IL-2 Variant Research Articles

Association of the TNF-α, IL-2, and IL-2RB gene variants with susceptibility to psoriasis in a Turkish cohort.

Aim of the studyThe aim of this study was to investigate the role TNF-α, IL-2, and IL-2RB variants in psoriasis (Ps) and to evaluate the association between these variants and clinical features.Material and methodsA total of 74 psoriatic patients and 74 healthy individuals were genotyped for these variants by PCR and/or RFLP.ResultsThe AA genotype of TNF-α (–308) was significantly more common in the patients (p = 0.013). TNF-α (–238) AA genotype was significantly increased in the patients (p = 0.028), while the GG genotype was decreased in the patient group, compared to the controls (p = 0.016). IL-2 (–330) variant GG and TT genotype was more common in the patients (p = 0.037, p = 0.009, respectively), while IL-2 (–330) GT genotype was increased in the control subjects (p = 0.001). IL-2 (–330) GG genotype frequency was significantly decreased (p = 0.021) and the TT genotype frequency was significantly increased among patients with psoriatic arthritis in comparison with Ps patients (p = 0.014). IL-2RB TC genotype frequency was significantly decreased and TT genotype frequency was significantly increased in the patients with positive family history of Ps compared to those who had a negative family history (p = 0.017, p = 0.014, respectively). Also, IL-2RB CC genotype was significantly increased among the patients with late-onset Ps in comparison with the early onset Ps group (p = 0.009). The frequency of IL-2 (–330) TT genotype was significantly higher in mild Ps patients than moderate-severe patients (p = 0.043).ConclusionsOur data suggest a potential role of these genes as candidate genes for susceptibility to Ps in a Turkish cohort.

Abstract 1594: Enhancement of the anti-tumor activity of CEA TCB via combination with checkpoint blockade by PD-L1 and interleukin-2 variant immunocytokine

Abstract Cancer immunotherapy represents a promising therapeutic approach to extend the overall survival of cancer patients. However, several mechanisms within the tumor microenvironment orchestrate the suppression of host immune response requiring combination strategies to prolong the durability of effect. Among suppressive pathways, up-regulation of PD-1 ligand (PD-L1) and its interaction with PD-1 receptor plays a key role in suppression of T-cell activity, a mechanism also called adaptive immune resistance. Therefore, approaches able to inhibit the tumor immune suppressive mechanisms along with those expanding the frequency of intra-tumor T-cells and enhancing/prolonging their functionality are required. CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent and in combination with atezolizumab in Phase 1/1b studies in patients with advanced and/or metastatic CEA-expressing tumors (NCT02324257; NCT02650713). CEA TCB treatment leads to increased intra-tumoral T cell infiltration and T-cell activation along with up-regulation of PD-1/PD-L1 suppressive pathway. Here we show that combination of CEA TCB with PD-L1 blocking antibody in vitro enhances T cell activation as detected by increased CD3 signaling and secretion of pro-inflammatory cytokines. Combination in vivo performed in both stem cell humanized NOG mice (HSC mice engrafted with MKN45) and fully immunocompetent human CEA transgenic C56BL/6 mice (hCEA Tg mice engrafted with MC38-hCEA) demonstrated significantly improved anti-tumor activity of combination as compared to activity of single agents, yielding to increased number of tumor-free animals. Randomization of animals that progressed to CEA TCB monotherapy revealed that combination of CEA TCB with PD-L1 blocking antibody is required to control tumor outgrowth, as tumors treated with corresponding monotherapy arms progressed to treatment. Efficacy of CEA TCB was also potentiated when administered in combination with a half-life-extended IL-2 variant (untargeted (IgG-IL2v) or fibroblast-activating protein-targeted IL-2 variant (FAP-IL2v)), resulting in stronger tumor growth inhibition in MKN45-bearing HSC mice or prolonged survival in PanCO2-hCEA-bearing hCEA Tg C56BL/6 mice. Synergy likely reflects ability of IL2v to enhance anti-tumor efficacy by increasing number of effector T cells in tumors. In conclusion, CEA TCB treatment leads to intra-tumoral T cell infiltration and T-cell activation. This is accompanied by up-regulation of PD-1/PD-L1 suppressive pathway, which can be overcome by combination therapy with a PD-L1 inhibitor. In vivo efficacy of CEA TCB is further potentiated when administered in combination with immunotherapies that increase the pool of available tumor-infiltrating effector cells. Citation Format: Marina Bacac, Sara Colombetti, Linda Fahrni, Tanja Fauti, Valeria Nicolini, Johannes Sam, Petros Papastogiannidis, Marine Le Clech, Xavier Miot, Inja Waldhauer, Karolin Rommel, Christian Gerdes, Christian Klein, Pablo Umaña. Enhancement of the anti-tumor activity of CEA TCB via combination with checkpoint blockade by PD-L1 and interleukin-2 variant immunocytokine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1594. doi:10.1158/1538-7445.AM2017-1594

Effects of Soy Product Intake and Interleukin Genetic Polymorphisms on Early Gastric Cancer Risk in Korea: A Case-Control Study.

