David Y. Jackson, Michael Nathaniel Alonso, and Arthur Lee
- Bolt Biotherapeutics, Inc., Redwood City, CA
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 which comprises an ethylene glycol group or glycine residue. Methods for treating cancer with the immunoconjugates of the invention are also described.
CovalX Technology Used
Immunoconjugates I-II were prepared by reacting Imidazoquinoline 1 (1-(4-aminobutyl)-2-propyl-1H-imidazo[4,5-c]quinolin-4-amine) with 2,5-dioxopyrrolidin-1-yl 4-((2-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)amino)-2-oxoethyl)carbamoyl)cyclohexane-1-carboxylate to form NHS-Gly2-CC-1. Next, Imidazoquinoline 1 was converted to NHS-EG-CC-1 analogously. Finally, Imidazoquinoline 1 and aldehyde 2 were reacted with sodium borohydride to form an intermediate that was then esterified with N-hydroxysuccinimide, forming NHS-EG-1.
At 1-5 mg/ml, the antibody was resuspended in phosphate buffered saline (PBS) before being reacted with a 10-fold molar excess of one of the 3 converted forms of Imidazoquinoline 1 (NHS-Gly2-CC-1, NHS-EG-CC-1, NHS-EG-1) for 30 minutes at room temperature. Using 3 washes of PBS, the immunoconjugates that formed were purified and then equilibrated.
In order to determine the adjuvant to antibody ratio, samples were desalted and buffer exchanged using columns into deionized water. A MALDI target plate was prepared by spotting a sinapic acid matrix on the plate and allowing the spots to dry before proceeding. Samples were mixed in a 1:1 ratio with or without bovine serum albumin (BSA) standard (0.25-1μM BSA) and then spotted on top of the dried matrix spots on the plate. The plates were allowed to dry again and the samples were analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system. The HM1 system allows the mass spectrometer to detect protein sizes in the range of a fully intact IgG antibody, thus increasing the sensitivity and resolution of the data.