Sven Berger

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings.

A New Anti-CXCR4 Antibody That Blocks the CXCR4/SDF-1 Axis and Mobilizes Effector Cells

The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell–derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor–mediated G-protein activation and β-arrestin-2 recruitment following CXCR4 activation. The binding of the hz515H7 antibody to CXCR4 inhibits the SDF-1–induced signaling pathway, resulting in reduced phosphorylation of downstream effectors, such as Akt, Erk1/2, p38, and GSK3β. Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal blood cells, induces both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7 displays antitumor activities with multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context. Furthermore, hz515H7 binds to primary tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our results demonstrate two major mechanisms of action, making hz515H7 unique in this regard. Its potential as a best-in-class molecule is currently under investigation in a phase I clinical trial. Mol Cancer Ther; 15(8); 1890–9. ©2016 AACR.

Abstract 3673: Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager

Background. 4-1BB (CD137) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome toxicity and efficacy limitations of current 4-1BB targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific based on Anticalin® technology. We have previously reported on the generation and characterization of PRS-343 with regard to preclinical proof-of-concept and basic drug-like properties. 1 Here, we describe the preclinical dataset supporting initiation of a first-in-patient trial. Methods and Results. The pharmacology of PRS-343 was investigated by ex vivo assays based on mixed culture of human PBMC and tumor cell lines. We find that 4-1BB costimulated T cells prominently increase production of IL-2, GM-CSF, TNF-α and IFN-γ. Using a set of immortal cancer cell lines spanning a range of HER2 surface copy numbers, we identified a copy number threshold above which PRS-343 reliably elicited T cell costimulation with a high potency and an EC50 in the subnanomolar range. PRS-343 was well tolerated in a repeat-dose study in cynomolgus monkeys, with no overt toxicity and no significant drug-related toxicological findings. Pharmacokinetic assessment confirmed dose-proportional exposure of the animals during the course of the study. In a mouse model of human PBMC-induced xenograft-vs-host disease (xGvHD), PRS-343 did not show an acceleration of xGvHD development, in contrast to a 4-1BB targeting benchmark. Again utilizing ex vivo assays, we found no PRS-343 induced T cell costimulation in a panel of primary cells, showing that physiological levels of HER2 are insufficient for activation. In a cytokine release assay, proinflammatory cytokine induction by PRS-343 in the absence of a primary TCR stimulation was negligible. Conclusion. The ex vivo experiments described indicate that HER2 expression level is expected to be a reliable marker for patient stratification for PRS-343. The toxicology assessment of PRS-343 indicates that the benign toxicity profile of trastuzumab is retained in PRS-343 with regard to HER2 targeting, and that PRS-343 is expected to elicit its costimulatory effects strictly on T cells also receiving a primary TCR signal and strictly localized to HER2-positive tumors. This is in agreement with in vivo mouse model data showing PRS-343 leads to tumor-localized CD8+ T cell expansion, 1 and supports the potential of PRS-343 as an efficacious yet well tolerable 4-1BB costimulating agent. The reported data is the basis for the trial design of a first in patient Phase 1 trial with PRS-343. 1 Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Thomas Jaquin, Sven Berger, Manuela Durr, Corinna Schlosser, Andrea Allersdorfer, Christine Rothe, Louis A. Matis, Shane A. Olwill. Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager [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 3673. doi:10.1158/1538-7445.AM2017-3673

Abstract B016: Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in humanized mouse model

Background: CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a CD137/HER2 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods: Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. We have shown previously that the bispecific fusion PRS-343 targets CD137 and HER2 in a bispecific manner and efficiently activates T cells ex vivo in the presence of HER2-positive cells. Here, in vivo proof of concept data is presented utilizing a humanized mouse model in immunocompromised mice and the SK-OV-3 cell line as a HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 for three weeks. An IgG4 isotype antibody served as the negative control, while a CD137-targeting benchmark antibody and trastuzumab with an engineered IgG4 backbone (“tras-IgG4”) served as controls for monospecific targeting of CD137 and HER2, respectively. Results: PRS-343 activity was investigated at four different weekly doses of PRS-343 (4μg, 20μg, 100μg and 200μg). We found that PRS-343 dose-dependently led to strong tumor growth inhibition compared to treatment with the isotype control, and that the tumor response was accompanied by a significantly higher tumor infiltration with human lymphocytes (hCD45+). Interestingly, the anti-CD137 benchmark neither displayed tumor growth inhibition nor enhanced lymphocyte infiltration into tumors compared to isotype. The tras-IgG4 control was also devoid of lymphocyte infiltration into the tumor, but displayed a tumor growth inhibition comparable to PRS-343. Taken together, these data show that PRS-343 provided dual activity by both increasing the frequency of tumor-infiltrating lymphocytes by bispecific targeting of CD137 and HER2 as well as mediating direct tumor growth inhibition by the direct, monospecific targeting of HER2. Notably, the tumor growth inhibition provided by targeting HER2 did not require any antibody directed cellular cytotoxicity (ADCC) as both PRS-343 and the tras-IgG4 control lack the ability to interact with Fc-gamma receptors on NK cells that ADCC would require. The animal model also allowed investigating the potential safety of PRS-343: While the anti-CD137 benchmark accelerated the onset of graft-versus-host-disease and led to stronger expansion of CD8+ T cells in the peripheral blood compared to the isotype control group, both of these effects were absent for PRS-343. The data therefore support the envisaged mode of action of selective, tumor-localized costimulatory T cell activation, as well as the concept that such an approach may lead to higher efficacy and reduced systemic toxicity compared to conventional anti-CD137 mAbs. Conclusion: We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the CD137/HER2 bispecific PRS-343. In a humanized mouse model, PRS-343 displays dual activity based on monospecific HER2-targeting and bispecific, tumor-localized costimulation of CD137. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The direct, monospecific HER2-targeting activity may provide an additional therapeutic benefit and work in synergy with local CD137 costimulation. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Thomas Jaquin, Andrea Allersdorfer, Sven Berger, Alexander Wiedenmann, Gabriele Matschiner, Julia Schuler, Ulrich Moebius, Christine Rothe, Shane A. Olwill. Costimulatory T-cell engagement by PRS-343, a CD137 (4-1BB)/HER2 bispecific, leads to tumor growth inhibition and TIL expansion in humanized mouse model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016.