PurposeThe current study investigated whether the combined effects of soy intake and genetic polymorphisms of interleukin (IL) genes modify gastric cancer risk.Materials and MethodsA total of 377 cases and 754 controls of Korean origin were included in the analysis. Soy consumption was assessed using a semi-quantitative food frequency questionnaire. Seven variants of IL10 (rs1800871), IL2 (rs2069763 and rs2069762), IL13 (rs6596090 and rs20541), and IL4R (rs7205663 and rs1805010) were genetically analyzed. To analyze the combined effect of soy intake and genetic polymorphisms, a low-intake group and high-intake group of each type of soy were categorized based on the intake level of the control group. Interactions between soy products and these genetic variants were analyzed by a likelihood ratio test, in which a multiplicative interaction term was added to the logistic regression model.ResultsA higher intake of nonfermented soy products was associated with a reduced cancer risk (odds ratio [OR], 0.62; 95% confidence interval [CI], 0.43 to 0.90), and the reduced risk was only apparent in males (OR, 0.44; 95% CI, 0.27 to 0.71). None of the IL genetic polymorphisms examined were independently associated with gastric cancer risk. Individuals with a minor allele of IL2 rs2069762 and a higher intake of nonfermented soy food had a decreased risk of gastric cancer (OR, 0.46; 95% CI, 0.31 to 0.68) compared to those with a lower intake (pinteraction=0.039).ConclusionBased on the genetic characteristics of the studied individuals, the interaction between IL2 rs2069762 and nonfermented soy intake may modify the risk of gastric cancer.

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines

ABSTRACTWe developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor–host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.

31P - Schedule optimization of a novel tumor-targeted IL-2 variant immunocytokine by integration of human in vivo immune cell kinetics and functional imaging

31P - Schedule optimization of a novel tumor-targeted IL-2 variant immunocytokine by integration of human in vivo immune cell kinetics and functional imaging

IL2 Variant Circumvents ICOS+ Regulatory T-cell Expansion and Promotes NK Cell Activation.

Clinical responses to high-dose IL2 therapy are limited due to selective expansion of CD4+CD25+Foxp3+ T-regulatory cells (Treg), especially ICOS+ Tregs, rather than natural killer (NK) cells and effector T cells. These ICOS+ Tregs are highly suppressive and constitutively express high levels of IL2Rα (CD25) and CD39. Here, we characterized the effect of a mutant form of IL2 (F42K), which preferentially binds to the lower affinity IL2Rβγ with reduced binding to CD25, on Tregs, effector NK cells, and T-cell subsets. Unlike wild-type (WT) IL2, F42K did not efficiently induce the expansion of highly suppressive ICOS+ Tregs in peripheral blood mononuclear cells (PBMC) from healthy controls and melanoma patients. Instead, it promoted the expansion of CD16+CD56+ NK cells and CD56hiCD16- NK cell subsets in both short- and long-term cultures, with enhanced Bcl-2 expression. Stimulation of PBMCs with F42K induced expression of more NK cell activation molecules, such as NKp30, NKp44, DNAM-1, NKG2D, 4-1BB/CD137, and Tim-3, than WT IL2. F42K induced greater upregulation of TRAIL, and NK-mediated cytolytic activity was increased against both autologous and HLA-mismatched melanoma cells compared with WT IL2. Gene expression analysis revealed distinct gene expression profiles stimulated by F42K, WT IL2, and IL15. F42K therapy in vivo also induced a dramatic reduction in the expansion of ICOS+ Tregs, promoted NK cell expansion, and inhibited melanoma tumor growth more efficiently than WT IL2 and more effectively than anti-CTLA-4. Our findings suggest that F42K could be a potential substitute for WT IL2 as a cytokine therapy for cancer. Cancer Immunol Res; 4(11); 983-94. ©2016 AACR.

