CovX-BODIES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 552

CovX-Bodies were subjected to trypsin digestion and the
resulting peptide fragments were analyzed by electrospray
LC/MS/MS. The LC/MS/MS data was compared to the
predicted fragmentation pattern and processed using Waters
Protein Lynx Global server. Near complete identification of
predicted peptide fragments was achieved along with con-
firmation that only the peptide fragment containing LysH93
was modified with the AZD linked peptide. A site directed
mutant of CVX-2000 was expressed wherein the LysH93
was mutated to AlaH93. No fusion reaction was detected
when LysH93Ala mutant of CVX-2000 was treated with a
3:1 stoichiometric excess of AZD-linked peptides, thereby
confirming that LysH93 is required for the fusion reaction.
Direct proof of the molecular specificity of the AZD conju-
gation to LysH93 was obtained when the crystal structure of
the Fab fragment of CVX-2000 in complex with a peptide
with an AZD linker was determined at 1.8 A resolution.
Consistent with peptide mapping results, each CVX-2000
Fab fragment was found to bind one AZD linker (Figure
38.7A). LysH93 was located at the bottom of a 12 A long
lipophilic tunnel, forming a covalent amide bond with the
linker (Figure 38.7B) providing structural proof of the site of
conjugation. This result perfectly agrees with the reaction
mechanism of a nucleophilic attack of the LysH93 e -amino
group on the AZD carbonyl carbon, leading to ring opening
and covalent conjugation via a b -alanine bond. As predicted,
LysH93 was surrounded by nonpolar residues consistent
with an environment contributing to the reduced pKa and
increased reactivity for the LysH93

with a water molecule that was stabilized by the hydroxyl
groups of SerH106 and TyrL41. The side chains of TyrH95,
PheH96, TyrH97, TrpH33, TyrL101, and TyrL37 lined the
wall of the hydrophobic tunnel, consistent with the good
stability of the covalently linked therapeutic conjugates.
The reaction between CVX-2000 and 1,3-diketone linkers
is very rapid and often complete within a matter of minutes.
Reaction of the -amino group of LysH93 with 1,3-diketone

results in the formation of a vinylogous amide that can be
detected spectroscopically. In contrast, the reaction between
LysH93 and AZD linkers results in the formation of a b -ala-
nine bond that lacks a characteristic spectroscopic signature.
Resultantly, the kinetics of the fusion reaction of the AZD
linkers are usually studied using a SEC/MS method. The
SEC/MS method is capable of detecting and calculating the
ratios of various species during the course of the conjugation
reaction. At the start of the conjugation reaction, only the
unconjugated CVX-2000 is detected; a few minutes after the
onset of the fusion process, three major species are observed
withmasses consistent with, (1) unconjugated CVX-2000; (2)
monovalent fusion product; and (3) divalent fusion product.
The fusion reaction is deemed complete when only the
divalent product is detected by SEC/MS. The reaction with
AZD linker is slower than with the 1,3-diketone linkers but is
usually complete within a few hours. The physicochemical
properties of the payload being conjugated have an influence
on the kinetics of the fusion reaction.

e

-amino group. TyrH95
and TyrL37, which occlude the binding site in the apo-
crystal structure, adopt an open conformation in the covalent
conjugate structure thereby enabling access to LysH93. The
LysH93

e

38.2.3 Choice of Payload

The range of payloads that have been fusedwith CVX-2000 to
generate CovX-Bodies include small molecules, linear and
cyclic peptides, cysteine knotted toxins, 20 kDa proteins, and -amino group also formed a 2.9 A hydrogen bond

e

FIGURE 38.7 Crystal structure of CVX-2000 Fab in complex with AZD linker. (A) The AZD
linker is shown in stick model with its omit (2Fo-Fc) electron density map contoured at 1.2 s level.
Linker atoms outside of the hydrophobic tunnel were disordered and not observed in the crystal
structure. (B) The covalent adduct between LysH93 and the AZD linker, as well as other key residues
lining the hydrophobic tunnel.