020-429e-9eef-af388e8a4d46","order_by":13,"name":"Hannah Thomas","email":"","orcid":"","institution":"Department of Pathobiology. University of Pennsylvania School of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Thomas","suffix":""},{"id":415996870,"identity":"9aa3b29a-29c1-4637-a825-89c59cc172ac","order_by":14,"name":"John Swain","email":"","orcid":"","institution":"Department of Pathobiology. University of Pennsylvania School of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Swain","suffix":""},{"id":415996871,"identity":"76fd7ee5-3ea3-44b6-bed3-5e7d92c024b8","order_by":15,"name":"Raimon Duran-Struuck","email":"","orcid":"","institution":"Department of Comparative Medicine, University of Washington School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Raimon","middleName":"","lastName":"Duran-Struuck","suffix":""},{"id":415996872,"identity":"030fb191-771c-4096-aa3b-6af9b7d5cd1a","order_by":16,"name":"Asunción Borrero Borrego","email":"","orcid":"https://orcid.org/0000-0002-0293-5614","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Asunción","middleName":"Borrero","lastName":"Borrego","suffix":""},{"id":415996873,"identity":"23100a4b-ae95-4fb2-92cc-1846fb06856f","order_by":17,"name":"Shujing Liu","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Shujing","middleName":"","lastName":"Liu","suffix":""},{"id":415996874,"identity":"b47a5da2-b648-43dd-adfe-57132b652468","order_by":18,"name":"Xiaowei Xu","email":"","orcid":"https://orcid.org/0000-0003-4098-7690","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Xu","suffix":""},{"id":415996875,"identity":"17e5208a-8fe3-475b-8c9c-fe6945dfd571","order_by":19,"name":"Meghana Sukthankar","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Meghana","middleName":"","lastName":"Sukthankar","suffix":""},{"id":415996876,"identity":"b2d38c78-b3a0-48a3-a9e2-f81ee567ab1c","order_by":20,"name":"Danielle Pappas","email":"","orcid":"https://orcid.org/0000-0003-0412-3588","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Danielle","middleName":"","lastName":"Pappas","suffix":""},{"id":415996877,"identity":"fa9cbac2-7762-4de1-8de8-a760d646d268","order_by":21,"name":"Terrence Park","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Terrence","middleName":"","lastName":"Park","suffix":""},{"id":415996878,"identity":"26a57b64-b901-425b-8fd5-94dd6517a329","order_by":22,"name":"Sindy Liao-Chan","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Sindy","middleName":"","lastName":"Liao-Chan","suffix":""},{"id":415996879,"identity":"69ae04a1-6808-4e6a-8a3d-c0fea3c0f566","order_by":23,"name":"Kelly Moynihan","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Moynihan","suffix":""},{"id":415996880,"identity":"69171247-c30c-4748-8cea-bcba4d878482","order_by":24,"name":"Yik Yeung","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Yik","middleName":"","lastName":"Yeung","suffix":""},{"id":415996881,"identity":"4580ee2b-700c-47d1-8592-35bd8418c697","order_by":25,"name":"Ivana Djuretic","email":"","orcid":"","institution":"Asher Biotherapeutics Inc.","correspondingAuthor":false,"prefix":"","firstName":"Ivana","middleName":"","lastName":"Djuretic","suffix":""}],"badges":[],"createdAt":"2025-02-05 04:25:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5962170/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5962170/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81965130,"identity":"df41b22c-e17a-4815-8513-fc6431cd514f","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2838572,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/bf8a93a2e79cfd201eb09c53.png"},{"id":81966396,"identity":"09892973-90b1-4450-92dd-72b5c8a90c7a","added_by":"auto","created_at":"2025-05-05 11:36:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3565403,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/4749aa2dd7ac36f119d20228.png"},{"id":81965141,"identity":"3710cc52-f79f-4150-9b20-5bc69f1b15fa","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3567929,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/363f1ad41fc6123defeec9d3.png"},{"id":81967838,"identity":"e2c4533e-86c3-4181-8322-4ed834c4d73e","added_by":"auto","created_at":"2025-05-05 11:44:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2559683,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/69710d0eb11a8ef2eb26c14b.png"},{"id":81965145,"identity":"83148ff0-34f0-4dc6-b4a7-19ab15dcc073","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2805155,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/be9e619896d5fefd4d2addbc.png"},{"id":81968959,"identity":"5e4e98f7-9adf-43c1-9e1c-daa21b561d09","added_by":"auto","created_at":"2025-05-05 12:00:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16522062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/4aec8096-4c91-4aa7-850d-60fbc407b7f6.pdf"},{"id":81965126,"identity":"7c2e6d12-b7ef-48f2-964d-c7c7edfdd5bb","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13595,"visible":true,"origin":"","legend":"Supplementary Table S1","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/274b6165ce218caaa94c48e2.docx"},{"id":81965127,"identity":"f996be6b-0331-4a1b-8d07-2e087803807a","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17480,"visible":true,"origin":"","legend":"Supplementary Table S2","description":"","filename":"SupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/1d93d6a717c14a9b9f2dfd2f.docx"},{"id":81966399,"identity":"83d51241-824f-4f27-b56e-93cdc4e47bda","added_by":"auto","created_at":"2025-05-05 11:36:10","extension":"ai","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1022009,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigure1.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/4969c82df23037fafcf58f8c.ai"},{"id":81968177,"identity":"3087bca2-a03d-4c1e-9ef9-e7ecdab97714","added_by":"auto","created_at":"2025-05-05 11:52:10","extension":"ai","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":729208,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigure2.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/8077e652bab985670b7f0e3f.ai"},{"id":81967836,"identity":"46d42350-0405-48db-bbfc-a147a27677a2","added_by":"auto","created_at":"2025-05-05 11:44:10","extension":"ai","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":742809,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigure3.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/c031a777adeecff39790e3ca.ai"},{"id":81965131,"identity":"95ed81c4-872f-4984-a87d-3e742673f19e","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"ai","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1211170,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigure4.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/010524a0144d7200fcdad66c.ai"},{"id":81966394,"identity":"0e30a577-8525-4d6c-bd4e-24fdead966b4","added_by":"auto","created_at":"2025-05-05 11:36:10","extension":"ai","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":879645,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigure5.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/75eacfb6a8fd290720df2243.ai"},{"id":81966402,"identity":"4a990476-8a3e-400f-b2e4-aaed9c2293b5","added_by":"auto","created_at":"2025-05-05 11:36:10","extension":"ai","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":334299,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig1.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/76408e0340c7c2f77b923c08.ai"},{"id":81967832,"identity":"df981a38-1665-4762-a71a-5fd17e9a9284","added_by":"auto","created_at":"2025-05-05 11:44:10","extension":"ai","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":936799,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig2.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/67349de24fdaa4c84422b2ac.ai"},{"id":81966404,"identity":"5164ef8c-2637-46d7-adb2-8631f0885ff2","added_by":"auto","created_at":"2025-05-05 11:36:10","extension":"ai","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1036824,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig3.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/fa22cd9711516cc65d116792.ai"},{"id":81965138,"identity":"6678c0c0-3bb9-4d6b-9149-f1e88576bcf3","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"ai","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":800005,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig4.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/c0c0181cbea14ae35c2c35b4.ai"},{"id":81967833,"identity":"4b35421f-e123-4501-9963-6a1e129e6a22","added_by":"auto","created_at":"2025-05-05 11:44:10","extension":"ai","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":807361,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig5.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/4af9aaab618a8f41f2ba7bd8.ai"},{"id":81965150,"identity":"c3bf1789-6bc0-4f16-b24c-1d29a32f39cd","added_by":"auto","created_at":"2025-05-05 11:28:11","extension":"ai","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":609504,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig6.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/16e280773ae99c129cb7f70f.ai"},{"id":81965134,"identity":"fd1e329a-28eb-4f9e-a4f4-880e25767e8d","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"ai","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":782525,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig7.