358O - 89Zr-labeled CEA-targeted IL-2 variant immunocytokine in patients with solid tumors: CEA-mediated tumor accumulation in a dose-dependent manner and role of IL-2 receptor-binding

358O - 89Zr-labeled CEA-targeted IL-2 variant immunocytokine in patients with solid tumors: CEA-mediated tumor accumulation in a dose-dependent manner and role of IL-2 receptor-binding

Abstract 2217: Combining CEA-IL2v and FAP-IL2v immunocytokines with PD-L1 checkpoint blockade

Abstract Introduction: CEA-IL2v (RG7813, RO6895882) and FAP-IL2v (RG7461, RO6874281) are novel CEA-/FAP-targeted immunocytokines based on a novel IL2 variant (IL2v) with abolished CD25 binding that are currently in clinical phase 1 trials. PD-L1 is found on the surface of cells in various tumors and is induced by interferon gamma (IFNg). It prevents the immune system from destroying cancer cells by interacting with the inhibitory receptors PD-1 and B7.1 on T cells. Atezolizumab blocks the interaction of PD- L1 with its receptors and is currently being investigated in pivotal clinical trials for various cancers. Materials and Methods: PD-L1 expression on A549 tumor cells was analyzed by flow cytometry after treatment with CEA-IL2v alone or in the presence of PBMCs at varying E:T ratios. The combination of muCEA-IL2v, a murinized version of CEA-IL2v, with a muPD-L1 specific surrogate antibody was investigated in syngeneic stably CEA expressing PancO2 and MC38 cell lines in human CEA transgenic C57BL/6 mice. As control an untargeted DP47-muIL2v control immunocytokine was applied in the PancO2-CEA model. The combination of muFAP-IL2v, a murinized version of FAP-IL2v, with a muPD-L1 specific surrogate antibody was investigated in a syngeneic stably FAP expressing MC38 cell line in C57BL/6 mice. Results: In vitro, co-cultures of human PBMCs and the human A549 lung cancer cell line showed that CEA-IL2v induces the up-regulation of PD-L1 on A549 tumor cells in a concentration-dependent manner mediated through release of IFNg. In the syngeneic s.c. MC38-CEA model in CEA/hCD16 transgenic C57BL/6 mice, muCEA-muIL2v (0.5 mg/kg, q1wx2) and the muPD-L1 antibody (10 mg/kg, q1wx2) resulted in superior combined efficacy compared to the respective single agent therapies with respect to tumor growth inhibition. In the syngeneic orthotopic Panc02-CEA model in CEA transgenic C57BL/6 mice, muCEA-muIL2v (0.25 mg/kg, q1wx4) and the muPD-L1 antibody (10 mg/kg, q1wx4) resulted in superior median and overall survival model compared to the respective single agent therapies. Notably, the combination of muCEA-muIL2v was superior to the combination of the untargeted DP47-muIL2v control immunocytokine (exposure matched dose: 0.2 mg/kg, q1wx4) with the muPD-L1 antibody. Similarly, in the syngeneic s.c. MC38-FAP model the combination of muFAP-muIL2v (2 mg/kg, q1w4) and the muPD-L1 antibody (10 mg/kg, q1w4) resulted in superior combined efficacy compared to the respective single agent therapies in terms of tumor growth inhibition as well as median and overall survival. Conclusions: Taken together these nonclinical combination data support the clinical testing of combinations of CEA-IL2v and FAP-IL2v with atezolizumab. A clinical Phase 1b study combining CEA-IL2v and atezolizumab is currently ongoing (NCT02350673). Citation Format: Valeria Nicolini, Inja Waldhauer, Anne Freimoser-Grundschober, Stefan Evers, Jose Saro, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Combining CEA-IL2v and FAP-IL2v immunocytokines with PD-L1 checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2217.

The role of polymorphism of interleukin-2, -10, -13 and TNF-α genes in cutaneous T-cell lymphoma pathogenesis.

IntroductionAs the pathogenesis of cutaneous T-cell lymphomas (CTCL) is not fully understood, inherited gene polymorphisms are considered to play a role in the development of lymphomas.AimTo investigate whether certain gene polymorphisms might be involved in the development of CTCL.Material and methodsIn the case-control study we compared the frequency of nine selected single nucleotide polymorphisms (SNP) of seven genes (rs1800587/–889 C/T of interleukin (IL)-1α, rs2069762/–330G/T) and rs2069763/+166G/T of IL-2, rs1800925/–1112 C/T of IL-13, rs1800896/–1082 A/G of IL-10, rs4073/–251 A/T of IL-8, rs5370/K198N, rs180054/–1370T/G of endothelin-1 and rs1800629/–308 G/A of TNF-α) in 43 CTCL and Polish cases using the amplification refractory mutation system polymerase chain reaction method.ResultsWe have found that two genotypes, –330GG of IL-2 and –1112TT of IL-13 both promoter variants associated with “hypertranscription phenotype”, were over-represented in CTCL patients compared to healthy controls, and they increase the risk of malignancy development (OR = 5.82, p = 0.001 for IL-2 –330 GG, and OR = 5.67, p = 0.0024 for IL-13 –1112 TT). On the other hand, high transcription –308A allele of the TNF-α gene and –1082GG of IL-10 genotype is less frequent in lymphoma patients and has protective effects on the development of CTCL (OR = 0.45, p = 0.0466 for –308A of TNF-α, and OR = 0.35, p = 0.0329 for –1082GG of IL-10 genes).ConclusionsOur results indicate that hypertranscription promoter variants of IL-2 and IL-13 genes could be estimated as the risk factor for development of CTCL, while TNF-α and IL-10 variants have a protective effect.

