<p>PDF file - 62K, Internalization of GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) upon binding to (A) SU-DHL4 cells and (B, C) whole blood derived from two CLL patients. Samples were incubated for 0.5, 2, 4, or 7 h in (A) and for 0.5, 1, 2, 3 and 5 h in (B, C) with Alexa Fluor (trademark) 488-labeled GA101, rituximab, or ofatumumab at 37{degree sign}C, washed and incubated in the presence or absence of anti-Alexa Fluor 488 for 30 min at 4{degree sign}C. The remaining fluorescence indicates the amount of labeled antibody that is not accessible to the quenching anti-Alexa Fluor 488 antibody and thus corresponds to internalized antibody. The average fluorescence intensity and standard deviations were calculated from duplicates of the experiment with SU-DHL4 cells. Due to low number of primary CLL samples the average fluorescence intensity in B and C correspond to single values.</p>
Abstract 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.
Abstract MCSP/CSPG4 is a large transmembrane proteoglycan identified in melanomas as HMW-MAA. In the mouse it is known as neurite growth factor 2 (NG2), a marker of pericyte recruitment. MCSP has been used as a target for clinical imaging of (uveal) melanomas by immunoscintigraphy. MCSP shows uniform and abundant expression in ca. 60-80% of melanoma, and was described in lobular breast carcinoma, glioblastoma, osteo- & chondrosarcoma, and basal cell carcinoma. It is present at high levels on pericytes of tumor neovasculature, but down-regulated as vessels mature. Normal tissue expression is low and it is not detected on PBMCs. We have generated human/Cynomolgus cross-reactive antibodies against a membrane-proximal MCSP epitope by mouse immunization with a linear peptide derived from the membrane proximal D3 domain followed by boosting with melanoma cells. The mouse antibody LC007 was selected for humanization due to its potent induction of ADCC as a chimeric antibody, compared to antibodies to membrane distal epitopes of MCSP. LC007 as chimeric IgG1 and its humanized IgG1 derivative M4-3-ML2 are characterized by the following properties: i) Specific binding to the native epitope on MCSP+ melanoma cells, but no induction of internalization; ii) Specific IHC staining of MCSP+ cells in FFPET samples; iii) ca 10 nM monovalent affinity for hMCSP D3 domain. Moreover, glycoengineering of LC007 and M4-3-ML2 antibodies using GlycoMab technology resulted in increased binding affinity for hFcgRIIIa and enhanced ADCC potency and absolute killing of melanoma cell lines. As expected, neither up to 10 ug/mL wildtype, nor glycoengineered M4-3-ML2 induced relevant cytokine (IL-6, TNF-α, IFN-γ) release in human whole blood supporting that MCSP is not expressed there. Subsequently, we studied anti-tumoral efficacy of the chimeric antibody LC007 and the humanized antibody M4-3-ML2 in disseminated models of MV3 and MDA-MB435 melanoma after i.v. injection of tumor cells in hCD16 transgenic Scid mice, which express the functional human high affinity FcgRIIIa receptor on NK cells. Both, glycoengineered LC007 and M4-3-ML2 mediated efficacy in terms of enhanced median and overall survival in both disseminated xenograft models, and were superior to the respective non-glycoengineered antibodies. Taken together, our studies support MCSP/CSPG4 as an attractive target for antibody-based cancer immunotherapy. Further studies investigating the anti-angiogenic effect of MCSP antibodies via their action on pericytes/vascular smooth muscle cells are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-236. doi:1538-7445.AM2012-LB-236
Abstract Background: First generation T cell bispecifics (TCBs) were developed by utilizing high CD3 and tumor target binder affinities to induce a potent response. Instrumentalizing this, TCBs have clinically proven to elicit efficacious target cell killing and anti-tumor efficacy, but they encountered hurdles related to cytokine release. Recent studies suggested that reducing CD3 binder affinity could lead to an efficient tumor killing with lower cytokine levels. However, the answer to whether cytokine release is mainly dependent on CD3 binder affinity or a multifactorial phenomenon remains ambiguous. Here, we aim to characterize the efficacy-safety relationship of different TCBs through investigating the interplay between CD3 binder affinity and the affinity of the tumor target-specific binder. For that purpose, a series of TCBs with varying CD3 affinities, and two target binders of high and low affinities were generated in the 2:1 TCB format, resulting in a bivalent binding to the tumor antigen and a monovalent one to CD3, which offers a unique opportunity to enhance the activity of TCBs. Methods: Firstly, 2:1 TCBs comprising either the high or low affinity target binder and varying CD3 binder affinities were co-cultured in-vitro with human peripheral blood mononuclear cells (PBMCs), and human tumor cell lines, in order to assess T cell mediated cytotoxicity and cytokine release at different time points. Additionally, the impact on in-vivo efficacy, cytokine release, and pharmacokinetics was assessed in humanized mice implanted with a patient-derived xenograft model. Results: Based on the dose-response in the cytotoxicity assay, the CD3 binders were classified into high, intermediate, and low levels of affinity. As expected, we observed that the in-vitro T cell cytotoxicity, in-vivo tumor growth inhibition (TGI), and cytokine release levels were maximal with the highest affine CD3 binder. Simultaneously, we have observed a decrease in cytotoxicity response with decreasing CD3 affinity compared to the high affinity binder. However, this was accompanied by an atypical decrease in cytokine release. Particularly, the TCB comprising the intermediate CD3 affinity binder illustrated a comparable in-vitro cytotoxicity and in-vivo TGI to the high affinity binder with a marked decrease in cytokine release [4x reduction in-vitro and 6x reduction in-vivo]. Upon testing the TCBs comprising the low affinity target binder with the same CD3 binders, we observed an additional decrease in cytokine release with the decreasing CD3 affinity. In conclusion, we designed and generated TCBs with varying affinities in both the CD3 and target binding arms. Reducing cytokine release while maintaining adequate efficacy is feasible through CD3 binder affinity attenuation; however, the target binder affinity should be taken into consideration when interpreting our findings and designing new molecules. Citation Format: Omar Abdelmotaleb, Anneliese Schneider, Thomas Hofer, Johannes Sam, Martin Lechmann, Inja Waldhauer, Ali Bransi, Miro Eigenmann, Anne Freimoser-Grundschober, Christian Gassner, Alex Odermatt, Peter Brünker, Sara Colombetti, Christian Klein. Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1235.
<p>Figure S1. Related to Figure 1. Characterization of FAP-drozitumab BsAbs and antitumor efficacy in the presence of FAP-expressing fibroblasts in in vitro co-culture models; Table S1. BsAb binding to DR5 and FAP antigens; Table S2. Anti-tumor efficacy of FAP-drozitumab bispecific molecules is FAPdependent.</p>
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
