SPECIFICALLY TARGETING ANGIOPOIETIN-2 INHIBITS ......Jan 12, 2011 · FBS in a 5% CO2 incubator with 98% humidity to sub-confluence. 5x10 6 Colo-205 cells alone or 5x106 MDA-MB-435

1

SPECIFICALLY TARGETING ANGIOPOIETIN-2 INHIBITS ANGIOGENESIS, TIE2

EXPRESSING MONOCYTE INFILTRATION AND TUMOR GROWTH

Hanhua Huang, Jing-Yu Lai, Janet Do, Dingguo Liu, Lingna Li, Joselyn Del Rosario, Venkata R.

Doppalapudi, Steven Pirie-Shepherd, Nancy Levin, Curt Bradshaw, Gary Woodnutt, Rodney

Lappe, and Abhijit Bhat

CovX Research, Pfizer Inc. 9381 Judicial Dr. Suite 200, San Diego, CA 92121.

Running title: Development of Angiopoietin-2 Specific CovX-Bodies

Corresponding author: Abhijit Bhat, CovX Research, Pfizer Inc. 9381 Judicial Dr. Suite 200,

San Diego, Ca 92121. Phone: 858-964-2000. Fax: 858-964-2090. Email:

[email protected].

Current address for C. Bradshaw: Traversa Therapeutics, Inc. 10480 Wateridge Circle, San

Diego, CA 92121

Disclosure of potential conflicts of interest

Hanhua Huang, Jing-Yu Lai, Janet Do, Dingguo Liu, Lingna Li, Joselyn Del Rosario, Venkata R.

Doppalapudi, Steven Pirie-Shepherd, Nancy Levin, Gary Woodnutt, Rodney Lappe and Abhijit

Bhat are employees of CovX Research, Pfizer Inc. Curt Bradshaw is a former employee of

CovX Research.

Research. on April 12, 2021. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 13, 2011; DOI: 10.1158/1078-0432.CCR-10-2317

http://clincancerres.aacrjournals.org/

2

TRANSLATIONAL RELEVANCE

The application of peptides in drug development has been hindered by their poor in vivo stability,

which could be improved by technologies such as pegylation and fusion proteins. Here we

describe in detail a new strategy, irreversibly fusing peptides to a scaffold antibody with defined

regiochemistry and stoichiometry, resulting in metabolically stable bivalent fusion proteins,

CovX-Bodies. A successful example of anti-angiopoietin-2 (Ang2) CovX-Body CVX-060 is in

phase II clinical trial. Our data demonstrates that targeting Ang2 alone or in combination with

other standard of care agents could potentially lead to improved clinical benefit.

ABSTRACT

Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, while in

tumor Ang2 antagonizes Ang1’s function and promotes the initiation of the angiogenic switch.

Specifically targeting Ang2 is a promising anti-cancer strategy. Here we describe the

development and characterization of a new class of biotherapeutics referred to as CovX-Bodies,

which are created by chemical fusion of a peptide and a carrier antibody scaffold.

Experimental Design: Various linker tethering sites on peptides were examined for their effect

on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nM IC50s and

significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in

combination with standard of care agents. Tumor samples were analyzed for target engagement,

via Ang2 protein level, CD31 positive tumor vasculature and Tie2 expressing monocyte

penetration.

Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2-Tie2 interaction (IC50 < 1nM)

with dramatically improved pharmacokinetics (T½ > 100 hour). Using a staged Colo-205

xenograft model, significant tumor growth inhibition (TGI) was observed (40-63%, P < 0.01).




3

Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), while tumor

microvessel density (P < 0.01) and intratumor proangiogenic Tie2+CD11b+ cells (P < 0.05) were

significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan or

docetaxel, Ang2 CovX-Bodies produced even greater efficacy (~80% TGI, P < 0.01).

Conclusion: CovX-Bodies provide an elegant solution to overcome the PK-PD problems of

peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in

combination with standard of care agents.




4

INTRODUCTION

Angiogenesis is one of the hallmarks of tumor progression, allowing the tumor to expand

beyond the limit of oxygen and nutrient perfusion, and eventually metastasize to distant organs

(1, 2). Anti-angiogenic agents such as bevacizumab, sunitinib and sorafenib that target vascular

endothelial growth factor (VEGF) signaling pathways have demonstrated clinical utility in

treating solid tumors. However these agents result in only transient clinical responses and

tumors eventually develop resistance and escape the angiogenic blockade. The various

mechanisms of resistance can be attributed to the inherent heterogeneity of tumor cells. There

is a strong interest in targeting other proangiogenic factors along with VEGF as simultaneous

targeting of multiple pathways could translate into more robust therapeutic outcomes. The

angiopoietin-Tie system presents an attractive therapeutic target as it plays an important role in

tumor angiogenesis and provides a critical link between angiogenic and inflammatory pathways

(3).

