Kate Monroe, Helen Lam, Arnon Rosenthal, Seung-Joo Lee, Francesca Avogadri-Connors, and William Monteith
- Alector LLC; San Francisco, CA (US)
The present disclosure is generally directed to compositions that include antibodies, e.g., monoclonal, chimeric, humanized antibodies, antibody fragments, etc. that specifically bind on or more epitopes within a Siglec-7 protein, e.g., human Siglec-7 or a mammalian Siglec-7, and use of such compositions in preventing, reducing risk, or treating an individual in need thereof.
CovalX Technology Used
To determine the binding affinity of the Siglec-7 antibodies, they were tested with 15-mer or 25-mer peptides that spanned the entire human Siglec-7 or a reference Siglec-7 antibody. Using shotgun mutagenesis with an alanine-scanning library for the siglec-7 protein, the epitopes of anti-siglec-7 antibodies were mapped.
In order to identify conformational epitopes, a MALDI mass spectrometer that had been modified with a CovalX HM3 detection system was used. Antibody/antigen complexes were created by mixing equimolar solutions of Siglec-7 antigen and antibody (4 μΜ in 5 μ? each). 1 μ? of this complex was mixed with 1 μ? of matrix (recrystallized sinapic acid ( 10 mg/ml) in acetonitrile/water (1:1, v/v) with 0.1% TFA) from the CovalX K200 MALDI kit. 1 μ? of this final mixture was spotted on a MALDI plate, allowed to crystallize at room temperature and analyzed on a mass spectrometer. Following this analysis to determine the molecular weights of the antigen, antibody and complex, the potential competition of the conformational binding of the epitope with unstructured CIq peptides from proteolysis was analyzed. 25 μ? of the Siglec-7 ECD antigen (10 μM concentration) was digested with 5 μM immobilized pepsin and then incubated for 30 minutes at room temperature. The incubated samples were centrifuged and the supernatant was removed to allow for proteolysis to finish with high-mass MALDI. 5 μ? of the proteolysis generated antigen peptides were mixed with 5 μ? of antibodies (8 μM) before being incubated for 6 hours at 37 °C. 5 μ? of the incubated mixture was mixed with 5 μ? of the intact Siglec-7 antigen (4 μM) to create a final mixture of Siglec-7 (2 μM)/ antibody (2 μM)/ Siglec-7 antigen peptides (2.5 μM). The data from this high-mass MALDI analysis was analyzed using the CovalX Complex Tracker software.