Michael Nathaniel Alonso, Edgar George Engleman, Shelley Erin Ackerman, Justin Kenkel, Arthur Lee, David Y Jackson.


  1. The Board of Trustees of the Leland Stanford Junior University (Stanford, CA, US)
  2. Bolt Biotherapeutics, Inc. (Sunnyvale, CA, US)


The invention provides an immunoconjugate comprising an antibody construct which includes an antigen binding domain and an Fc domain, an adjuvant moiety, and a linker, wherein each adjuvant moiety is covalently bonded to the antibody via the linker. Methods for treating cancer with the immunoconjugates of the invention are also described.

CovalX Technology Used



After conjugation of the antibody-adjuvant immunoconjugate (Ab-SATA-SMCC-Adjuvant), the MALDI-TOF Mass Spectrometry (MS) containing CovalX high mass detector was used to find the average Drug to Antibody Ratio (DAR).

To prepare the samples and matrix for the MALDI process, the Ab-SATA-SMCC-Adjuvant samples were desalted and buffer exchanged into deionized water to be spotted onto the MALDI plate and allowed to dry. Then the sample was mixed at a 1:1 ratio with and without a bovine serum albumin (BSA) standard (0.25-1 μM BSA) and spotted onto the plate with the matrix to dry.

Both dried matrix and samples were analyzed using a MALDI TOF MS modified with the CovalX high mass detector, which enhances the sensitivity and resolution, to detect protein sizes in the range of a fully intact IgG antibody (˜150,000 kDa). The number of linkers present per antibody was determined using the mass difference between the SATA-modified and unmodified antibody. The average DAR was found using the mass difference between the SATA-modified antibody and the immunoconjugates.

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Categories : Patents, High-Mass MS