Tina Schwabe, Francesca Avogadri-Connors, Helen Lam, Ilaria Tassi, Seung-Joo Lee, and Arnon Rosenthal
- 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 a TREM2, e.g.,a mammalian TREM2 or human TREM2, 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 TREM2 antibodies, they were tested with 15-mer or 25-mer peptides that spanned the entire human TREM2 or mouse TREM2. Using shotgun mutagenesis, the epitopes of anti-TREM2 antibodies 9F5 (MAb), T21-9 (Fab), T22 (Fab), and T45-10 (Fab) were mapped.
Using the sequence from human TREM2, with a 14 residue, linear 15-mer peptides were synthesized. Linear 25-mer peptides were also synthesized based on the human or mouse TREM2 sequences. Using ELISA, the binding ability of the TREM2 antibodies to the synthesized peptides was tested.
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 TREM2 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 TREM2 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 TREM2 antigen (4 μM) to create a final mixture of TREM 2 (2 μM)/ antibody (2 μM)/ TREM2 antigen peptides (2.5 μM). The data from this high-mass MALDI analysis was analyzed using the CovalX Complex Tracker software.