The human angiopoietin-Tie system consists of two type-I tyrosine kinase receptors (Tie1 and

Tie2) and three secreted ligands (Ang1, Ang2 and Ang4) (3, 4). Of the three angiopoietin

ligands, Ang1 is constitutively expressed in many organs, while Ang2 is predominantly

expressed in tissues undergoing vascular remodeling (5, 6). Ang2 overexpression in many

cancers correlates with poor survival and more invasive cancers (3, 7). No clear correlation was

found between Ang1 expression and prognosis in several solid tumors, such as colorectal

carcinoma (8), bladder cancer (9), or non-small-cell lung cancer (NSCLC) (10). Recently, in

head and neck squamous cell carcinoma, high Ang1 was reported to correlate with better

prognosis (11). In a remodeling endothelium, such as those in tumors and diabetic retinopathy,

Ang2 acts as an Ang1 antagonist, promotes the dissociation of pericytes, resulting in unstable

vasculature (4). In addition to angiogenesis, Ang1 and Ang2 play a role in the inflammatory and




5

metastatic pathways through their ability to modulate the endothelial barrier and the integrity of

the blood vessels (3). High levels of Ang2 induce leaky vasculatures that facilitate the

extravasation of lymphocytes and promote the adhesion of rolling leukocytes to blood vessels

(12). Circulating Tie2+ monocytes have been shown to contribute to tumor angiogenesis (13-16).

Tumors with overexpressed Ang2 are more vascularized and have more Tie2 expressing

monocyte penetration (14). These findings suggest that increased Ang2 over Ang1 promotes

tumorigenesis and blocking Ang2 is an attractive strategy for cancer treatments. A selective

Ang2 trap significantly inhibits tumor growth, whereas treatment with a selective Ang1 trap has

no effect on tumor growth in a Colo-205 xenograft model (17). The peptibody AMG-386 in

various clinical trials traps both Ang1 and Ang2 and inhibits tumor growth preclinically (18, 19).

Herein we describe the development and characterization of peptide-antibody fusion proteins

referred to as CovX-Bodies, for selectively targeting Ang2 as a potential antiangiogenic

anticancer agent.




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MATERIALS AND METHODS

Synthesis of peptide phamacophore with the azetidinone linker and preparation of CovX-

Bodies

All peptides were synthesized on a Symphony peptide synthesizer using Rink Amide resin

(Nova-Biochem, 100-200 mesh). Briefly, peptides were made using sequential deprotection and

coupling with Fmoc protected amino acids. During the peptide synthesis ε-Mtt-Fmoc-Lys-OH

was used at the desired linker tethering site. Once the peptide sequence was assembled on the

resin, the ε-amino group of Lys was unmasked by treatment with 1% TFA in dichloromethane,

and coupled to the azetidinone (AZD) linker. The tethered peptide was cleaved off the resin by

brief exposure to a solution of 95% TFA in water and purified by preparative reverse phase

HPLC using a Zorbax SB-C18 column. The purity of the final products was assessed by

analytical reverse-phase LC/MS.

Peptide phamacophores with AZD linkers were combined at a 3:1 molar ratio with CVX-2000, a

humanized IgG1 monoclonal aldolase antibody, used at 20 mg/mL in a buffer of 10 mM histidine,

10 mM glycine, 2% sucrose, pH 6.5. The AZD linker was designed to be recognized and

covalently bound only by the reactive lysine in the binding site of CVX-2000. After incubating at

room temperature overnight, free pharmacophore was removed from the reaction solution by

size exclusion chromatography (SEC) using a Superdex 200 10/300 GL column on an

AKTAPurifier (GE healthcare). The SEC buffer was 0.5 M sodium chloride, 0.1 M sodium

phosphate, pH 6.5. CovX-Bodies were exchanged into PBS using centrifugal filters and their

valency was confirmed by LC-MS. This process is referred to as programming hereafter and the

resulting peptide antibody fusions are termed as CovX-Bodies (Figure 1).