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/972cb6acd75a7aee4a459ebe.ai"},{"id":81965148,"identity":"f5eb9399-92a9-4d00-b28b-0327942f8648","added_by":"auto","created_at":"2025-05-05 11:28:10","extension":"ai","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":594926,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig8.ai","url":"https://assets-eu.researchsquare.com/files/rs-5962170/v1/4e893676bfbab8b141bbdcb3.ai"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nS.G. has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. S.G. is a scientific co-founder and holds equity in Interius Biotherapeutics and Carisma Therapeutics. S.G. is a scientific advisor to Carisma, Currus, Interius, Kite, NKILT, Mission Bio, and Vor Bio. J.F. holds patents and intellectual property in T-cell-based cancer immunotherapy with royalties, has received funding from Tmunity Therapeutics and Danaher Corporation, consults for Retro Biosciences, and serves on the scientific advisory boards of Cartography Bio, Shennon Biotechnologies Inc., CellFe Biotech, OverT Bio, Inc., and Tceleron Therapeutics, Inc. P.B., C.K., M.S., D.C.P., T.P., S.L., and K.D.M are employees of Asher Biotherapeutics. Y.A.Y. and I.D. are employees of and own stock in Asher Biotherapeutics.","formattedTitle":"Selective support of engineered T cells using a cis-targeted interleukin-2 enhances anti-tumor activity and obviates the need for lymphodepletion","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficacy in the treatment of B cell and plasma cell malignancies. Complete remission (CR) rates for B-ALL receiving CD19-targeted CAR T cell therapy exceed 80%, while NHL and MM patients achieve CR rates between 30% and 60% (\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The response rate to CAR-T therapy is highly correlated with the in vivo expansion and persistence of CAR-T cells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, strategies that can improve the durability and function of CAR-T cells have the potential to improve patient outcomes.\u003c/p\u003e \u003cp\u003eOne strategy for enhancing persistence involves providing cytokine support to the CAR-T cells. Currently, this is accomplished through lymphodepletion, which stimulates the production of homeostatic cytokines and transiently removes cellular \u0026ldquo;sinks\u0026rdquo; that compete with the adoptively transferred cells (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). However, lymphodepletion is associated with a significant incidence and severity of cytopenias and their attendant complications, such as susceptibility to infection (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Therefore, an approach that could enhance CAR-T cell activity while minimizing the need for lymphodepleting chemotherapy would likely be of general interest and appeal.\u003c/p\u003e \u003cp\u003eThe cytokine interleukin (IL)-2 is crucial for the activation and expansion of CD4 and CD8 T cells. However, a demonstrable pharmacologic effect requires the administration of high doses of IL-2, as given in tumor-infiltrating lymphocyte (TIL) therapy, where it is accompanied by dose-limiting cardiovascular and respiratory toxicities. In addition, IL-2 promotes the proliferation of regulatory T (Treg) cells, which may restrain its anti-tumor effect (\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Thus, rhIL-2 therapy for cancer is limited by toxicity and by stimulation of undesirable immunosuppressive cell populations, which has limited its broader utility in combination with T-cell therapies.\u003c/p\u003e \u003cp\u003eThe intermediate-affinity IL-2 receptor complex is a heterodimer of the β chain (CD122) and the common γ chain (CD132), and is expressed broadly, including on resting effector T cells and natural killer cells. The high-affinity IL-2 receptor complex is a heterotrimer of the α chain (CD25) along with CD122 and CD132 and is expressed constitutively on Treg cells and innate lymphoid cells, and transiently on activated conventional T cells (Tconv) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Previous efforts to selectively activate effector T cells without stimulating Tregs led to the development of engineered IL-2 variants with attenuated affinity for IL-2Rα (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) (\u0026ldquo;not-α\u0026rdquo; IL-2 variants) or with enhanced affinity for IL2Rβ/γ (IL-2 \u0026lsquo;superkines\u0026rsquo;) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, these variants are not completely selective for effector and memory T cells, since natural killer (NK) cells, innate lymphoid cells and endothelial cells also express IL-2 receptors and may contribute to toxicities of these variants in patients. Indeed, NK cells were found to mediate toxicities of the not-α IL-2 variants in mice (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and clinical data with engineered IL-2 variants suggest that substantial improvements in IL-2\u0026rsquo;s therapeutic index could not be achieved in patients when signaling is delivered broadly to IL2Rβ/γ\u0026thinsp;+\u0026thinsp;cells (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWe set out to overcome the limitations of wild-type (WT) IL-2 and previously engineered IL-2 molecules in enhancing the potential of cell therapies by developing an approach that selectively delivers IL-2 stimulation to engineered T cells, without activating endogenous cells. We recently showed that a CD8\u0026thinsp;+\u0026thinsp;T cell-selective IL-2 molecule, consisting of an attenuated IL-2 mutein linked to an antibody targeting the CD8b molecule, can enhance anti-tumor and anti-viral immunity while improving tolerability compared to \u0026ldquo;not-α\u0026rdquo; IL-2 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Building on this concept of cell type-selective targeting (cis-targeting) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), here we engineered a cis-targeted IL-2 that is comprised of an IL-2 variant that does not bind to IL2Rα (CD25) and has reduced affinity for IL2Rβ (CD122), linked to a targeting antibody against a surface molecule specifically expressed on engineered cells. We show that targeting IL-2 to a truncated, non-signaling EGFR tag (EGFRt) expressed in CAR T cells can selectively and safely stimulate engineered human and non-human primate CAR-T cells resulting in substantially improved anti-tumor efficacy in mouse models, and prolonged B cell aplasia in non-human primates in the absence of lymphodepleting chemotherapy. Collectively, these data provide proof-of-concept enabling IL-2\u0026rsquo;s utility in combination with cell therapy and promise to enhance the efficacy of T cell therapies while avoiding the life-threating toxicities associated with wild-type IL-2 and lymphodepletion.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCis-targeted IL-2 fusion molecules deliver selective IL-2 stimulation to CAR-T cells in vitro\u003c/h2\u003e \u003cp\u003eTo develop an IL-2 molecule that can signal selectively on CD19 CAR-T cells, we fused a CAR-T cell-targeting antibody to an attenuated IL-2 mutein with no IL-2R⍺ binding and reduced IL-2Rβ binding (Fig.\u0026nbsp;1a). Attenuation of IL-2 affinity to both IL-2R⍺ and IL-2Rβ was required to avoid activation of endogenous Tregs and NK cells that express high levels of IL-2R⍺ and IL-2Rβ, respectively (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Indeed, IL-2 mutein (IL2m1) fused to a control antibody did not activate conventional T cells in human PBMCs at concentrations below 100nM and regulatory T cells and NK cells below 10nM (Extended Data Fig.\u0026nbsp;1a, Supplementary Table\u0026nbsp;1). IL-2 muteins were targeted to CAR-T cells either in an idiotype-specific manner, using an antibody against the FMC63 clone of the CAR constructs employed in commercial CART19 products; or in a reporter-specific manner, using an antibody based on panitumumab directed against a non-signaling truncated EGFR (EGFRt) tag that is co-expressed with the CAR as used in lisocabtagene ciloleucel (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Whereas WT IL-2 stimulated all cells expressing its receptor, cis-targeted IL-2 fusion molecules targeting the CAR (CAR-IL2m1) or EGFRt (panit-IL2m1) mediated selective IL-2 signaling due to their preferential stimulation of cells that express both the IL-2R and the targeting antigen (CAR or EGFRt) (Fig.\u0026nbsp;1b-d). Using STAT5 phosphorylation as a readout, stimulation of CAR-T cells with the cis-targeted IL-2 muteins led to \u0026gt;\u0026thinsp;100-fold increase in selectivity compared with WT IL-2, suggesting that IL-2 fusion binding to a cell surface antigen in cis compensated for its low affinity binding to IL-2R (Fig.\u0026nbsp;1d; Extended Data Fig.\u0026nbsp;1b). Consistent with selective IL-2 signaling in vitro, CAR-IL2m1 and panit-IL2m1 induced robust and selective in vivo expansion of CAR-T cells in immunodeficient NOD-SCIDγc-/- (NSG) mice (Fig.\u0026nbsp;1e). To demonstrate the potential of CAR-T cells stimulated with IL-2 mutein to mediate an anti-tumor effect in vivo, we engrafted NSG mice with the B-ALL cell line NALM6 and down-titrated the CART19 dose. We found that the addition of CAR-IL2m1 could rescue sub-therapeutic doses of CART-19 (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnti-idiotype CAR cis-targeting induces antigen-independent cytokine release and toxicity\u003c/h3\u003e\n\u003cp\u003eChimeric antigen receptors contain intracellular CD3z and costimulatory signaling domains that become activated upon binding to tumor antigen (\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Given that anti-idiotype CAR targeting of IL-2 brings in proximity two signaling receptors (CAR and IL-2R) which could induce their potential cross talk (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), we were concerned that CAR-IL2m1 could activate CAR-T cells in the absence of CD19 antigen. Indeed, CAR-IL2m1 stimulation of human CART19 cells induced the production of interferon-γ (IFN-γ), tumor necrosis factor (TNF) and IL-5 in an antigen-independent manner at levels comparable to that induced by anti-CD3 and anti-CD28 stimulation (Extended Data Fig.\u0026nbsp;1c). In contrast, Panit-IL2m1 targeting the non-signaling truncated EGFR tag induced significantly lower cytokine secretion and this was further attenuated when the Panit-targeting antibody was fused with IL2m2, an IL-2 mutein with 5-10-fold lower affinity than IL2m1 (Extended Data Fig.