Abstract IA07: Enhancing immune effector function via antibody engineering

Abstract Therapeutic antibodies of the IgG1 isotype have been established as important modality for cancer therapy in the clinic. They mediate their anti-tumoral efficacy largely by direct effects such as apoptosis/cell death induction or signaling inhibition, and by recruiting innate immune effector cells such as NK cells, macrophages and monocytes for antibody dependent cellular cytotoxicity and phagocytosis (ADCC, ADCP). Recently, methods to improve the immune effector function of therapeutic antibodies have been developed by enhancing FcgRIII binding including Fc-engineering and engineering the carbohydrate portion of the antibody. We have developed and applied a technology known as “glycoengineering” to improve the immune effector function of therapeutic antibodies. Obinutuzumab (GA101) is a glycoengineered Type II anti CD20 monoclonal antibody of the IgG1 isotype derived by humanization and elbow hinge optimization of the parental B-Ly1 mouse antibody that has recently been approved by the FDA and EMA for first line therapy of chronic lymphocytic leukemia (CLL) patients in combination with chlorambucil. In nonclinical studies obinutuzumab mediates enhanced direct (caspase independent) cell death induction with a concomitant reduction of complement dependent cytotoxicity compared to rituximab or ofatumumab by virtue of being a Type II CD20 antibody. As a consequence of glycoengineering the Fc region the core fucosylation of obinutuzumab is reduced and it has enhanced affinity for low and high affinity FcγRIIIa receptors resulting in enhanced NK cell mediated antibody dependent cellular cytotoxicity (ADCC). Recently, we demonstrated that glycoengineering not only enhances ADCC, but also results in enhanced antibody dependent cellular phagocytosis (ADCP) as compared to non-glycoengineered antibodies when physiological amounts of immunoglobulins are present. Furthermore, through enhanced affinity for FcγRIIIb that is expressed on neutrophils, obinutuzumab also mediates enhanced activation of neutrophils and neutrophil mediated phagocytosis. Finally, we have shown that in contrast to rituximab or non-glycoengineered obinutuzumab, ADCC mediated by obinutuzumab is largely unaffected by inhibitory KIR/HLA interactions. Thus, the modification of the Fc fragment of obinutuzumab appears as an effective strategy to enhance ADCC and ADCP by NK cells, macrophages/monocytes and neutrophils independent of inhibitory KIR expression. In whole blood assays derived from healthy donors and chronic lymphocytic leukemia (CLL) patients these properties result in superior depletion of normal and malignant B cells in terms of potency and absolute B cell depletion as compared to rituximab. In order to further enhance the efficacy of ADCC competent therapeutic antibodies we have developed a novel class of tumor-targeted IL2v immunocytokines with the aim to activate and induce the proliferation of innate and adaptive immune effector cells in the tumor. In the FAP-IL2v immunocytokine an IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of one of the Fc chains of a tumor stroma-targeted FAP antibody. FAP-IL2v lacks binding to CD25, but retains IL-2Rßγ binding so that is no longer preferentially activates Treg cells and reduces sensitivity for Fas-mediated apoptosis as compared to wildtype IL-2-based FAP-IL2wt immunocytokine. In C56BL/6 mice, FAP-IL2v strongly expands and activates NK cells and CD8+ T cells, and skews the CD4:CD8 ratio towards CD8+ T cells. In vitro, FAP-IL2v induces the proliferation and activation of NK cells and enhances the cytotoxic activity of NK cells in combination with ADCC competent antibodies. Similarily, in xenograft (hCD16 transgenic SCID mice) and BALB-neuT (immunocompetent ratHER2 transgenic mice) models, FAP-IL2v strongly enhances the antitumoral efficacy and survival mediated by HER2 antibodies making it a promising combination partner for ADCC-competent antibodies in future clinical investigation. Citation Format: Christian Klein. Enhancing immune effector function via antibody engineering. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA07.

