Anti-CD33 Antibodies and Methods of Use Thereof

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December 15, 2016

Inventors

Kate Monroe, Helen Lam, Francesca Avogadri-Connors, Seung-joo Lee, William Monteith, Herve Rhinn, and Arnon Rosenthal

Assignees

Alector LLC; San Francisco, CA (US)

Abstract

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 CD33 protein, e.g. , human CD33 or a mammalian CD33, and use of such compositions in preventing, reducing risk, or treating an individual in need thereof.

CovalX Technology Used

HM3

K200

Complex Tracker

Outcomes

To determine the binding affinity of the CD33 antibodies, they were tested with 15-mer or 25-mer peptides that spanned the entire human CD33 or a reference CD33 antibody. Using shotgun mutagenesis, the epitopes of anti-CD33 antibodies 2E12, 2F5, 6C7, and 6A3 were mapped using an alanine-scanning library for the CD33 protein.

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 CD33 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 CD33 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 CD33 antigen (4 μM) to create a final mixture of CD33 (2 μM)/ antibody (2 μM)/ CD33 antigen peptides (2.5 μM). The data from this high-mass MALDI analysis was analyzed using the CovalX Complex Tracker software.