\u0026nbsp;1a; Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eWe administered a single dose of CAR-IL2m1 or panit-IL2m2 one day after CART-19 to compare their ability to safely enhance CAR-T cell function in vivo (Fig.\u0026nbsp;1f). Although both molecules prolonged the survival of tumor-bearing mice compared to mice that received control CAR-T cells, Panit-IL2m2 showed better tumor control than CAR-IL2m1 and induced median survival of 78 days vs 57 days, respectively (p\u0026thinsp;=\u0026thinsp;0.028) (Fig.\u0026nbsp;1g-h). In addition, transient body weight loss indicative of toxicity was observed in mice receiving CAR-IL2m1, suggesting that targeting of IL-2 to CAR has a narrower therapeutic window than targeting to a non-signaling tag such as EGFRt (Fig.\u0026nbsp;1i). Thus, by selecting the optimal combination of targeting antigen (EGFRt instead of CAR) and of IL-2 mutein affinity (IL2m2 instead of IL2m1), we developed a cis-targeted IL-2 that can substantially and safely enhance the anti-tumor activity of CAR-T cells.\u003c/p\u003e\n\u003ch3\u003eDevelopment of IL-2 fusion molecule that is selective for truncated EGFR over EGFR\u003c/h3\u003e\n\u003cp\u003eThe targeting antibody in panit-IL2m2 is based on panitumumab and binds the full-length EGFR as well as the truncated EGFR that is used as an extracellular tag in CAR-T cells. To prevent the binding of cis-targeted IL-2 to cells that express full-length EGFR (such as epithelial cells), which could have unintended off-target toxicity, we developed an antibody that is selective for EGFRt over EGFR. The EGFRt-selective antibody Ab43 recognizes only the truncated EGFR expressed in CAR-T cells and does not bind to HE293 cells that express full length EGFR (Fig.\u0026nbsp;2a-b). When fused to IL2m2, this EGFRt-selective molecule, referred to as EGFRt-IL2, stimulated CAR-T cells with equivalent potency to panit-IL2m2 in vitro (Fig.\u0026nbsp;2c) and in vivo (Fig.\u0026nbsp;2d). Furthermore, CAR-T cells stimulated with the EGFRt-IL2 one day after infusion were able to selectively re-expand following a prolonged rest of 2 months in NSG mice when stimulated with a second dose of EGFRt-IL2 (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eTo confirm that EGFRt-IL2 can bind and stimulate patient-derived CAR-T cells, we obtained residual cells saved from a lisocabtagene ciloleucel product, a commercial CART19 co-expressing EGFRt (Supplementary Fig.\u0026nbsp;3a). While exposure to WT IL-2 used at a high concentration of 10nM mediated superior overall T cell expansion (likely due to the fact that WT IL-2 induces IL2Rα and becomes more potent by binding to the trimeric high affinity receptor), only stimulation with EGFRt-IL2 led to a selective expansion of CAR\u0026thinsp;+\u0026thinsp;T cells in the CART19 product (Supplementary Fig.\u0026nbsp;3b-d).\u003c/p\u003e\n\u003ch3\u003eEGFRt-IL2 mediates strong CD19 CART expansion and anti-leukemia activity without toxicity\u003c/h3\u003e\n\u003cp\u003eWe next sought to define the in vivo anti-tumor activity of EGFRt-IL2 when administered early (day 1) or delayed (day 7) after CD19 CAR-T cells. NSG mice bearing NALM6 leukemia cells were treated with a \u0026ldquo;stress dose\u0026rdquo; of CART19, followed 1 or 7 days later by a single dose of EGFRt-IL2 (Fig.\u0026nbsp;2e). EGFRt-IL2 treatment on day 1 induced strong anti-tumor activity with 5/5 complete tumor regressions and no toxicity as measured by weight loss (Fig.\u0026nbsp;2f; Extended Data Fig.\u0026nbsp;2a,b). EGFRt-IL2 strongly and preferentially expanded CAR-T cells reaching a peak of over 2,400-fold expansion by day 13 in mice that received both CAR-T cells and EGFRt-IL2 compared to mice that received only CAR-T cells (Fig.\u0026nbsp;2g-i). Early EGFRt-IL2 treatment resulted in improved survival exceeding 100 days (Fig.\u0026nbsp;2j). While delayed treatment with EGFRt-IL2 did potentiate CART19 anti-leukemia activity, the magnitude of this effect was less than with treatment on day 1 (Fig.\u0026nbsp;2f, j). Delayed administration of the cytokine was associated with lower peak expansion of CAR-T cells (Fig.\u0026nbsp;2g-i). EGFRt-IL2-treated mice that had cleared tumors were re-challenged with NALM6 140 days post CAR-T infusion and showed moderate tumor control compared to na\u0026iuml;ve mice suggesting remaining CAR-T activity even 140 days after initial infusion (Extended Data Fig.\u0026nbsp;2c,d). These results suggest that early (pre-emptive) administration of EGFRt-IL2 may be preferable to delayed administration, at least in the setting of a fast-growing leukemia xenograft.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEGFRt-IL2 enhanced CAR-T cell expansion and survival of mice using a sub-optimal dose of CAR19 T cells manufactured from a lymphoma patient PBMCs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe above experiments were largely performed using CART19 made from healthy donor T cells. To assess whether EGFRt-IL2 could potentiate the activity of CAR-T cells manufactured from a cancer patient, CART19 cells were prepared from cryopreserved PBMCs that had been collected from a patient with B cell lymphoma (Supplementary Fig.\u0026nbsp;4a-b). NALM6-bearing NSG mice were treated with 0.25x10\u003csup\u003e6\u003c/sup\u003e CAR-T cells followed by EGFRt-IL2 1 day later (Fig.\u0026nbsp;3a). Mice treated with EGFRt-IL2 experienced improved tumor control, attributed to marked expansion of patient-derived CAR-T cells (Fig.\u0026nbsp;3b-d). To test whether repeated administration of EGFRt-IL2 could provide ongoing CAR-T cell expansion and further enhance their anti-tumor activity, we treated one group with 3 doses of EGFRt-IL2, on day 1, day 21 and day 49 post-CAR-T cell infusion (every 3 to 4 weeks) and found EGFRt-IL2 dependent re-expansion in majority of these mice (Fig.\u0026nbsp;3d), with a trend toward delayed tumor growth and prolonged survival. (Fig.\u0026nbsp;3b, c).\u003c/p\u003e \u003cp\u003eAmong the mice treated with EGFRt-IL2, a pre-defined group was sacrificed on day 14 to assess CAR-T cell engraftment and tumor burden in the spleen. We found a significantly higher number of CAR-T cells in mice treated with EGFRt-IL2 compared to the CART-only group (Fig.\u0026nbsp;3e). In addition, CAR-T cells from mice treated with EGFRt-IL2 displayed lower levels of PD1 and LAG3 exhaustion markers as compared to CAR-T cells that received PBS alone (Fig.\u0026nbsp;3f). To gain further insight regarding the impact of EGFRt-IL2 treatment in vivo, CD45\u0026thinsp;+\u0026thinsp;cells from CART19\u0026thinsp;+\u0026thinsp;PBS and CART19\u0026thinsp;+\u0026thinsp;EGFRt-IL2 treatment groups were harvested from the bone marrows on day 14 for single cell transcriptomics. Clustering of all CAR-T cells identified 2 different populations for mice treated with CART19 only vs CAR19 with the EGFRt-IL2 (Fig.\u0026nbsp;3g). Compared with CART19 alone, CART19 exposed to EGFRt-IL2 had higher expression of some genes associated with activation (ZNF683, CD40LG, CD52, TIMP1, ANXA1), adhesion (ITGA1, ITGB1), the immune synapse (TAGLN2), and metabolism (ALOX5AP); and lower expression of transcription factors associated with exhaustion (ID3, TOX2, IKZF3), inhibitory molecules (CRTAM, CD38, CD200, TNIP3, LAG3, IL10) (Fig.\u0026nbsp;3h). T cell subset analysis showed more proliferating CD8\u0026thinsp;+\u0026thinsp;T cells in the control group (Fig.\u0026nbsp;3i) which may be related to a higher tumor burden than in EGFRt-IL2 group at the time of analysis (47% vs 13% respectively). In addition, slightly higher effector memory CD8\u0026thinsp;+\u0026thinsp;T cells were observed in EGFRt-IL2 group compared to PBS group (35% vs 25% respectively). These findings show that EGFRt-IL2 enhances the anti-tumor activity of patient-derived CAR-T cells in a xenograft model by promoting expansion, reducing exhaustion, and improving tumor control. Transcriptomic and pathway analyses revealed increased activation and reduced exhaustion markers in CAR-T cells treated with EGFRt-IL2. These results suggest that EGFRt-IL2 can sustain CAR-T cell function and improve therapeutic outcomes.\u003c/p\u003e\n\u003ch3\u003eEGFRt-IL2 converts B7H3 CART cells into a curative therapy in a solid tumor model\u003c/h3\u003e\n\u003cp\u003eTo evaluate the potential of EGFRt-IL2 in the context of CAR-T cell therapy for solid tumors, we utilized a human A375 melanoma NSG xenograft model that expresses the B7H3 tumor antigen. A375 melanoma-bearing mice were treated with 4x10\u003csup\u003e6\u003c/sup\u003e CAR-T cells co-expressing an anti-B7H3 CAR along with the EGFRt tag with or without EGFRt-IL2 given either weekly or biweekly (Fig.\u0026nbsp;4a). WT IL-2 in combination with anti-B7H3 CAR-T cells was also tested. In this model, 4x10\u003csup\u003e6\u003c/sup\u003e CAR-T cells alone resulted in only a minor tumor growth delay, whereas complete tumor rejection occurred in all mice treated with either weekly or biweekly administration of EGFRt-IL2 (Fig.\u0026nbsp;4b-c, Extended Data Fig.\u0026nbsp;3a), with no apparent toxicity (Fig.\u0026nbsp;4d). In contrast, although the WT IL-2 combination induced a similar level of tumor regression as EGFRt-IL2 initially (Fig.\u0026nbsp;4b), significant toxicity developed in mice treated with WT IL-2 within 20 days, resulting in greater than 20% weight loss in all mice and treatment-related mortality (Fig.\u0026nbsp;4c-d).\u003c/p\u003e \u003cp\u003eWe further characterized the effects of EGFRt-IL2 on anti-B7H3 CAR-T cells by isolating peripheral blood and tumors from some mice that received PBS or weekly doses of EGFRt-IL2 at day 18 post initial CAR-T dose (Fig.\u0026nbsp;4a). In the blood, EGFRt-IL2 treatment selectively expanded CAR\u0026thinsp;+\u0026thinsp;T cells by approximately 80-fold over PBS control, resulting in the enrichment of CAR\u0026thinsp;+\u0026thinsp;T cells to over 90% compared to 20% in the inbound CAR-T product (Fig.