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Supplementary Figure from A Novel EGFRvIII T-Cell Bispecific Antibody for the Treatment of Glioblastoma

Supplementary Methods and Figure Legends.

Purpose: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ϵ upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts.Experimental Design: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB.Results: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell–dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells.Conclusions: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB–mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417–27. ©2016 AACR.See related commentary by Teijeira et al., p. 4277

In vitro live cell confocal imaging of LS174T RFP cells co-cultured with CFSE-labeled T-cells (E:T = 3:1) in the presence of CEA or a control TCB, respectively.

Supplementary Figure S1. CEA TCB leads stimulates the expression of CD25, a T-cell activation marker, on both T-helper (CD4+) and cytotoxic T-cells (CD8+) after incubation with LS174T tumor cells. Supplementary Figure S2. CEA TCB induces efficient MKN45 tumor cell lysis and T-cell activation in the presence of these tumor cells. Supplementary Figure S3. Both CD4+ and CD8+ T-cells contribute to CEA-TCB mediated killing. Supplementary Figure S4. Ex vivo analysis of in vivo tumor targeting. Supplementary Figure S5. Analysis of T-cell migratory persistence index.

2P time-lapse acquisition of LS174T / human PBMC cografts on day 4 after implantation (baseline), 24 h after control and 24 h after CEA TCB treatment. LS174T RFP tumor cells (red), CFSE labeled T-cells (green). Scale bar indicates 50 μm.

Ex vivo analysis of the CEA TCB (magenta) mediated cross-linking of CFSE labeled T-cells (green) to tumor cells. 3D reconstructions of confocal z-stacks acquired on tumor sections obtained from huPBMC (CFSE labeled T-cells)/LS174T RFP co-grafts, 24 h after intravenous A647 labeled CEA TCB injections are shown. Bar, 10 μm. Cell nuclei (DAPI) are shown in blue.

TYRP1-TCB is a CD3 T-cell bispecific (CD3-TCB) antibody for the treatment of advanced melanoma. A tumor growth inhibition (TGI) model was developed using mouse xenograft data with TYRP1-TCB monotherapy or TYRP1-TCB plus anti-PD-L1 combination. The model was translated to humans to inform a refined clinical strategy. From xenograft mouse data, we estimated an EC

T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2–7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rβγ > IL-2 Rβγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rβγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2-10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

Purpose: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ϵ upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts. Experimental Design: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB. Results: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell–dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells. Conclusions: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB–mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417–27. ©2016 AACR. See related commentary by Teijeira et al., p. 4277

In the past decade, many checkpoint inhibitors (CPI) have been approved for melanoma, demonstrating the increasing potential of immunotherapy as a lifesaving treatment for this devastating disease. However, many patients relapse or need to discontinue treatment due to adverse effects. Patients failing CPI have very limited treatment options and very poor outcomes. This supports the need for novel and more effective immunotherapies, including combination approaches. Bispecific antibodies like blinatumomab have been approved for hematologic malignancies but positive benefit in solid tumors has been more challenging to demonstrate. Given the relative high T cell content in melanoma, we considered this indication highly promising to investigate whether synthetic immunity also works in solid tumors. Here we present a novel potent treatment approach based on an off the shelf T cell engaging bispecific antibody directed against a melanoma associated target. The tyrosinase-related protein 1 antigen (TYRP1), an enzyme expressed in normal melanosomes and involved in melanin synthesis, is overexpressed in more than 50% of metastatic melanoma patients. TYRP1-TCB is a new 2+1 format T cell bispecific antibody targeting TYRP1 on tumor cells and CD3 on T cells. CD3 bispecific molecules do not depend on HLA expression or antigen presentation and could therefore overcome immune evasion mechanisms in patients failing CPI; as such, T cell redirection via TYRP1-TCB has the potential to be the foundation for new and improved CIT combinations. In vitro TYRP1-TCB mediated strong killing of patient derived melanoma cells from a dermatology cell bank as observed by LDH release, T cell activation and cytokine secretion with EC50 ranging from 30-1500 pM. Pre-clinical investigation in vivo with a murine surrogate molecule showed potent efficacy in subcutaneous as well as aggressive lung metastatic B16 melanoma mouse models that do not respond to anti-PD1 or anti-PD-L1 therapy. Furthermore, the combination with immunomodulatory agents like FAP-IL2v and FAP-4-1BBL molecules demonstrated enhanced efficacy as compared to the respective single agents. These preclinical data support the clinical evaluation of TYRP1 targeted CD3 bispecific antibody as a single agent and in combination in metastatic relapsed/refractory melanoma patients. Citation Format: Valeria G. Nicolini, Inja Waldhauer, Anne Freimoser-Grundschober, Marine Richard, Linda Fahrni, Esther Bommer, Christina Claus, Johannes Sam, Sara Colombetti, Roger Sutmuller, Mitchell P. Levesque, Anja Irmisch, Conrad Bleul, Neil Campbell, Marina Bacac, Ekkehard Moessner, Christian Klein, Pablo Umana. Combination of TYRP1-TCB, a novel T cell bispecific antibody for the treatment of melanoma, with immunomodulatory agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-389.