Abstract 556: Costimulatory T-cell engagement by the HER2/CD137 bispecific PRS-343 leads to strong antitumor effect in humanized mouse model

Background. CD137 (4-1BB) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. While multiple lines of evidence show that CD137 is a highly promising therapeutic target in cancer, current mAb-based approaches are not designed to achieve a tumor-target driven activation and may display toxicity and a limited therapeutic window due to peripheral T cell and NK cell activation. To overcome this limitation, we generated PRS-343, a HER2/CD137 bispecific that is designed to promote CD137 clustering by bridging CD137-positive T cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T cells. Methods. Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. PRS-343 was obtained by genetic fusion of the CD137-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. Results. The bispecific fusion PRS-343 targets CD137 and HER2 with nearly identical affinities compared to the parental building blocks, and is capable of binding both targets simultaneously. We show ex vivo that T cells are efficiently activated when incubated with PRS-343 and HER2-positive cells, and that the activation is HER2-dependent. The in vivo activity of PRS-343 was investigated utilizing a humanized mouse model with a tumor cell-line-derived, HER2-positive xenograft. When tumors had reached a predefined size, mice received human PBMC via an intravenous route and weekly intraperitoneal injections of PRS-343 or controls for three weeks. We found that PRS-343 led to strong tumor growth inhibition and a significantly better response compared to either isotype control or anti-CD137 benchmark mAbs. The data, which include phenotyping of peripheral and intra-tumoral lymphocytes, support the envisaged mode of action of tumor-localized costimulatory T cell activation. Conclusion. We report potent costimulatory T-cell engagement of the immunoreceptor CD137 in a HER2-dependent manner, utilizing the HER2/CD137 bispecific PRS-343. Compared to known CD137-targeting antibodies in clinical development, this approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity. The positive functional ex vivo and in vivo data of PRS-343 as well as the excellent developability profile support investigation of its anti-cancer activity in clinical trials. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Corinna Schlosser, Alexander Wiedenmann, Andrea Allersdorfer, Gabriele Matschiner, Sven Berger, Ulrich Moebius, Christine Rothe, Shane A. Olwill. Costimulatory T-cell engagement by the HER2/CD137 bispecific PRS-343 leads to strong antitumor effect in humanized mouse model. [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 556.

Abstract C56: Characterization of hz515H7, a novel humanized anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo antitumor activity.

Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. The chemokine receptor CXCR4 and its ligand Stromal cell-derived factor-1 (SDF-1) play a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel humanized monoclonal antibody hz515H7 was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. Its antitumor activity was investigated in vivo using several human tumor models. Materials and Methods: CHOK1 and NIH3T3 cells were stably transfected with human CXCR4. [125I]SDF1 and [35S]GTP S binding assays were performed on cell membranes containing CXCR4, using SPA-WGA beads. Calcium mobilization was monitored using Fluo-4NW dye. BRET assays were developed upon genetic fusion of CXCR4 to Rluc and β-arrestin-2 to YFP and transient co-expression in HEK293 cells. Xenograft model: 10.106 Ramos cells (B-cell lymphoma) were implanted s.c. into the right flank region of each SCID mouse and allowed to grow to the designated size before administration of antibodies. The mice were followed twice a week for the observation of xenograft growth. Tumor volume was calculated using the formula: π/6 × length × width × height. Results: Hz515H7 Mab was found to strongly inhibit SDF-1 binding, Gα protein activation and -arrestin-2 recruitment. It also inhibited calcium release, and constrained by itself CXCR4 homodimers conformation. Hz515H7 reduced SDF-1-induced cell migration in vitro. Moreover we also demonstrated that hz515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice. Altogether, the data demonstrate that Mab hz515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors. 285-286 C57 Targeting stromal platelet-derived growth factor receptorα (PDGFRα) inhibits lung cancer growth independent of tumor cell PDGFRα expression. Colleen Burns1, David Gerber2, Puja Gupta2, Michael T. Dellinger2, Jason E. Toombs2, Michael Peyton2, Inga Dunignan1, Jennifer Malaby1, Timothy Bailey1, Rolf A. Brekken2, Nick Loizos1. 1ImClone Systems, New York, NY; 2University of Texas Southwestern Medical Center, Dallas, TX. Platelet-derived growth factor receptor alpha (PDGFRα) is a type III receptor tyrosine kinase that is normally expressed on cells of mesenchymal origin (e.g. fibroblasts and smooth muscle cells) as well as on a variety of tumor types. In lung cancer, PDGFRα is expressed frequently by tumor associated stromal cells and, in a subset of tumors, by cancer cells themselves. The anti-human PDGFRα mAb, IMC-3G3, blocks the binding of PDGF-AA, -BB & - CC to PDGFRα, and was previously shown to inhibit tumor growth of glioblastoma, prostate, and sarcoma xenografts in mice. In our analysis, IMC-3G3 administered to mice 2× per week at 40 mg/kg significantly inhibited the growth of human H1703 non-small cell lung cancer (NSCLC) tumors with a T/C value of 35%. The PDGFRα-positive H1703 lung cancer line contains both PDGFRα and PDGF-CC gene amplifications and shows a co-dependency on this axis for proliferation. Co-dependence of this kind is a rare event in human lung cancer cell lines, suggesting that only a limited number of lung tumor patients might benefit from PDGFRα inhibition. However, this experimental system does not account for the potential therapeutic effects of stromal PDGFRα inhibition. In tumor stroma, the PDGF-PDGFRα axis functions in fibroblast activation, modulation of tumor interstitial pressure, and production and secretion of vascular endothelial growth factor (VEGF). To target stromal PDGFRα, an anti-mouse PDGFRα mAb (1E10) was generated and shown to inhibit PDGF-AA from binding to murine PDGFRα with an IC50 of 8.51 × 10−9M. MAb 1E10 inhibited PDGF-AA-induced phosphorylation of mouse PDGFRα but not human PDGFRα in cell-based assays; thus demonstrating the species specificity of 1E10. Tumor xenograft studies were performed using the PDGFRα-negative NSCLC cell lines Calu-6, H1993, and A549. MAb 1E10 treatment attenuated the tumor growth of A549 and Calu-6 xenografts in nude mice with T/C values of 67% and 51%, respectfully. In Calu-6 xenografts, the combination of 1E10 plus an anti-VEGF antibody (S12) significantly inhibited tumor growth compared to control. Microvessel density was significantly decreased by both 1E10 and S12 given alone or in combination. MAb 1E10 enhanced the anti-tumor activity of cisplatin-gemcitabine chemotherapy in A549 xenografts. However, MAb 1E10 monotherapy or when combined with cisplatin-gemcitabline chemotherapy showed no anti-tumor effect on H1993 xenograft growth. PDGF-AA and VEGF-A levels were determined in cell lines resistant and sensitive to 1E10 in vivo treatment. Elevated cancer cell expression of PDGF-AA and low expression of VEGF were associated with response to stromal PDGFRα targeting. Specifically, sensitive cell lines had relatively low VEGF-A expression (VEGF-A/PDGF-AA ratio 1.96 in Calu-6 cells and 10.87 in A549 cells), while the resistant cell line H1993 had elevated VEGF-A (VEGF-A/PDGF-AA ratio 65.86) when grown in cell culture. This difference may suggest that the 1E10-sensitive tumors have a relatively greater dependence on PDGF-AA-induced production of stromal VEGF. Therefore, inhibition of stromal PDGFRα represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. 286 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C56.

Abstract 4571: 515H7, a novel anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo anti-tumor activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. Chemokine receptor CXCR4/[Stromal cell-derived factor (SDF)-1] axis plays a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel monoclonal antibody (Mab 515H7) was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. It was able to efficiently bind CXCR4 on both transfected cells and human tumor cell lines. It was found to strongly inhibit SDF-1 binding, Gα protein activation and beta-arrestin2 recruitment. It also blocked second messenger release (calcium, cAMP) and constrained by itself the formation of CXCR4 homodimers. It also inhibits SDF-1-induced cell migration in vitro. 515H7 Mab antitumor activities were also investigated in vivo using several human tumor models. We demonstrated that 515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice. In addition to its effect on tumor growth, 515H7 Mab was also able to significantly improve mice survival in the lethal U937 model. The herewith data demonstrate that Mab 515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4571. doi:10.1158/1538-7445.AM2011-4571