\u0026nbsp;4e). Within the tumor, CAR\u0026thinsp;+\u0026thinsp;T cells from both PBS and EGFRt-IL2 groups expanded compared to CAR- T cells and were enriched to over 90% in both groups. However, EGFRt-IL2 treatment resulted in over 86-fold more CAR\u0026thinsp;+\u0026thinsp;T cells in the tumor compared to PBS controls (Fig.\u0026nbsp;4f). Furthermore, close to 60% of CD8\u0026thinsp;+\u0026thinsp;CAR\u0026thinsp;+\u0026thinsp;T cells in the tumor from PBS-treated mice were of the exhausted phenotype, as defined by co-expression of PD1, LAG3, and TIM3, compared to only 25% in EGFRt-IL2-treated mice (Fig.\u0026nbsp;4g, Extended Data Fig.\u0026nbsp;3b-c)). Similar pattern was observed with CD4\u0026thinsp;+\u0026thinsp;CAR\u0026thinsp;+\u0026thinsp;T cells of which 30% co-expressed the 3 inhibitory markers in EGFRt-treated mice compared to 45% in PBS-treated mice (Fig.\u0026nbsp;4g). This likely explains the lack of meaningful anti-tumor activity observed in the CAR-T alone treatment group. Overall, the results presented here suggest that EGFRt-IL2 selectively and strongly expanded CAR\u0026thinsp;+\u0026thinsp;T cells and reduced the expression of inhibitory/exhaustion markers, dramatically enhancing CAR-T cells' anti-tumor activity in a solid tumor setting.\u003c/p\u003e \u003cp\u003eNext, we sought to selectively stimulate tumor-infiltrating lymphocytes (TILs) expressing EGFRt using our EGFRt-IL2 molecule. To this end, we generated EGFRt\u0026thinsp;+\u0026thinsp;TILs from an excisional biopsy of a patient with metastatic melanoma (Extended Data Fig.\u0026nbsp;4a). These cells were added to an autologous organoid model (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) derived from the same patient\u0026rsquo;s melanoma tissue. Flow cytometry analysis revealed a pronounced selective expansion of EGFRt\u0026thinsp;+\u0026thinsp;TILs by day 7 post-stimulation with EGFRt-IL2, compared to WT-IL2 (Extended Data Fig.\u0026nbsp;4b,d), while the anti-tumor effect in this short-term assay was similar between TILs stimulated with WT-IL2 and EGFRt-IL2 (Extended Data Fig.\u0026nbsp;4c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEGFRt-IL2 allows autologous anti-CD20 CAR to mediate B cell aplasia without lymphodepleting chemotherapy in non-human primates.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCART19 do not cross-react with non-human primate (NHP) B cells. We therefore generated autologous anti-CD20 CAR-T cells from 2 adult rhesus macaques (RM) that recognize rhesus CD20 and co-express EGFRt (Supplementary Fig.\u0026nbsp;5a-c). We also selected a new EGFRt-IL2 molecule for rhesus macaque CAR-T cell experiments (Ab43-IL2m3) since Ab43-IL2m2 bound and stimulated rhesus CART cells poorly due to the binding specificity difference of the IL-2 mutein toward rhesus and human IL-2 receptors (Supplementary Fig.\u0026nbsp;6). On day 0, both animals were injected with 2.5 x 10\u003csup\u003e6\u003c/sup\u003e CAR-T cells/kg. RM#1 received 0.3 mg/kg of EGFRt-IL2 IV 30 minutes post CAR-T cell infusion while RM#2 received CAR-T cells alone (Fig.\u0026nbsp;5a, b). B cell counts began to fall on day 3 in RM#1 and remained undetectable in the blood until day 35 post infusion (Fig.\u0026nbsp;5c, Supplementary Fig.\u0026nbsp;7). In addition, bone marrow flow cytometry showed B cell aplasia in RM#1 through day 35. (Fig.\u0026nbsp;5d). This was accompanied by a transient increase in CD3\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;5e) of which approximately 14% expressed the CAR on day 8 (Extended Data Fig.\u0026nbsp;5a). Notably, on day 7 RM#1 developed hyperthermia, cutaneous erythema of face and neck, and was found to have biochemical features (marked renal azotemia and elevations of transaminases) consistent with dehydration induced by cytokine release syndrome (CRS) (Fig.\u0026nbsp;5f), while hematologic parameters were relatively stable (Fig.\u0026nbsp;5g). The animal was therefore treated on day 7 with intravenous fluids and dexamethasone (3mg/kg), and on day 8 with the IL-6 receptor antagonist tocilizumab (8mg/kg) and a second dose of dexamethasone (2mg/kg). These interventions resulted in prompt clinical and laboratory improvement (Fig.\u0026nbsp;5f,g). These clinical findings were associated with marked increase in MIP1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e and modest elevation in IFN-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\gamma\\:\\)\u003c/span\u003e\u003c/span\u003e and IL-6, which could correspond to CRS (Extended Data Fig.\u0026nbsp;5b). At the first indication of B cell recovery, RM#1 was re-dosed with EGFRt-IL2 on day 48 without additional CART cells. This time there were no clinical or lab abnormalities, no reduction in B cells and no increases in T cells (Fig.\u0026nbsp;5c, e-g). Retrospective analysis of animal serum showed the development of anti-CAR antibodies starting from day 28, coinciding with B cell recovery (Fig.\u0026nbsp;5h). Notably, antibodies directed against the EGFRt-IL2 molecule did not develop until 62 (14 days post re-dosing) (Supplementary Fig.\u0026nbsp;8a).\u003c/p\u003e \u003cp\u003eIn contrast to RM#1, RM#2 received autologous CART-20 without EGFRt-IL2. This animal did not show any evidence of B cell reduction, and no clinical or laboratory changes up to 14 days post CAR-T infusion (Fig.\u0026nbsp;5i,j; Extended Data Fig.\u0026nbsp;5c,d). The animal then received EGFRt-IL2 on day 14 post CART-20, again with no meaningful changes in B cell count (Fig.\u0026nbsp;5i,j), despite mild clinical changes including mild facial edema, photophobia and mild tremors that were treated with diphenhydramine and levetiracetam on day 14, and 1 dose of dexamethasone (3mg/kg). In RM#2, anti-CAR antibodies were detected from day 12 onwards and may have contributed to the lack of CAR-T cell expansion and activity from day 7 onward (Fig.\u0026nbsp;5k). Anti EGFRt-IL2 antibodies were not detected through day 166 (Supplementary Fig.\u0026nbsp;8b).\u003c/p\u003e \u003cp\u003eThese results suggest that administration of a single dose of EGFRt-IL2 along with autologous CAR-T cells can obviate the need for lymphodepletion and achieve B cell aplasia at the dose provided. ADA against CAR-T cells, which contain non-rhesus sequences, may have limited the potential for the repeat dose of EGFRt-IL2 to induce CAR-T re-expansion in this setting as re-dosing was performed well after the initiation of the ADA response against CAR-Ts.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eExpansion and persistence are considered crucial to the immediate and durable anti-tumor effects of adoptively transferred cells such as CAR-T cells and TILs (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The IL-2 cytokine family plays a key role in mediating T cell survival, proliferation, and memory formation (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). While current CAR-T cell therapy is not accompanied by exogenous cytokine administration, it is thought that lymphodepleting chemotherapy works, in part, by stimulating the production of homeostatic cytokines and by transiently depleting cell populations that can compete with the adoptively transferred T cells for these cytokines. IL-2 was the first cytokine used in cancer immunotherapy, with early clinical trials showing its effectiveness in stimulating \u0026ldquo;lymphokine activated killer cells\u0026rdquo; against solid tumors. However, the cytokine has a short half-life and the high doses required in TIL therapy are dose-limiting (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In addition, the pleiotropic nature of IL-2 cytokine may hamper the immune response due to the activation of regulatory T cells and toxicities, both of which were linked to IL-2 binding to IL-2R\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Consequently, efforts have been made to engineer IL-2 molecules with a bias towards IL-2R\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\gamma\\:\\)\u003c/span\u003e\u003c/span\u003e, thus avoiding both immune suppression and toxicity and opening possibilities of combinations with T cell therapies (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). However, many of the recently engineered IL-2 variants have been tested in the clinic and have shown limited success, including preferential expansion of NK cells over T cells in patients. Therefore, there is a need to further improve IL-2 therapeutics, including in the setting of adoptive T cell therapies.\u003c/p\u003e \u003cp\u003eWe have recently shown that a protein engineering approach referred to as cis-targeting can be used to engineer a more selective IL-2 to use as an immunotherapy for cancer and viral infections (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). We previously developed AB248, a CD8-targeted IL-2, by utilizing an IL-2 mutein with attenuated affinity to IL-2R\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e and IL-2R\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e fused to a targeting antibody recognizing CD8b, a molecule that is specific for cytotoxic CD8\u0026thinsp;+\u0026thinsp;T cells with limited to no expression on NK cells. AB248 recapitulated the effect of IL-2 on CD8\u0026thinsp;+\u0026thinsp;T cells, induced their selective expansion in mice and primates, and resulted in superior anti-tumor and anti-viral activity and enhanced tolerability when compared to an IL-2Rβγ agonist. Efficacy in mouse models was associated with the emergence of a tumor-infiltrating effector population and the rescue of dysfunctional or exhausted T cells in ex vivo cultured human tumor fragments. AB248 is currently in clinical development for the treatment of cancer alone and in combination with anti-PD1 (NCT05653882).