Valeria Lifke

Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM+ and IgG+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

RG7116, a therapeutic antibody that binds the inactive HER3 receptor and is optimized for immune effector activation

The EGF receptor (EGFR) HER3 is emerging as an attractive cancer therapeutic target due to its central position in the HER receptor signaling network. HER3 amplifies phosphoinositide 3-kinase (PI3K)-driven tumorigenesis and its upregulation in response to other anti-HER therapies has been implicated in resistance to them. Here, we report the development and characterization of RG7116, a novel anti-HER3 monoclonal antibody (mAb) designed to block HER3 activation, downregulate HER3, and mediate enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) via glycoengineering of the Fc moiety. Biochemical studies and X-ray crystallography revealed that RG7116 bound potently and selectively to domain 1 of human HER3. Heregulin binding was prevented by RG7116 at concentrations more than 1 nmol/L as was nearly complete inhibition of HER3 heterodimerization and phosphorylation, thereby preventing downstream AKT phosphorylation. In vivo RG7116 treatment inhibited xenograft tumor growth up to 90% relative to controls in a manner accompanied by downregulation of cell surface HER3. RG7116 efficacy was further enhanced in combination with anti-EGFR (RG7160) or anti-HER2 (pertuzumab) mAbs. Furthermore, the ADCC potency of RG7116 was enhanced compared with the nonglycoengineered parental antibody, both in vitro and in orthotopic tumor xenograft models, where an increased median survival was documented. ADCC degree achieved in vitro correlated with HER3 expression levels on tumor cells. In summary, the combination of strong signaling inhibition and enhanced ADCC capability rendered RG7116 a highly potent HER3-targeting agent suitable for clinical development.

A Robust High Throughput Platform to Generate Functional Recombinant Monoclonal Antibodies Using Rabbit B Cells from Peripheral Blood

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

CSF biomarkers of Alzheimer's disease concord with amyloid-β PET and predict clinical progression: A study of fully automated immunoassays in BioFINDER and ADNI cohorts

We studied whether fully automated Elecsys cerebrospinal fluid (CSF) immunoassay results were concordant with positron emission tomography (PET) and predicted clinical progression, even with cutoffs established in an independent cohort.

Abstract 2508: GE-huMab-HER3, a novel humanized, glycoengineered HER3 antibody with enhanced ADCC and superior preclinical in vitro and in vivo efficacy

HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. HER3 is a kinase dead receptor, but by forming heterodimers with other HER family receptors, HER3 works as amplifier for PI3 kinase driven tumorigenesis. It has been reported that tumors treated with EGFR-, HER2- or cMET-targeted therapies can escape via HER3 activation or upregulation. HER3 is expressed in a large variety of tumors for example in non-small cell lung cancer (NSCLC), head and neck, colorectal, gastric, pancreatic, breast, ovarian and prostate cancer. Anti-HER3 antibodies can work via various mechanisms including: (1) blocking ligand (HRGs) binding to the receptor, (2) blocking heterodimerization with other HER family members (HER1, 2 and 4), (3) downregulation of the receptor from the cell surface, and (4) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). The first three mechanisms lead to inhibition of HER3 phosphorylation and downstream signaling thereby resulting in tumor cell growth inhibition, while ADCC is a mechanism of direct target cell killing triggered by cross-linking of Fc receptors on immune effector cells (e.g. NK cells, macrophages). GE-huMab-HER3 is a novel humanized and glycoengineered IgG1 antibody that binds to HER3 with high affinity. This antibody prevents ligand binding and receptor heterodimerization thereby blocking receptor phosphorylation. In various tumor xenograft models treatment with this antibody leads to substantial tumor growth inhibition. E.g. GE-huMab-HER3 treatment achieved >50% tumor growth inhibition in 10 out of 17 NSCLC models and in some cases even resulted in complete tumor remission. However, these xenograft experiments only reveal part of this antibody9s therapeutic potential. A unique feature of GE-huMab-HER3 that differentiates it from other anti-HER3 antibodies, including AMG 888 and MM-121, is its ability to bind to human FcgRIIIa on immune effector cells with a 50-fold higher affinity than standard IgG1 antibodies, a property conferred by the engineered glycosylation of the antibody Fc region. Consequently, GE-huMab-HER3 exhibits superior potency and efficacy in ADCC, as shown in vitro using recombinant A549 cells and in vivo by its Fc mediated greater anti-tumor effect in A549 orthotopic mouse models compared to a non-glyco-engineered variant of the antibody, WT-huMab-HER3. The combination of strong signaling inhibition and enhanced ADCC capability renders GE-huMabHER3 a highly potent HER3-targeting agent. Phase I clinical testing of this promising novel compound is 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 2508. doi:1538-7445.AM2012-2508

