Johannes Sam

A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors

Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. Experimental Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA–expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1–positive tumors. Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286–97. ©2016 AACR.

In Vivo Fluorescence Imaging of the Activity of CEA TCB, a Novel T-Cell Bispecific Antibody, Reveals Highly Specific Tumor Targeting and Fast Induction of T-Cell-Mediated Tumor Killing.

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

Anti-tumoral, anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2

ABSTRACT Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.

CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies

Purpose: Despite promising clinical activity, T-cell–engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug. Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug. Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell–binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD. Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785–97. ©2018 AACR. See related commentary by Prakash and Diefenbach, p. 4631

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 3634: A novel tumor-targeted 4-1BB agonist and its combination with T-cell bispecific antibodies: an off-the-shelf cancer immunotherapy alternative to CAR T-cells

Immune cell costimulation via 4-1BB agonism has shown anti-tumor activity in the clinic and is an important element of next-generation chimeric-antigen-receptor (CAR) adoptive T-cell therapy approaches. However, the clinical development of first-generation, 4-1BB agonistic antibodies has been hampered by significant hepatic toxicity. Activity of such first-generation, 4-1BB agonistic antibodies typically depends on their hyperclustering via Fc-gamma-receptor (FcgR)-binding. Here we describe a next generation, tumor-targeted 4-1BB agonist whose activity is independent of FcgR-binding. The molecule consists of an IgG fusion protein composed of a trimeric, human 4-1BB ligand (4-1BBL), a targeting Fab moiety recognizing fibroblast activation protein (FAP), and a heterodimeric Fc region engineered to be devoid of interactions with FcgRs and C1q. The molecule mediates potent costimulation of CD8 T-, CD4 T- and NK-cells, but only in the presence of FAP-expressing cells, such as cancer associated fibroblasts, which are highly prevalent in many solid tumors. This FAP-targeted 4-1BB agonist is significantly more potent and efficacious than first generation, standard 4-1BB agonistic antibodies when compared side-by-side in preclinical models. We show its activity in a variety of preclinical models including reporter cell assays, assays with primary T- and NK-cells, ex-vivo assays with patient tumor-derived material including cancer cells, stroma cells and tumor-infiltrating lymphocytes, fully immunocompetent murine tumor models (employing a surrogate, murinized molecule targeting murine FAP and carrying murine 4-1BBL), and in human hematopoietic stem cell-humanized mice with human tumor xenografts. We also demonstrate its activity in combination with checkpoint inhibitors and with T-cell redirecting approaches, such as a CEA-CD3 T-cell bispecific antibody. We show that hepatic toxicity of first generation, standard 4-1BB antibodies is dependent on FgR interactions and the next generation, FcgR-independent and FAP-targeted molecule described here is safe and does not induce any hepatotoxicity in preclinical models including non-human primates where it was tested at doses of up to 50 mg/kg and where it showed a long circulatory half-life. Its combination with T-cell bispecific antibodies induces a massive T cell accumulation in the tumor, accompanied with an elevated CD8/Treg ratio, as compared to the respective monotherapies. Therefore, we conclude that the tumor-targeted cross-linking of 4-1BB provides a safe and effective way for the co-stimulation of T cells for cancer immunotherapy and its combination with T-cell bispecific antibodies may provide an alternative, but more convenient, off-the-shelf approach to CAR T-cell therapies. The molecule is scheduled to enter clinical trials soon. Citation Format: Christina Claus, Claudia Ferrara, Sabine Lang, Rosmarie Albrecht, Sylvia Herter, Maria Amann, Sandra Richards-Grau, Johannes Sam, Sara Colombetti, Marina Bacac, Christian Klein, Pablo Umana. A novel tumor-targeted 4-1BB agonist and its combination with T-cell bispecific antibodies: an off-the-shelf cancer immunotherapy alternative to CAR T-cells [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 3634. doi:10.1158/1538-7445.AM2017-3634

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.