\u003c/p\u003e \u003cp\u003eHere we used a similar cis-targeting technology to demonstrate that IL-2 can be specifically directed to engineered T cells, in order to limit any off-target effects that may arise from long-term stimulation of endogenous T cells. We directed engineered IL-2 molecules to CAR-T cells by linking them to antibodies directed against the CAR idiotype or against the EGFRt extracellular tag. The molecules tested here showed high selectivity for engineered T cells, using pSTAT5 assays, proliferation, cytokine production, and tumor cell killing in vitro; as well as in vivo proliferation and persistence, durable tumor responses and prolonged survival in xenografted mice, including those treated with CAR-T cells made from the blood of a lymphoma patient. The IL-2 molecule directed specifically to the CAR led to antigen-independent cytokine release, likely due to induced proximity of the two signaling receptors, CAR and IL-2R, and this translated to toxicity in mice. We therefore prioritized anti-tag IL-2 molecules, ultimately generating a first-in-class antibody selective for the EGFRt tag. This construct was able to stimulate CAR-T cells from a patient treated with lisocabtagene (a commercial CART19 product that contains an EGFRt tag), and does not cross-react with the wild type EGFR expressed on epithelial cells, which should translate into a further improvement in therapeutic index in patients.\u003c/p\u003e \u003cp\u003eWe then showed in a second tumor model that the anti-EGFRt-IL2 molecule can enhance the activity of anti-B7H3 CART cells in a melanoma xenograft model, while reducing toxicity compared with wild-type (un-engineered) IL-2. Our data suggest that a single cis-targeted IL-2 mutein could be used to partner with different adoptive cell therapies, as long as these contain a specific extracellular tag. Lifileucel is an autologous TIL therapy that recently obtained FDA approval for melanoma, and that must be combined with high-dose IL-2. While TIL products are usually not genetically engineered, we demonstrated here that TILs can be transduced to express a tag and that administration of the EGFRt-IL2 molecule can further enhance their function, and perhaps this approach could be tested in future to improve TIL function without exposing the patient to the toxicity of high-dose IL-2.\u003c/p\u003e \u003cp\u003eFinally, we reasoned that potent IL-2 stimulation could obviate the need for lymphodepleting chemotherapy. In rhesus macaques we found that EGFRt-IL2 could indeed mediate B cell aplasia. As expected, in the absence of chemotherapy there was no hematopoietic toxicity. Notably, in this model we found that B cell depletion was profound yet transient, lasting approximately 6 weeks. We note that this is similar to the B cell depletion that occurs in patients recently treated with LD chemotherapy and CART19 cells for autoimmune diseases (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). B cell recovery was accompanied by the development of ADA, initially anti-CAR antibodies, and later, antibodies against EGFRt-IL2. The early occurrence of anti-CAR antibodies may be attributable to the human and murine components of the CAR construct. Future studies may evaluate earlier redosing of EGFRt-IL2 prior to the development of ADA which may result in even longer B cell aplasia. Whether such ADA will occur in humans is unknown. Biweekly dosing of EGFRt-IL2 alongside CAR-T cells may be a feasible approach in patients to maintain long term B cell and tumor cell depletion.\u003c/p\u003e \u003cp\u003eThere are other approaches to obviate the need for lymphodepletion. These include constitutive STAT5 activation, or administration of synthetic IL-2 or IL-2 family cytokines (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The orthogonal IL-2/IL-2R system showed selective stimulation of engineered CAR T cells in murine models (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). However, orthogonal IL-2/IL-2R pairs require further genetic modification of the T cells, and therefore, could not be readily added to current standard of care CAR T therapeutics.\u003c/p\u003e \u003cp\u003eIn conclusion, our data show that combining the EGFRt-IL2 with CAR T cell therapy markedly enhances the anti-tumor efficacy of the CAR T cell therapy in two preclinical xenograft models models. We also showed that human melanoma TILs can be transduced with an extracellular tag and stimulated using EGFRt-IL2, and their anti-tumor function augmented against autologous melanoma organoids. EGFRt-IL2 eliminated the need for conditioning lymphodepletion chemotherapy prior to an autologous CART cell treatment in an NHP model. Together, these studies support cis-targeted IL-2 as a valuable addition to multiple aspects of therapeutic adoptive T cell therapy, which should be tested in appropriately designed clinical trials.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of interleukin (IL)-2 molecules\u003c/h2\u003e \u003cp\u003eAntibodies against the FMC63 and EGFRt were generated via hybridoma or phage display. The amplified cDNA fragments of heavy and light chain V-domains were inserted in frame into a human IgG1 construct with knob-into-hole modification in the IgG CH3 domains (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). IL-2 portions of the constructs were cloned in frame with the \u0026ldquo;hole\u0026rdquo; heavy chain using a flexible linker between the C-terminus of the IgG heavy chain and the N-terminus of IL-2. To abolish FcγR binding/effector function and prevent FcγR co-activation, the following mutations were introduced into the CH2 domain of each of the IgG heavy chains: L234A/L235A/G237A (EU numbering). Molecules were expressed in HEK293 cells and then purified using Protein A affinity chromatography, followed by ion-exchange chromatography and then size exclusion chromatography. SDS-PAGE analyzed the purity, integrity, and monomeric state of the fusion constructs. The protein concentration of purified IL-2 fusion constructs was determined by measuring the optical density at 280 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHuman T-cell transduction\u003c/h2\u003e \u003cp\u003eHealthy donor T cells were obtained from the Human Immunology Core (HIC) at the University of Pennsylvania, or from the Stanford Blood Bank. Deidentified lymphoma patient PBMCs were obtained from the Stem Cell and Xenograft Core at the University of Pennsylvania. Cells were expanded with antiCD3/CD28 beads (ThermoFisher, Catalogue# 11132D) for up to 14 days (until cell size is less than 350fL) and transduced with concentrated lentivirus from HEK293 T cells transfected with anti-CD19-41BB- CD3ζ plasmid DNA beginning on day\u0026thinsp;+\u0026thinsp;1 at MOI of 3. CAR-T cells utilized in mouse pharmacodynamic studies were generated using a construct generated with either an anti-CD19 [FMC63] scFv or anti-B7H3 \u0026ldquo;clone CD276.MG, (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e)\u0026rdquo; scFv fused to human CD8 transmembrane region that was upstream of 41BB costimulatory molecule and the CD3zeta signaling domain. This CAR cassette was encoded upstream of a self-cleaving P2A peptide that drove expression of truncated epidermal growth factor receptor (EGFRt), consisting of only the transmembrane region and extracellular domain 3 and domain 4 of EGFR, and cloned into a lentiviral vector under control of EF-1⍺ promoter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines\u003c/h2\u003e \u003cp\u003eThe NALM6 cell line was obtained from the ATCC and maintained in RPMI media supplemented with 10% fetal calf serum, penicillin, and streptomycin (R10). The cells were transduced with a luc2-EGFP lentiviral construct and sorted twice by positive-selection flow cytometry to \u0026gt;\u0026thinsp;99% purity. Cells were viably cryopreserved in 90% fetal calf serum and 10% dimethylsulfoxide until required for use. For all functional studies, NALM6 cells were thawed at least 12 hours before analysis and rested overnight at 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e per mL in R10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro cytokine release assay\u003c/h2\u003e \u003cp\u003eAntigen-independent cytokine release assays were performed by incubating 5 x 104 thawed human CAR-T cells with the indicated stimulus in X-VIVO 15 media (Lonza) in a 96-well flat bottom plate (Corning) for 72 hours. Cell culture supernatants were collected and stored at -80\u0026deg;C until analysis was performed by MSD U-plex plates (Mesoscale Discovery) to quantify IFN\u0026#120574;, TNF⍺, IL-5, MIP1⍺, and IL-6 following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eAnti-human antibodies were purchased from BioLegend, and Thermofisher. Cells were first added to ACK lysing buffer, then they were stained with Fc block. They were then stained with our flow cytometry panel. Counting beads were used to quantify the concentration of cells from peripheral blood as per manufacture\u0026rsquo;s protocol (Invitrogen, CountBright Absolute Counting Beads). In all analyses, the population of interest was gated based on forward vs side scatter characteristics followed by singlet gating, and live cells were gated using Live Dead Aqua (Invitrogen). Analysis of pSTAT5 activity assays were performed using FlowJo v10.9. Briefly, CD3\u0026thinsp;+\u0026thinsp;T cells were gated based on CD8\u0026thinsp;+\u0026thinsp;high population, shown to consistently exhibit pSTAT5 activity in response to WT IL-2. Cells were further gated upon staining with anti-FMC63 idiotype antibody (Acro Biosystems) to determine the percent or gMFI of pSTAT5 activity in CAR\u0026thinsp;+\u0026thinsp;or non-CAR (CAR-) T cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHuman pSTAT5 Assay\u003c/h2\u003e \u003cp\u003eSTAT5 phosphorylation was assessed on a mixed population of CD3\u0026thinsp;+\u0026thinsp;T cells consisting of CAR-T cells (30\u0026ndash;50% CAR+) and non-CAR T cells in the same well of a 96-well plate. Briefly, blood was treated for 25 minutes with indicated molecules at 37\u0026deg;C at a 2X final dilution in unsupplemented RPMI-1640 media. Surface staining antibodies were added and incubated at 4\u0026deg;C for 10 minutes and cells were washed twice with PBS containing 2% BSA. Pre-warmed Lyse/Fix (BD Biosciences) was added and incubated at 37\u0026deg;C for 10 minutes. Following fixation, cells were washed and resuspended in chilled BD Perm Buffer III (BD Biosciences) and incubated for 1 hour at -20\u0026deg;C. Cells were then washed and stained for remaining surface and intracellular markers in TFP Perm/Wash buffer (BD Biosciences) for 45 minutes at 4\u0026deg;C. After washing, cells were resuspended in FACS buffer and analyzed by flow cytometry as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo murine models\u003c/h2\u003e \u003cp\u003eMale and female 8\u0026ndash;12 week old NOD-SCID-IL2rγ\u0026minus;/\u0026minus; (NSG) mice were purchased from Jackson laboratories or bred in-house. All experiments were performed on protocols approved by the institutional animal care and use committees of the University of Pennsylvania. These studies were conducted in accordance with the Declaration of Helsinki. The experimental outline schema of the used xenograft models is available in Fig.