7

Animal Models and Dosing agents

For pharmacokinetic studies, male Swiss Webster mice or Sprague Dawley rats (Charles Rivers,

Hollister, CA) were used. For rats, jugular vein and carotid arterial cannulae were surgically

implanted at Charles River Laboratories and animals were housed separately. 10 mg/kg CovX-

Bodies were injected intravenously through the tail vein for mice and via implanted jugular vein

cannulae for rats. For tumor studies, young adult female Nu-Foxn1nu mice (Charles River

Laboratories, MA) were housed 10 per cage in a temperature- and light-controlled vivarium.

Tumor cell lines Colo-205 and MDA-MB-435 were cultured in RPMI 1640 medium with 10%

FBS in a 5% CO2 incubator with 98% humidity to sub-confluence. 5x106 Colo-205 cells alone or

5x106 MDA-MB-435 cells mixed with matrigel (1:1, BD Bioscience, CA) were injected

subcutaneously into the upper right flank of each mouse. The tumor volume was calculated

using the formula: Volume = (Length x Width2) x π/6. All animal experiments were conducted

after protocol approval by the CovX Institutional Animal Care and Use Committee.

Ang2-Tie2 competition and pharmacokinetic ELISA

Competition ELISA was used to determine the in vitro potency of CovX-Bodies in blocking the

Ang2-Tie2 interaction. Human Tie2-Fc, human Ang2 protein, biotinylated anti-human Ang2

antibody and streptavidin HRP were from R&D Systems. Briefly, high-binding half-well plates

were coated with 50 ng Tie2-Fc in PBS. Compounds were diluted in the presence of 50 ng/mL

Ang2. Bound Ang2 was detected by a biotinylated anti-Ang2 antibody used at 2 μg/mL. For

pharmacokinetic analysis of CovX-Bodies, after intravenous administration of CovX-Bodies,

blood samples (~0.08 mL/bleed) were taken from mice via retro orbital sinus bleeds into

capillary blood collection tubes (BD Biosciences, Franklin Lakes, NJ) with protease inhibitor

cocktail (Sigma, St. Louis, MO) starting at 5 min up to 7 or 14 days. The blood samples were

allowed to clot on ice for 30 minutes. Samples were then centrifuged at 12000 rpm for 10




8

minutes at 4°C to collect serum and immediately stored at -80 °C until analysis. The detection

assay conditions were optimized for reproducibility and sensitivity (limit of detection was as low

as 10 ng/mL). 50 ng Ang2 protein in PBS was coated as a capture reagent overnight at 4°C.

The plates were blocked with Superblock (Scytek, Logan, UT) for one hour at room temperature.

Samples and standards were prepared in 10% mouse serum in Superblock, added to blocked

plates, and incubated for one hour. A HRP anti-human IgG (1:2000, Bethyl Laboratories, TX)

was used to detect CovX-Bodies. Data analysis was undertaken with WinNonlin version 4.1

(Pharsight Corporation) using a weighted 1/Y2 two compartment model.

Tumor xenograft studies

Subcutaneously inoculated tumors staged to the desired volume (average about 200 mm3) were

randomized and dosed accordingly. Ten animals were used per group. Bevacizumab

(AvastinTM), docetaxel (TaxotereTM), irinotecan (CamptosarTM), and 5-FU (fluorouracil) were

diluted to the desired dosing concentrations with either phosphate-buffered saline (for

bevacizumab) or 5% dextrose in water (for docetaxel, irinotecan, and 5-FU), immediately prior

to administration, and were administered intraperitoneally in a volume of 0.2 ml/mouse. CovX-

Bodies were administered intravenously in a volume of 0.2 ml/mouse. 16 mg/mL sorafenib

(Biomol, PA) was prepared fresh every 3-4 days in cremophor/ethanol (50:50; Sigma cremophor

EL, 95% ethanol). The final dosing solution (4 mg/mL) was prepared by diluting with water.

Sunitinib was prepared in 0.5% polysorbate 80, 10% polyethylene glycol 300 and 1:1.02 molar

ration of hydrochloric acid to sunitinib, pH 3.5. Sorafenib and sunitinib were dosed by oral

gavage. For combination studies, the control groups were given vehicles used for both agents.

Tumor growth data were analyzed using two-way ANOVA with Bonferroni post-tests between

groups using Graphpad Prism Ver 5.0, and depicted as mean + SEM.