MULTICENTER EVALUATION OF THE ANALYTICAL CHARACTERISTICS OF THE ELECSYS® TOTAL-TAU CEREBROSPINAL FLUID (CSF) AND ELECSYS® PHOSPHO-TAU (181P) CSF IMMUNOASSAYS

recovery relative to later denaturation (Mean (SD) CSF Ab142collection1⁄41061(476) pg/mL, CSF Ab1-42Aliquot1⁄4996(414) pg/ mL; vs. CSF Ab1-42Analysis1⁄4822(385) pg/mL, CSF Ab142LoBind1⁄4899(376) pg/mL (p<0.0002)). The Ab1-42/Ab1-40 ratio reduced recovery variability due to collection condition, and improved discrimination between control and dementia groups. The assay was stable for 2 years, but CSFAb peptide measurements and the Ab1-42/Ab1-40 ratio were not longitudinally stable for the study period. CSF Ab1-42 was stable for 15months (“Collection”,“Aliquot”) or 18months (“Analysis”). The Ab1-42/Ab1-40 ratio was stable for 8 months (“Collection”,“Aliquot”) and 11months (“Analysis”). Conclusions:When measured with a UP-RPLC-MS/ MS assay, earlier denaturation improved CSFAb peptide recovery. CSF Ab1-42, Ab1-40, Ab1-38, Ab1-34, and the Ab1-42/Ab1-40 ratio were not stable for the study period when samples were stored frozen. The Ab1-42/Ab1-40 ratio was useful for normalizing adsorption-related artifacts at baseline and for separating Dx groups, but was more sensitive to longitudinal instability. We recommend that UP-RPLC-MS/MS analysis of CSF Ab peptides from frozen samples occur within the cutoffs specified or close to the time of collection.

A UNIFIED PRE-ANALYTICAL PROTOCOL FOR HANDLING OF CSF SAMPLES BEFORE ANALYSES OF AD BIOMARKER LEVELS

O2-09-01 CSF, PLASMA AND MRI BIOMARKER TRAJECTORIES DURING THE DEVELOPMENT OFALZHEIMER’S DISEASE Sebastian Palmqvist, Niklas Mattsson, Olof Strandberg, Philip Insel, Shorena Janelidze, Erik Stomrud, Oskar Hansson, Department of Neurology, Sk ane University Hospital, Lund, Sweden; Clinical Memory Research Unit, Lund University, Malm€o, Sweden; Department of Neurology, Lund, Sweden; Clinical Memory Research Unit, Lund University, Lund, Sweden; Center for Imaging of Neurodegenerative Diseases, San Francisco Veterans Administration Medical Center, San Francisco, CA, USA; Lund University, Lund, Sweden. Contact e-mail: sebastian.palmqvist@med.lu.se

Abstract B67: Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo

Recent success of T cell based cancer immunotherapy approaches like chimeric antigen receptor T cells (CAR-Ts) and especially bispecific T cell engagers (BiTEs) has suggested great potential for cancer therapy. However, these technologies have several drawbacks like high risks for side-effects, such as autoimmunity, as well as inappropriate T cell activation due to a non-natural synapse. Hence, we sought to use antibody-mediated delivery of viral peptides to tumor cells in order to mimic viral infection and induce tumor cell lysis by virus-specific cytotoxic CD8+ T cells. Here, we generated immunoconjugates called antibody-targeted pathogen-derived peptides (ATPPs), where mature virus-derived MHC class I peptides from Influenza A or Epstein-Barr virus were conjugated to a tumor-associated antigen-specific antibody via a disulfide bond. Using optical imaging in combination with fluorescence resonance energy transfer, we revealed that after ATPP binding to the target antigen and subsequent internalization, the peptides are released in endosomes and loaded onto recycling MHC class I complexes. These peptide-MHC complexes are subsequently presented on the cell surface and mediate activation of human peptide-specific CD8+ T cells as shown by Interferon-γ release using enzyme-linked immunosorbent assay. Moreover, ATPP-loaded cancer cells from different tumor types were efficiently killed in vitro by human CD8+ T cells in a dose-dependent manner, reaching up to 100% killing after 24h at 0,13nM with an effector-to-target ratio of 3:1. Tumor cell death was assessed by the xCELLigence system as well as lactate dehydrogenase quantification in the supernatant. Interestingly, ATPPs were about 1000-times more potent than free peptide, while antibody alone, a non-cleavable ATPP construct, as well as peptides conjugated to a non-binding antibody exhibited no effects. Importantly, all used antibodies carried the P329G/LALA mutation for abolished Fc-receptor binding. In addition, we performed an experimental subcutaneous xenograft in vivo study with PDL1-expressing MDA-MB231 breast cancer cells in NOG mice in combination with anti-PD1 treatment. We observed that ATPPs successfully recruited adoptively transferred human peptide-specific CD8+ T cells into the tumor and induced 60% tumor growth inhibition of an established tumor after 3 weeks in the anti-PD1 combination setting. Altogether, we generated first in class immunoconjugates for antibody-targeted loading of cancer cells with foreign MHC-I restricted peptide antigens. Our results demonstrate potent ATPP-mediated antitumor efficacy in vitro and in vivo , by loading tumor cells with viral peptide antigens and redirecting virus-specific cytotoxic T cells against cancer. Citation Format: Julian Peter Sefrin, Lars Hillringhaus, Valeria Lifke, Alexander Lifke. Antibody-mediated delivery of viral peptides selectively activates CD8+ T cells to eliminate tumor cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B67.