A Novel Angiopoietin-2 Selective Fully Human Antibody with Potent Anti-Tumoral and Anti-Angiogenic Efficacy and Superior Side Effect Profile Compared to Pan-Angiopoietin-1/-2 Inhibitors

<div><p>There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.</p> </div

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

<div><p>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.</p></div

Functionality and sequence diversity of the recombinant antibodies per animals and bleeds.

*<p>”Functional” refers to the biochemical IL1RL1:IL33 inhibition assay with a cut off of ≥40% inhibition.</p

Concordance of VL and VH sequences of single <i>E. coli</i> colonies derived from pool-cloning shown for 8 different B-cell clones (ASCs).

<p>Concordance of VL and VH sequences of single <i>E. coli</i> colonies derived from pool-cloning shown for 8 different B-cell clones (ASCs).</p

Comparison of the classical and the ligation-independent expression cloning workflows.

<p>Comparison of the classical and the ligation-independent expression cloning workflows.</p

Results of the primary screening using primary supernatants (SN) of the B-Cell Cloning workflow containing monoclonal rabbit antibodies.

<p>The statistically confirmed thresholds were rabbit IgG >0.013 µg/ml, human IL1RL1 binding >OD (optical density) 0.195, human Fc binding ≤OD 0.125, cynomolgus IL1RL1 binding >OD 0.184, murine IL1RL1 binding >OD 0.164, and biochemical human IL1RL1:IL33 inhibition ≥40%.</p

Yield of IL1RL1-specific rabbit antibodies.

<p>Scatter Plots depicting the yield of the primary screening using all 7644 supernatants: (<b>A</b>) Human IL1RL1 binding (unit: optical density (OD)) versus IgG concentration; (<b>B</b>) human IL1RL1 binding versus cynomolgus IL1RL1 binding or versus (<b>C</b>) murine IL1RL1 binding. Scatter Plot showing the correlation of the biochemical inhibition assay with the cellular inhibition assay: (<b>D</b>) Threshold ≥40% inhibition, RSq: 0.36, (<b>E</b>) magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086184#pone-0086184-g003" target="_blank">Figure 3D</a> using the threshold of >90% inhibition, RSq: 0.9. The statistically confirmed cut off values for the calculation of the percentages were as follows: rabbit IgG-positive >0.013 µg/ml, human IL1RL1-positive >OD 0.195, human Fc-positive ≤OD 0.125, cynomolgus IL1RL1-positive >OD 0.184, murine IL1RL1-positive >OD 0.164. Green are the supernatants deriving from the pre-incubation only scenario and red are the SN after the protein panning step. The diamond, the circle and the cross indicate the three different animals. (<b>F</b>) Result of the two dimensional binding matrix identifying different binding epitopes on human IL1RL1. The colored numbers indicate different antigen specific antibodies. The black numbers describe the three antibody groups. The degree of antibody competition in the matrix is depicted by a 3-colour scale with green, black, red color indicating highest competition, mid or lowest competition, respectively.</p

Tumor necrosis in LC06 and LC08 treated Colo205 tumors.

<p>(<b>A</b>) Quantitative image analysis shows 27% of necrotic area in vehicle treated, 51% in LC06 treated and 43% in LC08 treated tumors (n = 5, *p<0.05 compared to control, Student's t-test). Results are expressed as mean ± SEM. Two independent experiments were performed to confirm the results. (<b>B</b>) Representative mosaic images (10x) of vehicle, LC06 or LC08 treatments. Scale bar represents 1.3 mm.</p

Tumor cell dissemination to the lungs in orthotopic KPL-4 xenograft tumors after LC06 and LC08 treatment.

<p>Treatment with LC06 and LC08 resulted in a significant reduction of disseminated tumor cells to the lungs (n = 10, *p<0.05 compared to control, Student's t-test). The results were confirmed in two independent experiments. Results are expressed as mean ± SEM.</p

Analysis of the VH-VDJ and VK-VJ sequences to assess clonality and diversity of the recombinant rabbit antibodies.

<p>The distribution of the (A) CDR-H3 length (amino acid count) and of the (B) amino acid replacement frequency within the VH region in comparison to VHa1 and VHa3 allotype germ line sequences. Dark grey: VH1a1 germ line gene; light grey: VH1a3 germ line gene. (C) Clustering of the rabbit antibodies according to their CDR-H3 and CDR-L3 sequence similarity. The bold numbers indicate the rabbits.</p