CEA-targeted engineered IL2: Clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation.

3016 Background: CEA-IL2v (RG7813) is an engineered IL-2 variant (IL-2v) with abolished IL-2Rα (CD25) binding fused to an antibody targeting Carcinoembryonic Antigen (CEA). The molecule was designed to improve the pharmacological and safety profile of IL-2 and enable tumor targeting. Methods: In an ongoing Phase I trial of CEA-IL2v (ranges 0.1 – 40 mg flat dose IV; schedule q1W and q2W) in patients (pts) with advanced and/or metastatic solid CEA+ tumors, extensive pharmacodynamic monitoring in peripheral blood as well as immunohistochemical (IHC) and flow cytometric (FC) analysis of pre- and on-treatment biopsy specimens (day + 4) were performed, including tests for effector (CD8+) and helper T cell (CD4+) percentage and density. In a sub-study, biodistribution was analyzed using PET imaging of 89Zr-CEA-IL2v at doses of 6 and 30 mg. Results: Of the 11 pts treated at 30 mg, 4 had sequential tumor biopsies evaluable both by IHC and FC. An increase in the median density (IHC) of CD8+ T cells from 62.5 (7 - 90) [median (range)] to 104.2 (7 - 255) cells/ mm2 was observed in 3/4 subjects after the 1st dose. No relevant change in the density of CD4+ T cells was detected. FC analysis confirmed the increase in CD8:CD4 ratio in the same 3/4 patients: from 0.88 (0.57 - 1.24) to 4.54 (0.36 - 7.47). Effects in peripheral blood included strong lymphocyte expansion that was mostly driven by Natural Killer (NK) and CD8+ T cells. Medians and ranges for the 11 pts before and 4 days after dosing were: NK: 168 (range: 52 - 337) vs. 655 (206 - 1569) cells/µl; CD8+: 340 (19 - 464) vs. 831 (78 – 3754) cells/µl. While there was no clear evidence of CEA-mediated tumor accumulation at 6 mg in the first 6 pts, intra-tumoral accumulation of 89Zr-CEA-IL2v was observed in 2/2 pts at 30 mg. Additional studies are ongoing. Conclusions: Collectively, these data support the concept that reduced affinity of CEA-IL2v for IL2R-alpha together with high-affinity binding to CEA enables tumor targeting. These findings suggest that CEA-IL2v is the first tumor-targeted cytokine, which increases immune infiltration and activates NK and T cells both in the periphery and within tumors. Clinical trial information: NCT02004106.

CD8+ T Cell Fate and Function Influenced by Antigen-Specific Virus-Like Nanoparticles Co-Expressing Membrane Tethered IL-2.

A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as ‘natural adjuvants’. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8+ T cell responses in vitro and in vivo. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8+ T cell effector functions in vitro and in vivo. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8+ T lymphocytes.

Predictive model for risk of severe gastrointestinal toxicity following chemotherapy using patient immune genetics and type of cancer: a pilot study.

Severe chemotherapy-induced gastrointestinal toxicity (CIGT) is common and results in treatment delays, dose reductions, and potential premature treatment discontinuation. Currently, there is no diagnostic marker to predict CIGT. Proinflammatory cytokines, produced via toll-like receptor signaling, are key mediators of this toxicity. Hence, this pilot study investigated the association between immune genetic variability and severe CIGT risk. Genomic DNA from 34 patients (10 with severe CIGT) who had received 5-fluoruracil-based chemotherapy regimens was analyzed for variants of IL-1B, IL-2, IL-6, IL-6R, IL-10, TNF-a, TGF-b, TLR2, TLR4, MD2, MYD88, BDNF, CRP, ICE, and OPRM1. Multivariate logistic regression created a prediction model of severe CIGT risk. There were no significant differences between patients with and without severe CIGT with regards to age, sex, type of cancer, or chemotherapy treatment regimens. The prediction model of severe CIGT risk included TLR2 and TNF-a genetic variability and cancer type (colorectal and gastric). This prediction model was both specific and sensitive, with a receiver operator characteristic area under the curve of 87.3%. This is the first report of immune genetic variability, together with cancer type, being predictive of severe CIGT risk. These outcomes are being validated in a larger patient population.

