Oliver Ast

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

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

ABSTRACT We 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.

Abstract 486: Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer.

IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the described IL-2-based immunocytokines has progressed beyond Phase II trials due to various constraints in their design: 1) pM affinity for IL-2Rαβγ on immune cells and pulmonary vascular endothelium compromising tumor targeting due to the fusion of two wildtype IL-2 moieties to the antibody, together with FcγR binding on the same cells; 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rαβγ binding; 3) Preferential activation of Tregs over immune effectors by wt IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Rα (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 targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA- and FAP-IL2v were recombinantly produced and induction of P-STAT5, proliferation, activation induced cell death (AICD), activation markers and cytokines were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting, pharmacodynamics and anti-tumor efficacy were analyzed in SCID and immunocompetent C57Bl/6 mice. FAP- and CEA-IL-2v completely lack binding to CD25, but retain IL-Rβγ binding. They do not bind to CD25 or preferentially activate Tregs, and induce lower degree of AICD. However, IL-2Rβγ bioactivity is retained and they activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers and proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards CD8+ T cells in vivo. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite of higher exposure and circulatory half-life than the corresponding wt IL-2 immunocytokine. MicroSPECT/CT imaging revealed FAP-mediated tumor targeting of FAP-IL2v with low normal tissue uptake with FAP-IL2v tumor targeting being similar to the parental FAP antibody with low accumulation in lymphoid tissues and clearly superior to an FAP-targeted wt IL-2 immunocytokine that shows preferential spleen targeting. Studies in tumor-bearing mice showed dose dependent anti-tumor efficacy of CEA- and FAP-IL2v in established xenograft and syngeneic mouse models. Thus, CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcγR binding, monovalency and high-affinity tumor-targeting as compared to classical immunocytokines. They retain capacity to activate NK and T‐effector cells through IL‐2Rβγ; in particular once targeted to the tumor microenvironment. These data support their investigation for the immunotherapy of CEA/FAP-positive tumors. Citation Format: Christian Klein, Inja Waldhauer, Valeria Nicolini, Claire Dunn, Anne Freimoser-Grundschober, Sylvia Herter, Edwin Geven, Otto Boerman, Tapan Nayak, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Peter Bruenker, Ralf Hosse, Sabine Lang, Sebastian Neumann, Hubert Kettenberger, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Pablo Umana. Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 486. doi:10.1158/1538-7445.AM2013-486

Abstract 2481: CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors

T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumor cells and T cells trigger strong T cell activation resulting in the killing of tumor cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumors (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumor antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumor and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro, CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumor cells with high expression of CEA. In vivo, CEA TCB induces dose- and time-dependent regression of CEA-expressing tumors with variable amounts of immune cell infiltrate. In fully humanized NOG mice, CEA TCB is efficacious in poorly-infiltrated tumors and converts non-inflamed into highly-inflamed tumors. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumors and/or expands pre-existing ones and is able to induce T cell re-localization from the tumor periphery into the tumor bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoral T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumor-targeted T cell bispecific antibody with promising anti-tumor activity and the novel ability to modify the tumor microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Johannes Sam, Valeria Nicolini, Nathalie Steinhoff, Oliver Ast, Peter Bruenker, Ralf Hosse, Thomas Hofer, Ekkehard Moessner, Christiane Jaeger, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2481. doi:10.1158/1538-7445.AM2015-2481

Abstract 3629: Engineering a novel asymmetric head-to-tail 2+1 T-cell bispecific (2+1 TCB) IgG antibody platform with superior T-cell killing compared to 1+1 asymmetric TCBs