9

Histological analysis

For tumor necrotic studies, four five-µm sections, five sections apart from each other, were

prepared and stained with H&E following standard histological procedures. To determine

necrotic and viable tumor area, whole field images of each stained tumor section were acquired

at 10x magnification using a Qimaging Micropublisher 5.0 RTV camera coupled with a Nikon

Eclipse 80i microscope equipped with an automatic stage. ImagePro 6.0 was used to quantify

the necrotic and viable area by using the tracing function and a common macro for all images.

For CD31 positive vasculature staining, snap-frozen tumor sections were oriented and

embedded in OCT blocks. Three sections (5 μm) from different depth of the tumor mass were

used for each tumor. CD31 antibody and anti-Rat IgG detection kit (Pharmingen, San Diego,

CA) were used for blood vessel staining. CD31 positive area in each tumor was imaged using a

Qimaging Micropublisher 5.0 RTV camera coupled with a Nikon Eclipse 80i microscope (20X).

Five images were taken across each tumor section where CD31 staining was greatest in the

viable rim so that a total of 15 images were taken for each tumor. The number of blood vessels

was determined by operators blinded to the study design and the total number of vessels from

the 15 images was presented as microvessel density (MVD). For Tie2+ macrophages, the

primary antibodies were mouse anti-Tie2 (Pharmingen, San Diego, CA) and rat anti-CD11b

(Abcam, Cambridge, MA). Three images (40x) from the area with the greatest CD11b staining

were taken and counted by operator blinded to the treatments. 9-10 animals were used per

group and one animal from the treatment group was lost during the study, which was not related

to the treatment since all other animals in the group were normal and not found in subsequent

studies. Histological data were analyzed by one way ANOVA followed by Dunnett’s posthoc

analysis.




10

RESULTS

Tethering at different sites of a peptide pharmacophore impacts the activity and

pharmacokinetics of CovX-Bodies

Fusing peptides to various scaffolds through their N- or C- termini has been studied extensively,

while our approach allows us to fuse peptides to a specially-designed scaffold not only through

their termini but also via middle residues. The AZD linker used here creates an irreversible

covalent bond between a peptide and the scaffold and is different from that used in previously

published CovX-Bodies based on a reversible diketone linker. The improvement on

pharmacokinetics with these reversible linkers was not sufficient for most clinical drug

development (20, 21). A series of CovX-Bodies were made as shown in Table 1 by moving the

tether attachment site across the length of the peptide. This was done by a serial substitution of

Lysine at each position of the peptide using the ε-amino group of the Lysine side chain as a site

for tethering the AZD linker (Figure 1). The resulting CovX-Bodies were tested in an in vitro

Ang2-Tie2 competition ELISA assay. The pharmacokinetic profile of active CovX-Bodies was

studied in mice in order to identify molecules with the best combination of potency and

pharmacokinetics. This tether walk exercise showed the dramatic effect of tether positioning on

the potency and pharmacokinetic profile of the resulting CovX-Bodies. CovX-Bodies linked

through either the N- or C-termini were active with IC50s of 1.83 and 0.22 nM, respectively.

Tethering at certain internal residues (4, 6, 7, 10 and 13) resulted in a complete loss of

antagonist activity (IC50 > 1000 nM) of CovX-Bodies, whereas tethering at positions 5, 14, 17

resulted in significantly compromised potency. All other conjugations had IC50s within the range

of the terminally linked ones (Table 1). The pharmacokinetic profile of the CovX-Bodies showed

a dependence on tether site as molecules bearing a tether at internal sites showed better half-

lives compared with the ones tethered near either terminus. Thus, CovX-Bodies with internal




11

tethers (9, 11, 16 and 18) resulted in much improved in vivo stability with a beta half life of 72-94

hours, when compared with terminal fusions, with a beta half life of about 20 hours (Table 1).

However, with different peptide pharmacophores (CVX-37 and CVX-51), no significant

improvement in pharmacokinetics was observed by linking through the middle residue (Table 1).

For compound CVX-32, changing the linker by modifying the PEG length or the linking amino

acid to diamino butyric acid (Dab) or diamino proprionic acid (Dap) did not have an effect on the

potency or pharmacokinetic profile (data not shown).