\u0026nbsp;1. NALM6 cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/ mouse) were injected in 200 \u0026micro;L of PBS into the tail veins of mice; tumor burden was assessed by live imaging of animals for bioluminescence. Mice were injected intraperitoneally (IP) with 150 \u0026micro;L of luciferin (15 mg/mL) and imaged starting on Day \u0026minus;\u0026thinsp;1 (6 days post NALM6 transplant) with a bioluminescence imager (Xenogen IVIS-200 Spectrum). Animals were imaged once weekly up to Day 110. Anti-CD19-redirected T cells (CART19), or untransduced (UTD) human T cells were injected in 200 \u0026micro;L of PBS at multiple doses ranging from 0.1 to 1x10\u003csup\u003e6\u003c/sup\u003e cells/mouse into the tail vein. Mice were monitored for signs of severe disease and euthanized when they met pre-specified endpoints according to IACUC protocol monitoring plan including losing\u0026thinsp;\u0026gt;\u0026thinsp;20% body weight or hind limb paralysis. Mice were also monitored for any GvHD signs including hair loss and cachexia.\u003c/p\u003e \u003cp\u003eMice were injected with PBS, WT IL-2, or the indicated targeted-IL2 fusion molecules at the doses described in the figure legends and/or main text. Peripheral blood was collected via retroorbital bleed and stained for Live/dead (ThermoFisher), anti-mouse CD45, and anti-human CD45, CD3, CD8, CD4, PD-1, LAG3, TIM3, and either anti-FMC63 (Acro Biosystems) to detect the CD19 CAR-T cells or anti-G4S linker antibody to detect the B7H3 CAR-T cells. Excised tumors were digested using the gentleMACS Octo dissociator and stained with the above panel for quantification of T cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell RNA-seq Analysis\u003c/h2\u003e \u003cp\u003eRaw single-cell RNA sequencing (scRNA-seq) data were processed using Cell Ranger (version 7.1.0, 10x Genomics). Transcript reads were aligned to the GRCh38 human reference genome, which was supplemented with the scFv domain sequence of the CAR19 transcript. Filtered gene expression matrices were further analyzed in R (version 4.3.1) using the Seurat package (version 5.1.0).\u003c/p\u003e \u003cp\u003eTo exclude low-quality cells, cells were filtered out if they met any of the following criteria: fewer than 20 expressed genes, abnormal total UMI counts (\u0026lt;\u0026thinsp;100 or \u0026gt;\u0026thinsp;30,000), or elevated mitochondrial gene content (\u0026gt;\u0026thinsp;10%). Doublets were identified and removed using DoubletFinder (version 2.0.3).\u003c/p\u003e \u003cp\u003eBatch correction and sample integration were performed using Seurat\u0026rsquo;s SCTransform workflow. Cell types were annotated prior to integration using the Azimuth package (version 0.5.0) with both the built-in peripheral blood mononuclear cells (PBMCs) reference dataset (version 1.0.0) and an external PBMC reference from the Tabula Rasa project, provided by the Chan Zuckerberg Foundation. Following annotation, all scRNA-seq samples were integrated into a single dataset.\u003c/p\u003e \u003cp\u003eDimensionality reduction was performed by calculating principal components (PCs) using the RunPCA function in Seurat. The first 10 PCs were used as input for Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction via the RunUMAP function, enabling visualization and clustering of cells in reduced dimensions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Expression Analysis\u003c/h2\u003e \u003cp\u003eAfter integration, the dataset was subset to include only the cells expressing CD3E and scFvCAR19. Differential gene expression between experimental conditions was assessed using the FindMarkers function in Seurat with the Wilcoxon rank-sum test. Only genes expressed in more than 5% of cells in at least one condition were considered for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of EGFRt\u0026thinsp;+\u0026thinsp;TILs\u003c/h2\u003e \u003cp\u003eTumor-infiltrating lymphocytes (TILs) are T cells isolated from melanoma tumor fragments obtained from the Tara Miller Melanoma Center at the University of Pennsylvania. Fresh melanoma tumor tissue was physically and enzymatically digested using a solution containing collagenase/hyaluronidase and DNase. The cells were then cultured in a medium containing IL-7 and IL-15. TILs were isolated using a CD45\u0026thinsp;+\u0026thinsp;selection kit and further analyzed by flow cytometry for the expression of CD3, CD4, and CD8 markers. The isolated TILs were stimulated with anti-CD3/anti-CD28 beads for activation and expansion. On day 2 post-stimulation, the TILs were transduced with an EGFRt lentiviral vector and kept in culture with 50U/ml of rhIL2. On day 14, they were harvested and used fresh for the in vitro organoid experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity Assay for NHP CART cells\u003c/h2\u003e \u003cp\u003eEffector Cells (CAR20 T cells) or untransduced (UTD) control T cells were thawed and rested 12\u0026ndash;24 hours in R10 media (2 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL; RPMI\u0026thinsp;+\u0026thinsp;10% FBS, 100 U/mL penicillin/streptomycin, 1% GlutaMAX). Within 24 hours effector cells were counted and resuspended to appropriate concentrations to set up co-cultures with target cells at multiple E:T ratios. Target cells were luciferase-expressing Raji target cells maintained in culture, counted, and resuspended to a concentration of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL in R10. We tested several E:T ratios, the highest being 5:1 and the lowest 0.075:1 in for NHP RM#1 CAR20 T cells. One hundred microliters of target cells (1 x 105 cells) were added to all wells containing varying amounts of effector cells in 100 \u0026micro;L to achieve the specified E:T ratios, including a triplicate set of wells with target cells only. Wells were gently mixed and incubated at 37\u0026deg;C for 24 and 48 hours. At both time points, 2 \u0026micro;L of diluted Luciferin (1:10 dilution in PBS) was added to each well to determine luciferase levels. Bioluminescence was assessed on the Synergy H4 plate reader on auto-gain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eNon-human primate studies\u003c/h2\u003e \u003cp\u003e The rhesus macaque (RM) study was reviewed and approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) committee. Animals were housed in state of the art AAALAC International accredited facilities. Animals were monitored twice daily (or more often) and kept in temperature and humidity controlled rooms. NHPs were fed twice daily with Lab diet 5038 (for NHPs) and provided daily fruits and vegetables. RM (n\u0026thinsp;=\u0026thinsp;1 per group, both males) were dosed intravenously with CAR20-EGFRt T cells (2.5 x 10\u003csup\u003e6\u003c/sup\u003e cells/kg) on day 0. Cells were infused in 20-30mL of sterile PBS. RM#1 was also injected with EGFRt-IL2m on day 0. RM#2 was dosed intravenously with EGFRt-IL2 on day 14. Cells and muteins were dosed over 10\u0026ndash;20 minutes and vitals were monitored. Peripheral blood samples were collected and PBMCs isolated by lysing RBCs from peripheral blood samples with ACK lysing buffer. PBMCs were analyzed via flow cytometry. Cell blood counts and serum chemistries were routinely monitored and submitted to the clinical pathology laboratory at Penn Vet. Absolute counts were determined by relating the percentage of each lymphocyte population as determined by flow cytometry to the absolute lymphocyte counts as determined by hematology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDetection of ADAs against CAR-T cells\u003c/h2\u003e \u003cp\u003eSerum ADAs against CAR-T cells were assessed by flow cytometry. Rhesus CAR-T cells or untransduced controls (7.5x10\u003csup\u003e4\u003c/sup\u003e each) were incubated with diluted serum samples (1:20, 1:100, 1:500) in 96-well plates for 30min at 4\u0026deg;C. Cells were washed, then incubated with a labeled ant-rhesus IgG for detection (20 min, at 4\u0026deg;C). Cells were also stained for viability and cell surface expression of CAR (using labeled panitumumab), CD45, CD3, CD4, and CD8. After a final wash, cells were analyzed on a flow cytometer.