Abstract 2579: Combination with the novel tumor-targeted CEA-IL2v immunocytokine enhances the activity of ADCC-competent and glycoengineered antibodies in vitro and in vivo

Abstract We recently described a novel monomeric tumor-targeted CEA-IL2v immunocytokine. CEA-IL2v comprises a single IL-2 variant (IL2v) with abolished CD25 binding that is fused to the C-terminus of a humanized CEA antibody with a heterodimeric Fc devoid of FcγR and C1q binding. CEA-IL2v recognizes a membrane-proximal epitope of CEA. Here we show that CEA-IL-2v is able to enhance the activity of ADCC competent antibodies and glycoengineered IgG1 antibodies in vitro and in vivo. The combination of CEA-IL2v with ADCC-competent IgG1 antibodies was investigated in vitro in ADCC assays and in vivo in xenografts in hCD16a transgenic SCID mice that express hFcγRIIIα on NK cells and the homologous muFcγRIV on macrophages/monocytes. Antibodies tested include trastuzumab, cetuximab and the ADCC-enhanced glycoengineered EGFR antibody GA201. In the spontaneous genetically engineered BALB-neuT breast cancer mouse model a surrogate tumor stroma Tenascin C A2 (TNCA2)-targeted IL2v immunocytokine was used in combination with a ratHER2 antibody. PBMCs stimulated by CEA-IL2v in co-cultures with tumor cells showed dose-dependent elimination of tumor cells and activation of NK cells. Addition of trastuzumab or cetuximab resulted in induction of activation markers and enhanced killing of tumor cells. As a single agent, CEA-IL2v showed minor tumor growth inhibition in orthotopic i.m.f.p. KPL4 BC and s.c. N87 GC xenografts. The combination of CEA-IL2v (1 mg/kg, q7d×3) with trastuzumab (10 mg/kg in KPL4 and 25 mg/kg in N87, q7d×3) resulted in superior inhibition of tumor growth compared to the respective single-agent treatments, combination therapy induced 6/9 complete tumor remissions in N87 and 3/5 in KPL4. In the i.v. A549 lung cancer and i.s. LS174T CRC xenografts, single-agent CEA-IL2v showed a moderate effect on median survival, whereas the combination of CEA-IL2v (1 mg/kg, q7d×3) with cetuximab (25 mg/kg, q7d×3) increased median/overall survival. Similarly, in the i.v. A549 NSCLC and i.s. LS174T CRC xenografts, the combination of CEA-IL2v (1 mg/kg for A549 and 2 mg/kg for LS174T, q7d×3) with GA201 (25 mg/kg, q7d×3) resulted in superior median survival compared to the respective single-agent treatments with long term survival of 10/10 animals in the A549 model and survival of 2/8 animals in the LS174T model. Finally in the spontaneous GEM BALB-neuT breast cancer model the combination of TNCA2-IL2v with a ratHER2 IgG1 antibody resulted in a strong and significant reduction of tumor formation in 6/7 animals as compared to the respective single agents. Combining CEA-IL2v or TNCA2-IL2v with ADCC competent/enhanced IgG1 antibodies results in enhanced anti-tumor efficacy in xenografts and in a spontaneous immunocompetent genetically engineered BALB-neuT mouse model. These data support the clinical investigation of CEA-IL2v with ADCC competent/enhanced IgG1 antibodies. Citation Format: Valeria Nicolini, Inja Waldhauer, Anne Freimoser, Sara Colombetti, Federica Cavallo, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Combination with the novel tumor-targeted CEA-IL2v immunocytokine enhances the activity of ADCC-competent and glycoengineered antibodies in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2014-2579

Abstract 2592: Tumor targeting and pharmacodynamics of the novel targeted immunocytokine FAP-IL2v in a tumor-bearing Rhesus monkey