T cell bispecific antibodies that recruit and engage T cells for tumor cell killing through binding to the T cell receptor (TCR) upon binding to a tumor antigen (TA) and subsequent crosslinking have attracted broad interest. Here, we describe a novel asymmetric head-to-tail 2+1 T cell bispecific antibody (2+1 TCB) platform characterized by the fusion of a flexible Fab fragment to the N-terminus of the CD3e Fab of a heterodimeric asymmetric bispecific TA-CD3e IgG1 antibody in head-to-tail geometry via a flexible linker. The resulting TCB is monovalent for CD3e (KD 70-100 nM) and binds bivalently with avidity to the TA on the target cell. Correct heavy chain pairing is enabled by knob-into-holes technology, correct light chain pairing by CrossMAb technology or using a common light chain. This enables production with standard processes in CHO cells. To exclude FcgR-mediated unspecific TCR and FcgR co-activation resulting in unspecific cytokine release, Fc- effector functions (ADCC, ADCP, CDC) are abolished by introduction of P329G LALA mutations while FcRn binding and IgG-like pharmacokinetic properties are retained as shown in mouse and Cynomolgus. For comparative profiling, the following TCBs were generated with specificity for the tumor antigens MCSP/CSPG4, FOLR1/FRalpha, CD19 and CD20: 2+1 TCBCD3-inside, 2+1 TCBCD3-outside, one-armed 1+1 TCBCD3-inside and a classical asymmetric 1+1 IgG TCB. In vitro Jurkat-NFAT, T cell killing, activation and proliferation assays show that both 2+1 TCB formats mediate superior potency of killing (for CSPG4, FOLR1, CD19, CD20) and superior absolute killing (for CSPG4, CD19) compared to the respective classical asymmetric 1+1 IgG TCB. Surprisingly, the 2+1 TCBCD3-inside format was found to be superior in potency compared to the 2+1 TCBCD3-outside format, although its binding affinity for CD3e is reduced. These data confirm that TCBs mediate extremely potent T cell killing with fM-pM EC50 values based on CD3e antibodies with affinities of only 70-100 nM. Notably, for CD19 both, 2+1 TCBCD3-inside and one-armed 1+1 TCBCD3-inside, mediate comparable potency and overall killing, and both were superior compared to the asymmetric 1+1 IgG TCB. These data underline the importance of the head-to-tail geometry with two Fabs on one arm attached to each other via a flexible G4S-linker. Finally, using 2+1 and 1+1 FOLR1 TCBs we demonstrate that bivalent binding allows better differentiation in killing of cells with high vs. low FOLR1 expression as compared to monovalent binding. Taken together, we demonstrate that the 2+1 TCBCD3-inside is the most potent, efficacious and versatile TCB design. Due to its orientation with the CD3e Fab inside, it allows the conversion of existing antibodies into potent TCBs without format restriction. Based on this platform, CEA CD3 TCB (RG7802, Phase I/Ib) and CD20 CD3 TCB (RG6026, Phase I) have entered clinical trials. Citation Format: Christian Klein, Christiane Neumann, Tanja Fauti, Tina Weinzierl, Anne Freimoser-Grundschober, Inja Waldhauer, Linda Fahrni, Sylvia Herter, Erwin van Puijenbroek, Sara Colombetti, Johannes Sam, Sabine Lang, Sherri Dudal, Wolfgang Schafer, Jorg T. Regula, Samuel Moser, Oliver Ast, Ralf Hosse, Ekkehard Mossner, Peter Brunker, Marina Bacac, Pablo Umana. Engineering a novel asymmetric head-to-tail 2+1 T-cell bispecific (2+1 TCB) IgG antibody platform with superior T-cell killing compared to 1+1 asymmetric TCBs [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 3629. doi:10.1158/1538-7445.AM2017-3629

Abstract PR8: Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer.