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDetection of ADAs against EGFRt-IL2\u003c/h2\u003e \u003cp\u003eAn electrochemiluminescent homogeneous bridging assay on the MSD platform was used to detect EGFRt-IL2 specific ADA response. Briefly, MSD GOLD streptavidin 96-well plates were blocked with 200 \u0026micro;L/well of assay buffer (2% BSA in 1X PBS) for 1 to 3 hours at room temperature with shaking at 700 rpm. Test rhesus monkey serum samples, negative control (NC \u0026ndash; drug-na\u0026iuml;ve pooled sera), and a positive control (PC \u0026ndash; 100 ng/mL anti-IL2 specific mAb prepared in drug-na\u0026iuml;ve pooled sera) were diluted 5-fold in assay buffer. The 5-fold diluted samples were further diluted 4-fold into a master mix solution of 50 \u0026micro;L 0.25 \u0026micro;g/mL of biotinylated EGFRt-IL2, 50 \u0026micro;L 0.5 \u0026micro;g/mL ruthenylated EGFRt-IL2, and 50 \u0026micro;L of assay buffer (or 40 \u0026micro;g/mL unlabeled EGFRt-IL2 to assess assay specificity) for a final minimum required dilution of 1:20, and incubated at room temperature for 1 hour in the dark with shaking at 700 rpm. The MSD plates were washed three times with 300 \u0026micro;L/well of wash buffer (1X TBST). The samples and controls in master mix were transfer 50 \u0026micro;L/well to the blocked streptavidin plate and incubated at room temperature for 1 hour in the dark with shaking at 700 rpm. Subsequently, the plates were washed three times with 300 \u0026micro;L/well of wash buffer, 150 \u0026micro;l MSD GOLD Read Buffer A was added and the electrochemiluminescence signal was then measured on a MESO Quickplex SQ120 (MSD).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSerum cytokine detection in NHP\u003c/h2\u003e \u003cp\u003eRhesus monkey serum samples were collected at the indicated time points pre-injection and post-injection of EGFRt-IL2. IFNγ, TNFα, GM-CSF, IL-5, IL-6, and MIP1a were concurrently quantified using a U-PLEX 6-assay kit for non-human primates from MSD following the manufacturers protocol. Briefly, plates were labeled with 50 \u0026micro;L of 1x multiplex coating solution and incubated overnight at 4\u0026deg;C with shaking at 650 rpm. The plates were washed three times with 400 \u0026micro;l of manufacturer recommended PBS-T (PBS with 0.05% Tween-20) wash buffer, followed by the addition of 25 \u0026micro;L of assay buffer and either 25 \u0026micro;L of calibrator standards or test serum. Plates were incubated for 1 hour at room temperature with shaking at 700 rpm, then washed three times with 400 \u0026micro;L PBS-T per well. Manufacturer Read Buffer (2x) was added to each plate with 150 \u0026micro;L per well and the plate was analyzed with MSD instrument (Rockville, Maryland) using Methodical Mind software.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eStatistical analysis and reproducibility\u003c/h2\u003e \u003cp\u003eAll statistical analysis was performed using GraphPad Prism, where comparisons between groups were performed using either unpaired t test or ordinary one-way ANOVA with Turkey\u0026rsquo;s multiple comparison test. Unless noted otherwise, data are reported as median\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Survival data were analyzed using the log-rank (Mantel-Cox) test.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data associated with this study are present in the manuscript. Materials used in this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Schemes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental schemes were generated using biorender.com.\u003c/p\u003e\n\u003ch2\u003eCompeting interests:\u003c/h2\u003e\n\u003cp\u003eS.G. has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. S.G. is a scientific co-founder and holds equity in Interius Biotherapeutics and Carisma Therapeutics. S.G. is a scientific advisor to Carisma, Currus, Interius, Kite, NKILT, Mission Bio, and Vor Bio. J.F. holds patents and intellectual property in T-cell-based cancer immunotherapy with royalties, has received funding from Tmunity Therapeutics and Danaher Corporation, consults for Retro Biosciences, and serves on the scientific advisory boards of Cartography Bio, Shennon Biotechnologies Inc., CellFe Biotech, OverT Bio, Inc., and Tceleron Therapeutics, Inc. P.B., C.K., M.S., D.C.P., T.P., S.L., and K.D.M are employees of Asher Biotherapeutics. Y.A.Y. and I.D. are employees of and own stock in Asher Biotherapeutics.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions:\u003c/h2\u003e\n\u003cp\u003eS.S., N.D.M, Y.A.Y, I.J. and S.I.G. conceptualized the project, analyzed the data and wrote the paper. S.S., T.H., N.D.M, F.S., O.S., R.D.S., A.B.B. and M.K. carried out the translational experiments. D.J. and J.F. performed antigen quantification on rhesus macaques peripheral blood samples. P.B., C.K. designed and generated the proteins. S.L.C. performed the testing of ADA. S.S., N.D.M., T.H., K.D.M, I.D., and M.S. visualized the data. S.I.G. supervised the project. J.S., H.T., and R.D.S handled the treatment and care of the rhesus macaques in the NHP model.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\n\u003cp\u003eWe would like to acknowledge the human immunology core, the cell center service core, and the stem cell and xenograft core at the University of Pennsylvania.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFraietta, J. A. \u003cem\u003eet al.\u003c/em\u003e Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. \u003cem\u003eNat Med\u003c/em\u003e 24, 563\u0026ndash;571 (2018). \u003c/li\u003e\n\u003cli\u003eCappell, K. M. \u003cem\u003eet al.\u003c/em\u003e Long-Term Follow-Up of Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy. \u003cem\u003eJCO\u003c/em\u003e 38, 3805\u0026ndash;3815 (2020). \u003c/li\u003e\n\u003cli\u003eGrover, P. \u003cem\u003eet al.\u003c/em\u003e Chimeric antigen receptor T-cell therapy in adults with B-cell acute lymphoblastic leukemia. \u003cem\u003eBlood Advances\u003c/em\u003e 6, 1608\u0026ndash;1618 (2022). \u003c/li\u003e\n\u003cli\u003eKochenderfer, J. N. \u003cem\u003eet al.\u003c/em\u003e Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels. \u003cem\u003eJCO\u003c/em\u003e 35, 1803\u0026ndash;1813 (2017). \u003c/li\u003e\n\u003cli\u003eNeelapu, S. S. \u003cem\u003eet al.\u003c/em\u003e Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. \u003cem\u003eN Engl J Med\u003c/em\u003e 377, 2531\u0026ndash;2544 (2017). \u003c/li\u003e\n\u003cli\u003eSchuster, S. J. \u003cem\u003eet al.\u003c/em\u003e Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas. \u003cem\u003eN Engl J Med\u003c/em\u003e 377, 2545\u0026ndash;2554 (2017). \u003c/li\u003e\n\u003cli\u003eMunshi, N. C. \u003cem\u003eet al.\u003c/em\u003e Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. \u003cem\u003eN Engl J Med\u003c/em\u003e 384, 705\u0026ndash;716 (2021). \u003c/li\u003e\n\u003cli\u003ePorter, D. L. \u003cem\u003eet al.\u003c/em\u003e Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. \u003cem\u003eSci Transl Med\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 303ra139 (2015).\u003c/li\u003e\n\u003cli\u003eMueller, K. T. \u003cem\u003eet al.\u003c/em\u003e Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia. \u003cem\u003eBlood\u003c/em\u003e\u003cstrong\u003e130\u003c/strong\u003e, 2317\u0026ndash;2325 (2017).\u003c/li\u003e\n\u003cli\u003eMelenhorst, J. J. \u003cem\u003eet al.\u003c/em\u003e Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e602\u003c/strong\u003e, 503\u0026ndash;509 (2022).\u003c/li\u003e\n\u003cli\u003eTennant, M. D., New, C., Ferreira, L. M. R. \u0026amp; O\u0026rsquo;Neil, R. T. Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling. \u003cem\u003eMolecular Therapy\u003c/em\u003e 31, 2591\u0026ndash;2599 (2023). \u003c/li\u003e\n\u003cli\u003eRemoval of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells | Journal of Experimental Medicine | Rockefeller University Press. https://rupress.org/jem/article/202/7/907/40182/Removal-of-homeostatic-cytokine-sinks-by.\u003c/li\u003e\n\u003cli\u003eKlebanoff, C. A., Khong, H. T., Antony, P. A., Palmer, D. C. \u0026amp; Restifo, N. P. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cell-mediated tumor immunotherapy. \u003cem\u003eTrends Immunol\u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e, 111\u0026ndash;117 (2005).\u003c/li\u003e\n\u003cli\u003eNorth, R. J. Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells. \u003cem\u003eJ Exp Med\u003c/em\u003e\u003cstrong\u003e155\u003c/strong\u003e, 1063\u0026ndash;1074 (1982).\u003c/li\u003e\n\u003cli\u003eRejeski, K. \u003cem\u003eet al.\u003c/em\u003e Immune effector cell-associated hematotoxicity: EHA/EBMT consensus grading and best practice recommendations. \u003cem\u003eBlood\u003c/em\u003e\u003cstrong\u003e142\u003c/strong\u003e, 865\u0026ndash;877 (2023).\u003c/li\u003e\n\u003cli\u003eSpolski, R., Li, P. \u0026amp; Leonard, W. J. Biology and regulation of IL-2: from molecular mechanisms to human therapy. \u003cem\u003eNat Rev Immunol\u003c/em\u003e 18, 648\u0026ndash;659 (2018). \u003c/li\u003e\n\u003cli\u003eOverwijk, W. W., Tagliaferri, M. A. \u0026amp; Zalevsky, J. Engineering IL-2 to Give New Life to T Cell Immunotherapy. \u003cem\u003eAnnu. Rev. Med.\u003c/em\u003e 72, 281\u0026ndash;311 (2021). \u003c/li\u003e\n\u003cli\u003eKo, B., Takebe, N., Andrews, O., Makena, M. R. \u0026amp; Chen, A. P. Rethinking Oncologic Treatment Strategies with Interleukin-2. \u003cem\u003eCells\u003c/em\u003e 12, 1316 (2023). \u003c/li\u003e\n\u003cli\u003eRaeber, M. E., Sahin, D. \u0026amp; Boyman, O. Interleukin-2\u0026ndash;based therapies in cancer. \u003cem\u003eSci. Transl. Med.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, eabo5409 (2022).\u003c/li\u003e\n\u003cli\u003eBriukhovetska, D. \u003cem\u003eet al.\u003c/em\u003e Interleukins in cancer: from biology to therapy. \u003cem\u003eNat Rev Cancer\u003c/em\u003e\u003cstrong\u003e21\u003c/strong\u003e, 481\u0026ndash;499 (2021).\u003c/li\u003e\n\u003cli\u003eKrieg, C., L\u0026eacute;tourneau, S., Pantaleo, G. \u0026amp; Boyman, O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e\u003cstrong\u003e107\u003c/strong\u003e, 11906\u0026ndash;11911 (2010).\u003c/li\u003e\n\u003cli\u003eReinhardt, J. \u003cem\u003eet al.