CovX-BODIES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 556

cost of each product, the requirement of multiple infusions
and possible compliance and regulatory hurdles. CVX-241 is
a novel, first-in-class bispecific antibody that neutralizes two
important tumor angiogenesis growth factors in vitro as well
as in vivo and shows preclinical anti-tumor efficacy equal to a
combinations achieved by mixing distinct VEGF and Ang2
inhibiting agents that are not part of the same molecule.
CVX-241 was evaluated for its ability to inhibit the
interaction between human VEGF and its receptor VEGFR2,
as well as the interaction between human Ang2 and its
receptor Tie-2 using competitive binding ELISAs. CVX-
241 inhibited the binding of human rVEGF165 to human
VEGFR2 with high potency, IC 50 ¼

other. In this way, the pharmacokinetics and pharmaco-
dynamics of the bi-specific CovX-Body can be optimally
balanced in a controlled manner. Furthermore, replacing one
or both of the peptide pharmacophores with a new peptide
pharmacophore targeting a different protein can lead to a
completely new bi-specific agent. For example, a novel bi-
specific CovX-Body, targeting a v b 3 integrin and Ang2, was
made by replacing the VEGF peptide of a bi-specific CovX-
Body with a a v b 3 integrin binding peptide and tailoring the
linker accordingly. By changing the peptide pharmacophores,
this technology allows creation of multiple bi-specific prod-
ucts from one scaffold antibody.

0.9 nM, comparable to
the monofunctional VEGF binding CovX-Body. It also inhib-
ited the binding of hAng2 to hTie-2with similar potency to the
Ang2 binding CovX-Body (CVX-060), IC 50 ¼

38.3.3 CVX-343, Fusion of a Small Protein
with CVX-2000

0.6 nM. Addi-
tional in vitro binding experiments showed that CVX-241was
able to simultaneously bind both Ang2 and VEGF. Mouse PK
studies with CVX-241 showed that the Ang2 binding activity
had a half life of 91 hwhereas the VEGF binding activity had a
life of 92 h. In xenograft studies, CVX-241 showed an anti-
tumor effect that was significantly greater and prolonged than
either mono-specific CovX-Bodies alone and comparable
with the physical combination of the two mono-specific
CovX-Bodies. These results clearly demonstrated the supe-
rior therapeutic potential of the bispecific CovX-Body, CVX-
241, over the corresponding monospecific CovX-Bodies and
comparable efficacy to that of physical combination of two
monospecific CovX-Bodies. To demonstrate that CVX-241
targets both Ang2 and VEGF in vivo, the effects of CVX-241
on tissue Ang2 levels and phosphorylated VEGFR2
(pVEGFR2) levels were assessed using immunofluorescence
in Colo205 xenograft tumors. To measure Ang2 expression
within the tumors, frozen sections of these tumors were
stained with a FITC-labeled anti-Ang2 monoclonal antibody.
In CVX-241-treated groups, Ang2 levels were significantly
reduced by

FGF21, a member of the FGF19 subfamily, plays an important
role in regulating glucose and lipid homeostasis in animal
models. Administration of FGF21 improves insulin sensitivity,
reduces body weight, and reverses hepatic steatosis in diabetic
rodents and monkeys. Due to its short half-life, daily injection
of the protein is required for in vivo bioactivity. CVX-343 was
created by covalently linking two FGF21 proteins to the Fab of
CVX-2000 IgG1 mAb via an AZD linker [34]. Similar to the
FGF21 protein, CVX-343 increased Glut1 mRNA expression
in 3T3-L1 adipocytes with an EC 50 of 3.4 nM. In ob/obmice, a
single SC administration of CVX-343 significantly reduced
body weight gain, improved glucose tolerance, and lowered
liver triglyceride levels in a dose-related manner compared
with the vehicle control up to 6 days post dose, demonstrating
the prolonged pharmacodynamics of this molecule in vivo.In
diet-induced obese mice, once-weekly administration of
10mg/kgCVX-343 for 2weeks caused7%weight losswithout
affecting food intake, significantly decreased glucose AUC by
17% in an oral glucose tolerance test, and lowered serum
triglycerides and NEFA levels by 48 and 69%, respectively,
in comparison with vehicle controls. In db/ db mice, once-
weekly administration of CVX-343 (10mg/kg) improved glu-
cose toleranceand increasedpancreatic b -cell mass by 2.4-fold
in the absence of a body weight effect. FGF21 CovX-Body
CVX-343 shows comparable potency as the wild type FGF21
and prolonged efficacy across multiple preclinical models.
This long-acting FGF21molecule has an attractive therapeutic
profile for type 2 diabetes and is an example of the application
of CovX-Body technology beyond small molecules and pep-
tides to therapeutically relevant small proteins.

70% in comparison with the vehicle-treated
group. To measure pVEGFR2 levels within the treated
tumors, frozen sections of these tumor samples were also
double-stained with a FITC-labeled VEGFR antibody and a
rhodamine-labeled pVEGFR2 antibody, and the pVEGFR2
immunoreactivity was quantified. pVEGFR2/VEGFR2 was
significantly reduced by CVX-241 treatment in a dose-depen-
dent manner in comparison with the vehicle treated group.
These data demonstrate that CVX-241 affects both Ang2 and
VEGF pathways in the Colo205 xenograft model.
Peptide payload modification allows design of a bispecific
CovX-Body with desired characteristics. As can be expected,
there is a complex interplay between the composition of each
peptide payload, the site of attachment of the tether to each
peptide payload and the composition and length of the
spacers. This versatility is a unique feature of the technology
in that it facilitates critical optimization of the target binding
affinity and pharmacokinetics of the payloads relative to each

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