Abstract FAP-IL2v is a novel monomeric FAP-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Ralpha (CD25) binding is fused to the C-terminus of an high affinity anti-FAP antibody (1). FAP-IL2v was designed to selectively bind to the tumor thereby avoiding the systemic toxicity inherent to systemic administration of cytokines. The goal of this study was to assess the tumor targeting and pharmacodynamic properties of FAP-IL2v in an appropriate tumor bearing animal model. Explanted tumor xenografts in mice do not have high expression of FAP and lack the complete immune system which is important to assess the safety, efficacy, and biodistribution of these targeted immunocytokines. Therefore, we elected to use monkeys with spontaneous tumors for this study. Herein, we report a first of kind study in monkeys with spontaneous tumors to better understand tumor targeting and pharmacodynamics of FAP-IL2v. Three monkeys with a history of cancer and suspected relapse were identified. Immunohistochemistry was done on archival tumor tissue to assess target (FAP) expression and FDG-PET performed to detect metastatic lesions. Based on the positive FAP expression on tumor tissue and detectable tumor lesions and lesion locations from FDG-PET scans, a rhesus monkey with metastatic breast cancer was selected for further experimentation with FAP-IL2v. The monkey was administered with 0.5 mg/kg of FAP-IL2v mixed with tracer amounts of 89Zr labeled FAP-IL2v for assessment of tumor targeting using PET imaging. Baseline and on-treatment blood samples and tumor biopsies were collected after single-administration for assessment of pharmacodynamic effects. The study revealed that FAP-IL2v selectively targets and accumulates in the tumor lesions with minimal uptake in the lungs, spleen and lymphoid tissues (major sink organs and potential risk factors). All three FDG-avid lesions showed accumulation of FAP-IL2v over 6 days with peak uptake of SUV= 3.1 at day 3. After single administration, transient lymphocytosis and peripheral expansion of immune cells were observed. Analyses of tumor biopsies suggested increased immune cell infiltration. No signs of distress or cytokine release were observed during the treatment period. In conclusion, using a monkey with spontaneous tumors, we demonstrated that the FAP-IL2v targeted immunocytokine can selectively be delivered to tumor and induce intended changes in immune microenvironment without major adverse events. (1) C. Klein et al., Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer, AACR 2013, Poster 486 Citation Format: Stefan Evers, Pradeep K. Garg, J. Mark Cline, Sudha Garg, Greg Dugan, Anne Freimoser-Grundschober, Natalie D. Keirstead, Christian Klein, Tapan Nayak. Tumor targeting and pharmacodynamics of the novel targeted immunocytokine FAP-IL2v in a tumor-bearing Rhesus monkey. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2592. doi:10.1158/1538-7445.AM2014-2592

The IL-2 cytokine family in cancer immunotherapy

The use of cytokines from the IL-2 family (also called the common γ chain cytokine family) such as interleukin (IL)-2, IL-7, IL-15, and IL-21 to activate the immune system of cancer patients is one of the most important areas of current cancer immunotherapy research. The infusion of IL-2 at low or high doses for multiple cycles in patients with metastatic melanoma and renal cell carcinoma was the first successful immunotherapy for cancer proving that the immune system could completely eradicate tumor cells under certain conditions. The initial clinical success observed in some IL-2-treated patients encouraged further efforts focused on developing and improving the application of other IL-2 family cytokines (IL-4, IL-7, IL-9, IL-15, and IL-21) that have unique biological effects playing important roles in the development, proliferation, and function of specific subsets of lymphocytes at different stages of differentiation with some overlapping effects with IL-2. IL-7, IL-15, and IL-21, as well as mutant forms or variants of IL-2, are now also being actively pursued in the clinic with some measured early successes. In this review, we summarize the current knowledge on the biology of the IL-2 cytokine family focusing on IL-2, IL-15 and IL-21. We discuss the similarities and differences between the signaling pathways mediated by these cytokines and their immunomodulatory effects on different subsets of immune cells. Current clinical application of IL-2, IL-15 and IL-21 either as single agents or in combination with other biological agents and the limitation and potential drawbacks of these cytokines for cancer immunotherapy are also described. Lastly, we discuss the future direction of research on these cytokines, such as the development of new cytokine mutants and variants for improving cytokine-based immunotherapy through differential binding to specific receptor subunits.

S41. Novel CEA-targeted IL2 variant immunocytokine for immunotherapy of cancer

Meeting abstracts Here we describe a novel class of monomeric tumor-targeted immunocytokines that comprise a single IL-2 variant (IL2v) with abolished CD25 binding that is fused to the C-terminus of a tumor specific antibody with a heterodimeric Fc devoid of FcγR and C1q binding. For tumor

Novel Tumor-Targeted, Engineered IL-2 Variant (IL2v)-Based Immunocytokines For Immunotherapy Of Cancer

