CovX-BODIES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 553

oligonucleotides. However, most of the clinical stage appli-
cations of CovX-Bodies to date involve fusion with peptidic
pharmacophores. Peptides hold tremendous therapeutic and
commercial promise because they possess enormous struc-
tural diversity that are highly specific and potent, and
generally have fewer safety issues relative to small mole-
cules. Despite these specificity and potency advantages,
peptides have been handicapped by their poor half-life,
which is mostly a result of rapid renal clearance and
proteolytic degradation by serum proteases. CovX-Body
technology provides a means to markedly extend the half-
life of peptide-based drugs without compromising their
specificity and potency. This also presents opportunities
to pursue and resurrect peptide leads that had previously
been abandoned due to poor pharmacokinetic properties.
There are precedents for the conjugation of peptides to
scaffold proteins such as albumin, transferrin, and Fc frag-
mentsthathaveresultedinmolecules that are presently
undergoing clinical evaluation [13-16]. In contrast with
peptide fusion constructs generated at the DNA level and
expressed in cell cultures, the CovX-Body technology
involves a chemical fusion approach that permits the use
of peptides with non-natural structural features such as
noncoded amino acids, peptoids, N- and C-terminus caps
and stapled or otherwise constrained backbones. In addi-
tion, chemical fusion allows tremendous flexibility in mod-
ifying the linker position and composition for optimal
efficacy, stability and physicochemical properties.

drug product. When working with negatively charged
peptides,thattendtoremainassociatedwithCovX-Bodies
as a result of the Gibbs-Danan equilibrium, a charged
membranecanbeaddedtothepurificationprocess.In
those situations, a cation exchanging filter has always been
highly efficient at capturing unreacted peptides in a single
pass while letting the CovX-Bodies flow through without
any adsorption.
Drug Product release is performed using conventional
assays classically used for the release of therapeutic mono-
clonal antibodies. Similarly, Drug Product sterility is
ensured by classic isolation and filtration techniques in
use with most parenteral products.

38.3 APPLICATIONS OF THE CovX-BODY
TECHNOLOGY

38.3.1 CVX-060, a Selective Angiopoietin-2 Targeting
CovX-Body

Angiopoietins are ligands of the endothelial receptor Tie-2
and play a crucial role in the tumor angiogenic switch. It is
generally accepted that Angiopoietin-1 (Ang1) and Angio-
poietin-2 (Ang2) have opposing effects on tumor angiogen-
esis, and that increased expression of Ang2 relative to Ang1
in tumors promotes angiogenesis in presence of other growth
factors such as VEGF and PDGF [17]. The product concept
for CVX-060 is based on the premise that selectively
removing Ang2 from a tumor will restore the Ang2/Ang1
balance and inhibit angiogenesis and tumor growth [18]. A
series of analogs of a selective Ang2 binding peptide were
synthesized for fusion with CVX-2000, see Table 38.1. The
effect of the tether attachment site on the properties of the
resulting CovX-Body was evaluated by moving the tether
position across the length of the peptide. This was done by a
serial substitution of Lys at each position of the peptide and
subsequently using the

38.2.4 Manufacture, Process, Characterization

The scaffold antibody, CVX-2000 is expressed in Chinese
hamster ovary cells and purified at kilogram scale for
clinical use by a standard three-column process that includes
protein-A, anion exchange, and cation exchange chromato-
graphic steps. AZD tethered peptides are readily synthesized
at process scales using standard Fmoc-solid phase peptide
synthesis methods. The manufacturing process of CovX-
Bodies is optimized for robustness and simplicity. AZD-
peptides are solubilized shortly before use in either aqueous
buffer or DMSO depending on their physical properties.
They are then added directly to CVX-2000 in long-term
storage buffer and left to react with the catalytic antibody,
generally overnight at room temperature. Reaction progress
between CVX-2000 and the AZD-peptide can be monitored
using multiple orthogonal methods, such as reduction of
CVX-2000 specific activity, displacement of bound ANS,
reduced availability of CVX-2000 to form enaminone bonds
with diketones, and presence of CovX-Body by LC/MS or
by standard HPLC such as HIC.
The CovX-Bodies resulting from the reaction of the
AZD-peptide and CVX-2000 are purified by tangential
flow filtration to remove unreacted peptides and other low
molecular weight process related impurities from the bulk

-amino group of the Lys side chain
as a site for tethering the AZD linker. The resulting CovX-
Bodies were tested in an in vitro Ang2-Tie2 competition
ELISA assay. The pharmacokinetic profile of CovX-Bodies
was studied in mice to identify molecules with the best
combination of potency and pharmacokinetics. This “tether
walk” exercise showed the dramatic effect of tether position-
ing on the potency and pharmacokinetic profile of CovX-
Bodies. The molecules linked through either the N- or
C-termini were active with IC50s of 1.8 and 0.2 nM, respec-
tively. Tethering at certain internal residues (4, 6, 7, 10, and
13) resulted in complete loss of antagonist activity of CovX-
Bodies, whereas tethering at positions 5, 14, and 17 resulted
in significantly compromised potency. All the other internal
sites of attachment resulted in active CovX-Bodies with
potencies comparable to N- or C- terminal tethering. The
pharmacokinetic profile of the CovX-Bodies showed a
dependence on the tether site as molecules bearing a tether

e