Rabbits transgenic for human IgG genes recapitulating rabbit B-cell biology to generate human antibodies of high specificity and affinity.

Transgenic animals incorporating human antibody genes are extremely attractive for drug development because they obviate subsequent antibody humanization procedures required for therapeutic translation. Transgenic platforms have previously been established using mice, but also more recently rats, chickens, and cows and are now in abundant use for drug development. However, rabbit-based antibody generation, with a strong track record for specificity and affinity, is able to include gene conversion mediated sequence diversification, thereby enhancing binder maturation and improving the variance/selection of output antibodies in a different way than in rodents. Since it additionally frequently permits good binder generation against antigens that are only weakly immunogenic in other organisms, it is a highly interesting species for therapeutic antibody generation. We report here on the generation, utilization, and analysis of the first transgenic rabbit strain for human antibody production. Through the knockout of endogenous IgM genes and the introduction of human immunoglobulin sequences, this rabbit strain has been engineered to generate a highly diverse human IgG antibody repertoire. We further incorporated human CD79a/b and Bcl2 (B-cell lymphoma 2) genes, which enhance B-cell receptor expression and B-cell survival. Following immunization against the angiogenic factor BMP9 (Bone Morphogenetic Proteins 9), we were able to isolate a set of exquisitely affine and specific neutralizing antibodies from these rabbits. Sequence analysis of these binders revealed that both somatic hypermutation and gene conversion are fully operational in this strain, without compromising the very high degree of humanness. This powerful new transgenic strategy will allow further expansion of the use of endogenous immune mechanisms in drug development.

Predicting clinical decline and conversion to Alzheimer's disease or dementia using novel Elecsys Aß(1-42), pTau and tTau CSF immunoassays.

We evaluated the performance of CSF biomarkers for predicting risk of clinical decline and conversion to dementia in non-demented patients with cognitive symptoms. CSF samples from patients in two multicentre longitudinal studies (ADNI, n = 619; BioFINDER, n = 431) were analysed. Aß(1-42), tTau and pTau CSF concentrations were measured using Elecsys CSF immunoassays, and tTau/Aß(1-42) and pTau/Aß(1-42) ratios calculated. Patients were classified as biomarker (BM)-positive or BM-negative at baseline. Ability of biomarkers to predict risk of clinical decline and conversion to AD/dementia was assessed using pre-established cut-offs for Aß(1-42) and ratios; tTau and pTau cut-offs were determined. BM-positive patients showed greater clinical decline than BM-negative patients, demonstrated by greater decreases in MMSE scores (all biomarkers: -2.10 to -0.70). Risk of conversion to AD/dementia was higher in BM-positive patients (HR: 1.67 to 11.48). Performance of Tau/Aß(1-42) ratios was superior to single biomarkers, and consistent even when using cut-offs derived in a different cohort. Optimal pTau and tTau cut-offs were approximately 27 pg/mL and 300 pg/mL in both BioFINDER and ADNI. Elecsys pTau/Aß(1-42) and tTau/Aß(1-42) are robust biomarkers for predicting risk of clinical decline and conversion to dementia in non-demented patients, and may support AD diagnosis in clinical practice.

Sensitization of Tumors for Attack by Virus-Specific CD8+ T-Cells Through Antibody-Mediated Delivery of Immunogenic T-Cell Epitopes.

Anti-tumor immunity is limited by a number of factors including the lack of fully activated T-cells, insufficient antigenic stimulation and the immune-suppressive tumor microenvironment. We addressed these hurdles by developing a novel class of immunoconjugates, Antibody-Targeted Pathogen-derived Peptides (ATPPs), which were designed to efficiently deliver viral T-cell epitopes to tumors with the aim of redirecting virus-specific memory T-cells against the tumor. ATPPs were generated through covalent binding of mature MHC class I peptides to antibodies specific for cell surface-expressed tumor antigens that mediate immunoconjugate internalization. By means of a cleavable linker, the peptides are released in the endosomal compartment, from which they are loaded into MHC class I without the need for further processing. Pulsing of tumor cells with ATPPs was found to sensitize these for recognition by virus-specific CD8+ T-cells with much greater efficiency than exogenous loading with free peptides. Systemic injection of ATPPs into tumor-bearing mice enhanced the recruitment of virus-specific T-cells into the tumor and, when combined with immune checkpoint blockade, suppressed tumor growth. Our data thereby demonstrate the potential of ATPPs as a means of kick-starting the immune response against "cold" tumors and increasing the efficacy of checkpoint inhibitors.

Elecsys® Total-Tau and Phospho-Tau (181P) CSF assays: Analytical performance of the novel, fully automated immunoassays for quantification of tau proteins in human cerebrospinal fluid.

BACKGROUND: Total tau (tTau) and phosphorylated 181P tau (pTau) are supportive diagnostic cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease. Manual CSF tau assays are limited by lot-to-lot and between-laboratory variability and long incubation/turnaround times. Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF immunoassays were developed for fully automated cobas e analyzers, allowing broader access in clinical practice and trials. METHODS: Analytical performance, reproducibility, method comparisons with commercially available assays, and lot-to-lot and platform comparability (cobas e 601/411) of the Elecsys® CSF assays were assessed. Tau distributions and concentration ranges were evaluated in CSF samples from two clinical cohorts. RESULTS: Both assays showed high sensitivity (limit of quantitation [LoQ]: 63 pg/mL [tTau]; 4 pg/mL [pTau]) and linearity over the measuring range (80-1300 pg/mL; 8-120 pg/mL), which covered the entire concentration range measured in clinical samples. Lot-to-lot and platform comparability demonstrated good consistency (Pearson's r: 0.998; 1.000). Multicenter evaluation coefficients of variation (CVs): repeatability, < 1.8%; intermediate precision, < 2.8%; between-laboratory variability, < 2.7% (both assays); and total reproducibility, < 6.7% (tTau) and < 4.7% (pTau). Elecsys® CSF assays demonstrated good correlation with commercially available tau assays. CONCLUSIONS: Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF assays demonstrate good analytical performance with clinically relevant measuring ranges; data support their use in clinical trials and practice.

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

INTRODUCTION: 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. METHODS: Cutoffs for Elecsys amyloid-ß1-42 (Aß), total tau/Aß(1-42), and phosphorylated tau/Aß(1-42) were defined against [18F]flutemetamol PET in Swedish BioFINDER (n = 277) and validated against [18F]florbetapir PET in Alzheimer's Disease Neuroimaging Initiative (n = 646). Clinical progression in patients with mild cognitive impairment (n = 619) was studied. RESULTS: CSF total tau/Aß(1-42) and phosphorylated tau/Aß(1-42) ratios were highly concordant with PET classification in BioFINDER (overall percent agreement: 90%; area under the curve: 94%). The CSF biomarker statuses established by predefined cutoffs were highly concordant with PET classification in Alzheimer's Disease Neuroimaging Initiative (overall percent agreement: 89%-90%; area under the curves: 96%) and predicted greater 2-year clinical decline in patients with mild cognitive impairment. Strikingly, tau/Aß ratios were as accurate as semiquantitative PET image assessment in predicting visual read-based outcomes. DISCUSSION: Elecsys CSF biomarker assays may provide reliable alternatives to PET in Alzheimer's disease diagnosis.

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.

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.

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.