Additional changes in the pharmacophore further improved in vivo stability

In an attempt to improve its potency and pharmacokinetic profile, CVX-32 was subjected to a

series of point mutations with both natural and unnatural amino acids including several non-

natural capping groups at the N-terminus. Most of the changes introduced failed to identify

significantly better molecules except when the Lys at position 9 was changed to acetyl Lysine,

the resulting molecule CVX-060 had a half life of 110 hours (Table 1). In rats, CVX-060 had a

beta half life of 80 hours, which was improved over CVX-32 and CVX-87 with beta half lives of

51 and 55 hours in rats, respectively (P<0.01). CVX-060 had no direct binding to human Ang4

or its mouse ortholog Ang3 protein coated on plates while Tie2 did bind to them (sFigure 1A and

B). CVX-060 interacted with mouse Ang2 with EC50 of 0.35 nM, compared to 0.04 nM for human

Ang2 (sFigure 1C and D). Ang1-Tie2 interaction was not affected by CVX-060 up to 1 uM

(sFigure 2A). CVX-2000, the antibody scaffold, had no effect on the Ang2-Tie2 interaction up to

5000 nM (sFigure 2C). CVX-060 block mouse Ang2 and mouse Tie2 interaction with an IC50 of

about 8 nM (sFigure 2D).

CovX-Body programming improved the bioactivity of the peptide pharmacophores

Ang2 CovX-Bodies efficiently blocked the Ang2-Tie2 interaction in the competition ELISA. When

compared with the activity of tethered peptide pharmacophores, programming significantly




12

improved in vitro potency (Table 1, sFigure 2B), which may be due to the bivalent nature of

CovX-Bodies, resulting in increased avidity of the pharmacophores. The two binding sites were

demonstrated by surface plasma resonance for CVX-060 and its AZD tethered pharmacophore.

The predominant binding site of CVX-060 for Ang2 surface had a KD of 1.8 μM, which

corresponded well to the KD of the pharmacophore itself at 4.0 μM. The higher affinity site had a

KD of 4.9 nM, which was the affinity of the bivalent complex (sFigure 3). With these data, we

have demonstrated that we can significantly improve the in vivo stability and bio-activity of

peptides.

Ang2 CovX-Bodies reduced the level of Ang2 protein in tumor mass and inhibited the

growth of staged xenografts

The anti-tumor activity of the Ang2 CovX-Bodies was evaluated in several staged xenograft

models. Tumors were allowed to establish to an average volume of 200 mm3 prior to initiation of

therapy. CVX-32, CVX-37, CVX-060, and CVX-87 inhibited the growth of staged Colo-205

tumors significantly (P <0.01), with TGI values ranging from 40-63% (Figure 2A). When the

AZD tethered peptide pharmacophore CVX-32T of CVX-32 (0.4 mg/kg) was given at the molar

equivalent of pharmacophore present in 10 mg/kg CVX-32, it had no effect on the tumor growth

demonstrating the benefit of the extended pharmacokinetics of CovX-Bodies (Figure 2B).

To explore if Ang2 CovX-Bodies reached the target Ang2 protein inside the tumor mass, snap-

frozen tumors were homogenized and the total human Ang2 level in these tumors were

determined by ELISA. CVX-37 and CVX-060 significantly reduced Ang2 protein level by about

50% in the tumor mass (P < 0.01, Figure 2C). This was also confirmed by

immunohistochemistry and no significant change in Ang1 protein level was found by either

ELISA or IHC (data not shown). While Ang2 is reported to be mostly secreted by endothelial

cells, our data and recent reports from other groups indicate that some tumor cells express high




13

levels of Ang2 protein (22-24). In addition, we examined CVX-32 dosed intravenously at 15

mg/kg, twice weekly, in staged A549, HT-29 and A431 models that did not express significant

amounts of human Ang2 by ELISA or IHC, we observed 17-35% TGI (data not shown),

suggesting that Ang2 CovX-Bodies could block mouse endothelial derived Ang2 and caused

tumor growth inhibition.

Tumor vasculature and viability were reduced in treated tumors

Reducing Ang2 from the tumor mass may affect the formation of new blood vessels. Frozen

tumor sections were stained for CD31, an endothelial marker. CD31 positive microvessels were

significantly (P< 0.01) reduced by CVX-37 and CVX-060 (Figure 2D and E). H&E staining of

paraffin sections of CVX-37 and CVX-060 treated tumors were analyzed and significant

necrosis (P < 0.05) was found in these tumors (Figure 3A and C). Reduction of Ang2 levels

resulted in less blood vessel formation and increased tumor cell death in the center. The

treatment however left a viable rim to the tumor, which is similar to other anti-angiogenic agents.