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., Cancer Immunology, Immunotherapy, 66 (1), ISSN:0340-7004, ISSN:1432-0851

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 Schäfer, Jörg T. Regula, Samuel Moser, Oliver Ast, Ralf Hosse, Ekkehard Mössner, Peter Brünker, 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

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

Recent results from clinical trials have shown that immune therapies, particularly immune checkpoint inhibitors, can extend the overall survival of cancer patients and lead to durable responses. Despite these promising results, current immune-based therapies are only effective in a proportion of patients and combination strategies are needed to improve therapeutic benefit. Programmed death-ligand 1(PD-L1) is found on the surface of immune and tumor cells and its expression is induced by interferon gamma (IFNg). It prevents the immune system from destroying cancer cells by interacting with the inhibitory programmed death-1 (PD-1) and B7.1 receptors on activated T cells, which results in a T-cell inhibitory signal. CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent in a Phase I study in patients with advanced and/or metastatic CEA-expressing tumors. CEA TCB-mediated killing of tumor cells led to T cell activation, IFNg secretion and subsequent up-regulation of the PD-1/PD-L1 immune suppressive pathway in vitro and in vivo, similarly to what happens during tumor adaptive immune resistance mechanisms where upon recognition of tumor antigens, TILs produce IFNg, which drives PD-L1 expression in the tumor microenvironment and delivers a suppressive signal to T cells upon binding to PD-1. Incubation of the high-CEA expressing gastric carcinoma cell line (MKN45) with human PBMCs (E:T 10:1) and increasing concentrations of CEA TCB, led to a dose-dependent up-regulation of PD-1 receptor on both CD4+ or CD8+ T cell subsets as well as of PD-L1 on surviving tumor cells as early as 24 h following treatment. PD-1 expression was specific as it did not occur in the absence of CEA-expressing tumor cells or upon treatment with un-targeted control TCB. In vivo studies performed using MKN45 tumor xenografts (MKN45) in fully humanized NOG mice demonstrated that CEA TCB treatment led to increased frequency of intra-tumoral T cells expressing PD-1 and to a strong induction of PD-L1 expression in tumors. Combination of CEA TCB with a PD-L1 blocking antibody showed significant increase of anti-tumor activity as compared to the respective single agents. These preclinical data indicate that CEA TCB treatment leads to up-regulation of the PD-1/PD-L1 immune suppressive pathway and that the combination of CEA TCB with PD-L1 blocking agents results in enhanced anti-tumor activity. Phase Ib clinical trials investigating the combination of CEA TCB and atezolizumab are currently ongoing. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Linda Fahrni, Valeria Nicolini, Christian Gerdes, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, 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 1494.