\u003c/em\u003e Distinguishing activated T regulatory cell and T conventional cells by single‐cell technologies. \u003cem\u003eImmunology\u003c/em\u003e 166, 121\u0026ndash;137 (2022). \u003c/li\u003e\n\u003cli\u003eParisi, G. \u003cem\u003eet al.\u003c/em\u003e Persistence of adoptively transferred T cells with a kinetically engineered IL-2 receptor agonist. \u003cem\u003eNat Commun\u003c/em\u003e 11, 660 (2020). \u003c/li\u003e\n\u003cli\u003ePtacin, J. L. \u003cem\u003eet al.\u003c/em\u003e An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism. \u003cem\u003eNat Commun\u003c/em\u003e 12, 4785 (2021). \u003c/li\u003e\n\u003cli\u003eRouanne, M., Zitvogel, L. \u0026amp; Marabelle, A. Pegylated Engineered IL2 plus Anti-PD-1 Monoclonal Antibody: The Nectar Comes from the Combination. \u003cem\u003eCancer Discov\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 1097\u0026ndash;1099 (2020).\u003c/li\u003e\n\u003cli\u003eLevin, A. M. \u003cem\u003eet al.\u003c/em\u003e Exploiting a natural conformational switch to engineer an interleukin-2 \u0026lsquo;superkine\u0026rsquo;. \u003cem\u003eNature\u003c/em\u003e 484, 529\u0026ndash;533 (2012). \u003c/li\u003e\n\u003cli\u003eRen, J. \u003cem\u003eet al.\u003c/em\u003e Interleukin-2 superkines by computational design. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e 119, e2117401119 (2022). \u003c/li\u003e\n\u003cli\u003eMerchant, R. \u003cem\u003eet al.\u003c/em\u003e Fine-tuned long-acting interleukin-2 superkine potentiates durable immune responses in mice and non-human primate. \u003cem\u003eJ Immunother Cancer\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, e003155 (2022).\u003c/li\u003e\n\u003cli\u003eMoynihan, K. D. \u003cem\u003eet al.\u003c/em\u003e IL2 Targeted to CD8+ T Cells Promotes Robust Effector T-cell Responses and Potent Antitumor Immunity. \u003cem\u003eCancer Discovery\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 1206\u0026ndash;1225 (2024).\u003c/li\u003e\n\u003cli\u003eSun, Z. \u003cem\u003eet al.\u003c/em\u003e A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+ T-cell response and effective tumor control. \u003cem\u003eNat Commun\u003c/em\u003e 10, 3874 (2019). \u003c/li\u003e\n\u003cli\u003eItaliano, A. et al. Clinical activity and safety of simlukafusp alfa, an engineered interleukin-2 variant targeted to fibroblast activation protein-\u0026alpha;, combined with atezolizumab in patients with recurrent or metastatic cervical cancer. Journal of Clinical Oncology (2021) doi:10.1200/JCO.2021.39.15_suppl.5510.\u003c/li\u003e\n\u003cli\u003eJanku, F. \u003cem\u003eet al.\u003c/em\u003e Abstract LB041: THOR-707 (SAR444245), a novel not-alpha IL-2 as monotherapy and in combination with pembrolizumab in advanced/metastatic solid tumors: Interim results from HAMMER, an open-label, multicenter phase 1/2 Study. \u003cem\u003eCancer Research\u003c/em\u003e\u003cstrong\u003e81\u003c/strong\u003e, LB041 (2021).\u003c/li\u003e\n\u003cli\u003eBoni, V. et al. ARTISTRY-1: Nemvaleukin alfa monotherapy and in combination with pembrolizumab in patients (pts) with advanced solid tumors. Journal of Clinical Oncology (2021) doi:10.1200/JCO.2021.39.15_suppl.2513.\u003c/li\u003e\n\u003cli\u003eAndreata, F. et al. CD8 cis-targeted IL-2 drives potent antiviral activity against hepatitis B virus. Sci Transl Med 16, eadi1572 (2024).\u003c/li\u003e\n\u003cli\u003eKaptein, P. et al. CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool. Cancer Discov 14, 1226\u0026ndash;1251 (2024).\u003c/li\u003e\n\u003cli\u003eTaylor, N. D., Way, J. C., Silver, P. A. \u0026amp; Cironi, P. Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity. Protein Eng Des Sel 23, 251\u0026ndash;260 (2010).\u003c/li\u003e\n\u003cli\u003eAbramson, J. S. et al. Lisocabtagene maraleucel as second-line therapy for large B-cell lymphoma: primary analysis of the phase 3 TRANSFORM study. Blood 141, 1675\u0026ndash;1684 (2023).\u003c/li\u003e\n\u003cli\u003eLindner, S. E., Johnson, S. M., Brown, C. E. \u0026amp; Wang, L. D. Chimeric antigen receptor signaling: Functional consequences and design implications. \u003cem\u003eScience Advances\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, eaaz3223 (2020).\u003c/li\u003e\n\u003cli\u003eGuedan, S., Calderon, H., Posey, A. D. \u0026amp; Maus, M. V. Engineering and Design of Chimeric Antigen Receptors. \u003cem\u003eMolecular Therapy Methods \u0026amp; Clinical Development\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 145\u0026ndash;156 (2019).\u003c/li\u003e\n\u003cli\u003eRohrs, J. A., Zheng, D., Graham, N. A., Wang, P. \u0026amp; Finley, S. D. Computational Model of Chimeric Antigen Receptors Explains Site-Specific Phosphorylation Kinetics. \u003cem\u003eBiophysical Journal\u003c/em\u003e\u003cstrong\u003e115\u003c/strong\u003e, 1116\u0026ndash;1129 (2018).\u003c/li\u003e\n\u003cli\u003eFernandes, R. A. et al. Immune receptor inhibition through enforced phosphatase recruitment. Nature 586, 779\u0026ndash;784 (2020).\u003c/li\u003e\n\u003cli\u003eOu, L. \u003cem\u003eet al.\u003c/em\u003e Patient-derived melanoma organoid models facilitate the assessment of immunotherapies. \u003cem\u003eEBioMedicine\u003c/em\u003e\u003cstrong\u003e92\u003c/strong\u003e, 104614 (2023).\u003c/li\u003e\n\u003cli\u003eJafarzadeh, L., Masoumi, E., Fallah-Mehrjardi, K., Mirzaei, H. R. \u0026amp; Hadjati, J. Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward. \u003cem\u003eFront Immunol\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 702 (2020).\u003c/li\u003e\n\u003cli\u003eGoff, S. L. et al. Randomized, Prospective Evaluation Comparing Intensity of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating Lymphocytes for Patients With Metastatic Melanoma. J Clin Oncol 34, 2389\u0026ndash;2397 (2016).\u003c/li\u003e\n\u003cli\u003eLotze, M. T., Frana, L. W., Sharrow, S. O., Robb, R. J. \u0026amp; Rosenberg, S. A. In vivo administration of purified human interleukin 2. I. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e 134, 157\u0026ndash;166 (1985).\u003c/li\u003e\n\u003cli\u003eAspuria, P.-J. \u003cem\u003eet al.\u003c/em\u003e An orthogonal IL-2 and IL-2R\u0026beta; system drives persistence and activation of CAR T cells and clearance of bulky lymphoma. \u003cem\u003eSci. Transl. Med.\u003c/em\u003e 13, eabg7565 (2021). \u003c/li\u003e\n\u003cli\u003eMackensen, A. \u003cem\u003eet al.\u003c/em\u003e Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. \u003cem\u003eNat Med\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 2124\u0026ndash;2132 (2022).\u003c/li\u003e\n\u003cli\u003eAvanzi, M. P. \u003cem\u003eet al.\u003c/em\u003e Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System. \u003cem\u003eCell Rep\u003c/em\u003e 23, 2130\u0026ndash;2141 (2018). \u003c/li\u003e\n\u003cli\u003eKalbasi, A. \u003cem\u003eet al.\u003c/em\u003e Potentiating adoptive cell therapy using synthetic IL-9 receptors. \u003cem\u003eNature\u003c/em\u003e 607, 360\u0026ndash;365 (2022). \u003c/li\u003e\n\u003cli\u003eZhang, Q. \u003cem\u003eet al.\u003c/em\u003e A human orthogonal IL-2 and IL-2R\u0026beta; system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia. \u003cem\u003eSci. Transl. Med.\u003c/em\u003e 13, eabg6986 (2021). \u003c/li\u003e\n\u003cli\u003eMerchant, A. M. et al. An efficient route to human bispecific IgG. Nat Biotechnol 16, 677\u0026ndash;681 (1998).\u003c/li\u003e\n\u003cli\u003eMajzner, R. G. \u003cem\u003eet al.\u003c/em\u003e CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. \u003cem\u003eClin Cancer Res\u003c/em\u003e\u003cstrong\u003e25\u003c/strong\u003e, 2560\u0026ndash;2574 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5962170/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5962170/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Adoptively transferred T cells require cytokine stimulation, which is achieved using lymphodepleting chemotherapy with or without administration of exogenous interleukin 2 (IL-2). Lymphodepleting chemotherapy (LDC) is associated with cytopenias and attendant complications, while high dose IL-2 causes severe infusion toxicity and can stimulate undesirable cell populations. To address these challenges, we developed cis-targeted IL-2 fusion molecules which are comprised of an IL-2 mutein with attenuated binding to IL-2Rα and IL-2Rβ linked to an antibody that targets a cell-surface molecule expressed specifically on engineered T cells. Using T cells from healthy donors as well as from lymphoma and melanoma patients, we selectively stimulated CAR-T cells or engineered TILs and enhanced their anti-tumor function in multiple tumor models. Finally, we eliminated the need for LDC by combining CAR-T cells with cis-targeted IL-2, leading to B cell aplasia in non-human primates.","manuscriptTitle":"Selective support of engineered T cells using a cis-targeted interleukin-2 enhances anti-tumor activity and obviates the need for lymphodepletion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 11:28:05","doi":"10.21203/rs.3.rs-5962170/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-cancer","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natcancer","sideBox":"Learn more about [Nature Cancer](http://www.nature.com/natcancer/)","snPcode":"","submissionUrl":"","title":"Nature Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fc51dce1-b3a8-4927-9da2-b32d3784226d","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44364404,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":44364405,"name":"Biological sciences/Immunology/Cytokines/Interleukins"},{"id":44364406,"name":"Biological sciences/Immunology/Translational immunology"}],"tags":[],"updatedAt":"2025-05-05T11:28:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 11:28:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5962170","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5962170","identity":"rs-5962170","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}