Introduction: IL-2 therapy can lead to durable responses in a modest proportion of cancer patients, but the treatment is associated with significant toxicity. Over the last decades, various IL-2-based immunocytokines have been generated by fusing IL-2 to tumor-targeting antibodies. However, none of these molecules have progressed beyond Phase II trials and they are hampered by various liabilities: 1) High functional affinity (low pM) for IL-2Rabg on immune cells and on pulmonary vascular endothelium (Krieg et al., PNAS, 2010) compromising preferential tumor targeting due to fusion of two IL-2 moieties to the antibody. This is further compounded when the immunocytokine binds to FcgRs on the same cells. 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rabg binding. 3) Preferential activation of Tregs over immune effectors due to use of wildtype IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Ra (CD25) binding is fused to the C-terminus of a tumor-specific hIgG1 antibody with a heterodimeric Fc-part. FcgR and C1q binding is completely abolished by a novel Fc mutation. For targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were chosen. Experimental procedures: CEA- and FAP-IL2v were recombinantly produced and characterized by surface plasmon resonance. Induction of P-STAT5, proliferation, activation induced cell death (AICD), various activation markers and cytokine release were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting by imaging, immune-pharmacodynamics and anti-tumor efficacy were analyzed in immunocompromised Scid and immunocompetent C57BL/6 mice. Results: IL-2v completely lacks binding to CD25, but retains IL-Rbg binding. In line with this, FAP- and CEA-IL2v do not bind to CD25 or preferentially activate Tregs, and do not cause AICD. However, IL-2Rbg bioactivity is retained and they are still able to activate NK, CD4 and CD8 T cells as shown by concentration dependent increase in activation markers and induction of proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards activated CD8 T cells in vivo. In C57BL/6 mice CEA- and FAP-IL2v demonstrate improved safety despite of ca. 2-fold higher exposure and t1/2 than a wildtype IL-2-based IgG immunocytokine. SPECT/CT imaging revealed FAP-mediated tumor targeting and accumulation of FAP-IL2v with low normal tissue uptake. Notably, FAP-IL2v tumor targeting was similar to the parental FAP antibody with low accumulation in lymphoid tissues; clearly superior to an FAP-targeted wt IL-2 immunocytokine that showed preferential homing to the spleen. Studies in tumor bearing mice showed dose dependent efficacy of CEA- and FAP-IL2v in established xenograft and immunocompetent syngeneic mouse models in terms of survival. Conclusion: CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcgR binding, monovalency and high-affinity tumor-targeting as compared to conventional immunocytokines. They retain the capacity to activate NK and T effector cells through IL 2Rbg; in particular once targeted and immobilized in the tumor microenvironment. These preclinical properties support further investigation for the immunotherapy of CEA/FAP-positive tumors. This abstract is also presented as Poster A23. Citation Format: Christian Klein, Waldhauer Inja, Valeria Nicolini, Dunn Claire, Anne Freimoser, Anne Freimoser, Sylvia Herter, Edwin Geven, Otto Boerman, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Ralf Hosse, Sabine Lang, Sebastian Neumann, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Ekkehard Moessner, Pablo Umana. Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR8.

p95HER2–T cell bispecific antibody for breast cancer treatment

T cell bispecific antibodies against an isoform of HER2 effectively target many HER2-expressing tumors but not normal tissues. Fine-tuning HER2 targeting HER2 receptor tyrosine kinase is frequently overexpressed in breast and gastric cancer. HER2-overexpressing tumors can be treated with trastuzumab, an antibody against this receptor, and additional methods of targeting HER2 are also being developed. Unfortunately, HER2 is also expressed in normal tissues, resulting in unacceptable toxicities when HER2-targeting therapies damage healthy organs. Rius Ruiz et al. now propose targeting p95HER2, a carboxyl-terminal fragment of HER2 that is expressed in almost half of HER2-positive tumors. The authors demonstrate the effectiveness of this approach, as well as its safety due to the lack of p95HER2 expression in nontumor tissues. T cell bispecific antibodies (TCBs) are engineered molecules that include, within a single entity, binding sites to the T cell receptor and to tumor-associated or tumor-specific antigens. The receptor tyrosine kinase HER2 is a tumor-associated antigen in ~25% of breast cancers. TCBs targeting HER2 may result in severe toxicities, likely due to the expression of HER2 in normal epithelia. About 40% of HER2-positive tumors express p95HER2, a carboxyl-terminal fragment of HER2. Using specific antibodies, here, we show that p95HER2 is not expressed in normal tissues. We describe the development of p95HER2-TCB and show that it has a potent antitumor effect on p95HER2-expressing breast primary cancers and brain lesions. In contrast with a TCB targeting HER2, p95HER2-TCB has no effect on nontransformed cells that do not overexpress HER2. These data pave the way for the safe treatment of a subgroup of HER2-positive tumors by targeting a tumor-specific antigen.