Tie2+ CD11b+ macrophages were reduced in Ang2 CovX-Body treated tumors

Circulating Tie2+ monocytes have been reported to contribute to tumor angiogenesis (13-16).

We explored the effect of Ang2 reduction on Tie2+ macrophages in tumors by staining for Tie2+

CD14+ or Tie2+ CD11b+ cells. CVX-37 and CVX-060 significantly reduced the number of Tie2+

CD11b+ cells inside the tumors (Figure 3B and D, P<0.001) and similar data was observed

when CD14 was assessed (data not shown). These data indicate that Ang2 may be responsible

for recruiting Tie2+ monocytes into tumors, and further contributing to tumor angiogenesis in

addition to its direct role on endothelial cells.




14

Combining Ang2 CovX-Bodies with sunitinib, sorafenib and bevacizumab significantly

inhibited tumor growth

Patients with high expression of both VEGF and Ang2 have much worse prognosis than those

with either factor alone, suggesting that targeting both of them may help these patients (25, 26).

Ang2 compromises anti-VEGFR2 vessel normalization effect by increasing vessel permeability

(27). However, Ang2 could have both pro and anti-tumor activities (3). Blocking VEGF function

is also known to affect Ang2 expression. In advanced renal cell carcinoma patients treated with

sunitinib, plasma Ang2 levels were initially decreased and later increased when patients

developed resistance to sunitinib (28). We examined the potential combination benefit of

blocking both Ang2 and VEGF pathways by combining CovX-Bodies with known VEGF pathway

inhibitors, sunitinib, sorafenib and bevacizumab, in Colo-205 and MDA-MB-435 xenograft

models. CVX-060 was dosed at 10 mg/kg weekly, sunitinib (Figure 4A) or sorafenib (Figure 4B)

shown) was dosed at 20 mg/kg daily, and bevacizumab (Figure 4C, D and E) was given at 3 or

10 mg/kg, alone or in combination. All the agents when dosed alone demonstrated significant

tumor growth inhibition. When they were combined with CVX-060, there was a further reduction

in tumor growth reflecting additive combination benefits (Figure 4). For the MDA-MB-435 model

(Figure 4E), the study was designed to determine the delay of tumors reaching a pre-

determined volume (2000 mm3). The tumors in the combination group did not reach the pre-

determined limit after three months, while all the other groups reached the limit approximately

two months after dosing. These data demonstrate a clear combination benefit of blocking the

two angiogenic factors.




15

Combining Ang2 CovX-Bodies with docetaxel and irinotecan significantly inhibited tumor

growth

Anti-angiogenic treatments could potentially improve the delivery of chemotherapy agents

through blood vessel normalization. As demonstrated above, blocking Ang2 reduced the

microvessel density in tumors and its effect on chemotherapy agents was further explored. 10

mg/kg CVX-060 was combined with 5-FU, docetaxel, or irinotecan. All the agents when dosed

alone reduced tumor growth (Figure 5). When the chemotherapies were combined with CVX-

060, there was a further reduction in tumor volume reflecting an almost complete cessation of

tumor growth for combinations with docetaxel or irinotecan. The groups were terminated when

the tumor volume reached a pre-determined volume (2000 mm3) and the combination groups of

docetaxel and irinotecan never reached this limit during the time of the study. These data

indicate that Ang2 specific agents could be combined with standard of care chemotherapy

agents with potential benefit for patients.




16

DISCUSSION

Peptides hold significant therapeutic potential given the readily accessible diversity in their

structural and biological properties. However the promise of peptides remains to be translated

into available therapies primarily because peptides are handicapped by their poor in vivo

stability. Carrier proteins such as Fc domains of an immunoglobulin have been used as

scaffolds to improve the metabolic stability. These constructs are often expressed genetically

and are limited to fusing through the N- or the C-terminus of the peptide. Recombinant

approaches are also unable to easily incorporate non-natural structural elements, such as end-

capping, amino acid surrogates, and amide bond isosteres, which can often improve the

potency and metabolic stability of peptidic pharmacophores. Peptides can also be fused to

higher molecular weight biopolymers or coupled to synthetic carriers such as PEG, HES,

polysialic acid, which do not have defined stoichiometry. In contrast, with CovX-Bodies, a

systematic SAR can be built around tethering site and linker composition for creating the optimal

fusion proteins, and the regiochemistry and stoichiometry of the peptide attachment is precisely