IntroductionIL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the known IL-2-based immunocytokines has yet progressed to pivotal clinical trials due to various constraints in their design; in particular, the fusion of two wild-type IL-2 moieties to the antibody and retained FcgR binding of IgG-based immunocytokines. This design results in 1) high affinity binding with pM affinity to IL-2Raβγ on immune cells compromising tumor targeting and inducing rapid systemic clearance and short half-life; 2) high affinity for CD25 (IL-2Ra) expressed on pulmonary vascular endothelium contributing to pulmonary toxicity; and 3) preferential activation of Tregs over immune effectors. Here we describe a novel class of monomeric tumor-targeted immunocytokines where a single, engineered IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For tumor targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA-IL2v recognizes a membrane proximal epitope of human carcinoembryonic antigen (CEA) and binds preferentially to membrane-bound CEA, but not shed CEA. MethodsCEA- and FAP-IL2v were produced as recombinant proteins and their activity tested oneffector cells by assessing the activation of P-STAT5, cell proliferation, sensitivity to Fas-induced apoptosis, expression of activation markers and cytokine release upon treatment. Safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were analyzed in SCID and fully immunocompetent C57Bl/6 mice as single agent and in combination with trastuzumab and cetuximab. Tumor targeting was investigated in the orthotopic syngeneic Renca renal cell cancer tumor model in Balb/c mice by SPECT imaging. ResultsFAP- and CEA-IL2v completely lack binding to CD25, but retain IL-Rβγ binding, and show pM binding affinity to respective antigens, FAP on fibroblasts and CEA on tumor cells. As consequence of abolished binding to CD25 these molecules do not preferentially activate Tregs. The treatment of effector cells with IL2v reduces their sensitivity for Fas-mediated apoptosis (also known as activation induced cell death) as compared to wild-type IL-2 based immunocytokine. IL-2Rβγ bioactivity was retained and FAP- and CEA-IL2v activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers, cell proliferation and cytokine release. Furthermore, CEA-IL2v and FAP-IL2v enhanced the cytotoxic activity of NK cells when combined with ADCC-competent antibodies. Mechanism of action studies in fully immunocompetent mice showed that the molecules strongly expand and activate NK, CD8+ T cells and gd T cells (up to 100-fold) and skew the CD4:CD8 ratio strongly towards CD8+ T cells in the peripheral blood, lymphoid tissues, and in the tumor. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite a higher exposure and circulatory half-life than the analogous IL-2 based immunocytokine. MicroSPECT/CT imaging with radioactively labeled FAP-IL2v revealed good FAP-mediated tumor targeting in the orthotopic syngeneic Renca model with low normal tissue uptake and low accumulation in lymphoid tissues, contrary to analogous IL-2 based immunocytokine that showed preferential targeting to lymphoid tissue. Studies in tumor-bearing mice showed dose-dependent anti-tumor efficacy of FAP-IL2v and CEA-IL2v in syngeneic models. Additional studies in xenograft models in SCID mice transgenic for human CD16A showed that CEA-IL2v strongly enhances the antitumor efficacy and/or survival mediated by ADCC-competentantibodies, including trastuzumab and cetuximab. ConclusionCEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 binding, monovalency and high-affinity tumor-targeting as compared to classical IL-2-based immunocytokines. They retain capacity to activate and expand NK and CD8+ effector T cells through IL-2Rβγ in the periphery and the tumor microenvironment. These data support their further nonclinical and clinical investigation for immunotherapy of cancer. Clinical trials with CEA-IL2v are foreseen in 2014. Disclosures:Klein:Roche Glycart AG: Employment. Waldhauer:Roche: Employment. Nicolini:Roche: Employment. Dunn:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Danny:Roche: Research Funding. Boerman:Roche: Research Funding. Nayak:Roche: Employment. Herter:Roche: Employment. Van Puijenbroek:Roche: Employment. Ast:Roche: Employment. Hofer:Roche: Employment. Hosse:Roche: Employment. Lang:Roche: Employment. Neumann:Roche: Employment. Kettenberger:Roche: Employment. Neubauer:Roche: Employment. Gorr:Roche: Employment. Tuerck:Roche: Employment. Evers:Roche: Employment. Gerdes:Roche: Employment. Levitsky:Roche: Employment. Bacac:Roche: Employment. Moessner:Roche: Employment. Umana:Roche: Employment, Equity Ownership.

Immunotherapeutic strategies for relapse control in acute myeloid leukemia

Despite that the initial phases of chemotherapy induce disappearance of leukemic cells in many patients with acute myeloid leukemia (AML), the prevention of life-threatening relapses in the post-remission phase remains a significant clinical challenge. Allogeneic bone marrow transplantation, which is available for a minority of patients, efficiently prevents recurrences of leukemia by inducing immune-mediated elimination of leukemic cells, and over the past decades, numerous immunotherapeutic protocols have been developed aiming to mimic the graft-versus-leukemia reaction for the prevention of relapse. Here we review past and present strategies for relapse control with focus on overcoming leukemia-related immunosuppression in AML. We envisage future treatment protocols, in which systemic immune activators, such as vaccines, dendritic cell-based therapies, engineered variants of IL-2, or IL-15, are combined with agents that counter immunosuppression mediated by, e.g., the PD/PDL interaction, CTLA-4, CD200, reactive oxygen species, IDO expression, CXCR4, or the KIR/class I interaction, based on characteristics of the prevailing malignant clone. This combinatorial approach may pave the way for individualized immunotherapy in AML.