defined. The peptide pharmacophores can carry a desired combination of natural and non-

natural structural elements. CovX-Bodies provide an elegant solution to overcome the PK-PD

limitations of peptidic pharmacophores. The antibody carrier scaffold prevents the peptides

from being rapidly cleared via renal filtration and the antibody scaffold also partially protects the

peptide from rapid proteolytic degradation. CovX-Bodies are also unique in the respect that the

scaffold antibody has no known targets in vivo and can be employed across multiple therapeutic

programs. The pharmacophore determines the targeting properties of CovX-Body whereas the

antibody scaffold provides bivalency and long circulating half-life, resulting in an improved

dosing regimen relative to the peptide alone. The application of this technology was

successfully demonstrated by generating long acting bioactive CovX-Bodies targeting Ang2, an

important target in tumor angiogenesis.




17

For angiogenesis to occur, pre-existing blood vessels are stimulated by several hypoxia-driven

angiogenic factors. VEGF and angiopoietins have been increasingly shown to play key roles in

this angiogenic switch process. Ang1 is constitutively expressed in normal tissues and is

deemed essential to the recruitment of pericytes and maturation of newly formed blood vessels.

Ang2 is a key player in the destabilization of pre-existing blood vessel though antagonizing the

function of Ang1. Ang1 activates Tie2 and results in recruitment of pericytes, anti-permeability

and anti-inflammatory effects on endothelial cells (29, 30). Conversely, the actions of Ang2 are

context dependent. Ang2 acts as a partial Tie2 agonist under certain experimental conditions,

such as in the absence of Ang1 (31), when used as a designed pentameric COMP-Ang2 variant

(32), or when present in high concentrations (33). A high Ang2/Ang1 ratio correlates with poor

prognosis in many solid tumors(7). Conditional overexpression of Ang2 in endothelial cells in

vivo confirmed that Ang2 can inhibit Tie2 phosphorylation (34). On the other hand, ectopic

overexpression of Ang2 in tumor cells inhibits glioma tumor growth (35, 36), which may be

caused by defective angiogenesis due to the death of Ang2 induced unstable blood vessels in

the absence of abundant VEGF. These data are consistent with the hypothesis that Ang2

initiates the angiogenic process by promoting the dissociation of pericytes and results in

unstable blood vessels. Our data here indicate that systemic and specific reduction of the Ang2

protein can inhibit angiogenesis and tumor growth, supporting Ang2’s role in the pathological

angiogenic initiation process.

In addition to all the angiogenic factors from tumor cells and tumor endothelial cells, other host

cells, such as vascular progenitor cells and monocytes, could play critical roles in tumor

angiogenesis. Cells that express various monocytic/dendritic cell markers, such as Tie2+

monocytes, among others are involved in tumor vascularization (13). Myeloid cells such as

monocytes/macrophages may trigger vessel growth by releasing angiogenic factors and may

incorporate directly into nascent blood vessels (37). We demonstrated here that by blocking the




18

Ang2-Tie2 interaction, CovX-Bodies reduced the number of Tie2+ CD11b+ cells inside the tumor

mass, indicating that Ang2 may be responsible for the recruitment of Tie2+ monocytes into the

tumor mass.

Since the identification of angiopoietins, there have been several attempts to interfere with this

pathway. Sequestering both Ang1 and Ang2 can inhibit tumor growth and there were no

significant side effects associated with pharmacologic reduction of Ang1 and Ang2 in mice (18).

In a phase I studies of AMG-386, a peptibody trapping both Ang1 and Ang2, and CVX-060, a

CovX-Body selectively trapping Ang2, significant tumor blood flow reduction was observed with

DCE-MRI and early clinical benefit were observed as well (19, 38). The common side effects of

these agents are mild, fatigue, proteinuria and peripheral edema for AMG-386 (19), fatigue and

proteinuria for CVX-060 (38). If the mild adverse event profile observed in the early clinical trials

persists through advanced clinical development, these agents will be very attractive partners in

combination with other anti-angiogenic or chemotherapeutic agents.

Almost all effective anti-cancer treatments will eventually fail due to resistance. It has been

shown preclinically and clinically that combining targeted treatments, particularly anti-angiogenic

agents, with chemotherapy agents could potentially improve efficacy. Significant benefit was

demonstrated when Ang2 CovX-Bodies were combined with standard of care chemotherapy

agents, such as docetaxel and irinotecan, while the combination benefit with 5-FU is not as

significant. High levels of both VEGF and Ang2 in breast cancer (39), NSCLC (10), ovarian

cancer (40) and AML (41) correlate with a worse prognosis than those with either one alone.

Targeting Ang2, which was reported to be involved in not only angiogenesis, but also in

metastasis and inflammation, may help improve the efficacy of anti-VEGF treatments.

Significant combination benefit was demonstrated when Ang2 CovX-Bodies were combined with

other anti-angiogenic agents, such as sunitinib, sorafenib and bevacizumab. In advanced renal

cell carcinoma patients treated with sunitinib, plasma Ang2 levels first decreased and later




19

increased at resistance to the treatment (28). These data suggest that an Ang2 CovX-Body

could be a very effective addition to the treatment of solid tumors.




20

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Table 1. The in vitro potency of CovX-Bodies tethered at different residues, indicated by K(0P),

in blocking Ang2 -Tie2 interaction and their mouse pharmacokinetics. ND: not determined. 0P:

no PEG unit between peptide and the AZD linker. K(Ac): acetylated Lysine. DFB: 1,5-

difluorobenzoyl. Compounds 1-10, 12-22, CVX-32, CVX-060, CVX-87, CVX-37, and CVX-51

were all tested as CovX-Bodies, while peptides 1, 2 and CVX-060T (the pharmacophores of

CVX-060) were also tested as controls. The IC50s were average data from at least three

independent preparations for each compound.

Compound# Sequence IC50 (nM) T1/2 (hrs) Peptide 1 QK(Ac)YQPLDELDK(Ac)TLYDQFMLQQG 98 0.1 1 K(0P)K(Ac)YQPLDELDK(Ac)TLYDQFMLQQG 1.8 24 2 QK(0P)YQPLDELDK(Ac)TLYDQFMLQQG 0.3 13 3 QK(Ac)K(0P)QPLDELDK(Ac)TLYDQFMLQQG 0.2 18 4 QK(Ac)YK(0P)PLDELDK(Ac)TLYDQFMLQQG >1000 ND 5 QK(Ac)YQK(0P)LDELDK(Ac)TLYDQFMLQQG 44.2 ND 6 QK(Ac)YQPK(0P)DELDK(Ac)TLYDQFMLQQG >1000 ND 7 QK(Ac)YQPLK(0P)ELDK(Ac)TLYDQFMLQQG >1000 ND 8 QK(Ac)YQPLDK(0P)LDK(Ac)TLYDQFMLQQG 0.3 35 9 QK(Ac)YQPLDEK(0P)DK(Ac)TLYDQFMLQQG 2.0 72 10 QK(Ac)YQPLDELK(0P)K(Ac)TLYDQFMLQQG >1000 ND CVX-32 QK(Ac)YQPLDELDK(0P)TLYDQFMLQQG 0.3 74 12 QK(Ac)YQPLDELDK(Ac)K(0P)LYDQFMLQQG 0.1 56 13 QK(Ac)YQPLDELDK(Ac)TK(0P)YDQFMLQQG >1000 ND 14 QK(Ac)YQPLDELDK(Ac)TLK(0P)DQFMLQQG 32.8 ND 15 QK(Ac)YQPLDELDK(Ac)TLYK(0P)QFMLQQG 0.2 66 16 QK(Ac)YQPLDELDK(Ac)TLYDK(0P)FMLQQG 0.3 94 17 QK(Ac)YQPLDELDK(Ac)TLYDQK(0P)MLQQG 19.5 ND 18 QK(Ac)YQPLDELDK(Ac)TLYDQFK(0P)LQQG 0.6 72 19 QK(Ac)YQPLDELDK(Ac)TLYDQFMK(0P)QQG 0.1 65 20 QK(Ac)YQPLDELDK(Ac)TLYDQFMLK(0P)QG 0.1 35 21 QK(Ac)YQPLDELDK(Ac)TLYDQFMLQK(0P)G 0.3 27 22 QK(Ac)YQPLDELDK(Ac)TLYDQFMLQQK(0P) 0.2 20 CVX-060 QK(Ac)YQPLDEK(Ac)DK(0P)TLYDQFMLQQG 0.5 110 CVX-060T QK(Ac)YQPLDEK(Ac)DK(0P)TLYDQFMLQQG 342 ND CVX-87 QK(Ac)YQPLDELDK(0P)TLFDQFMLQQG 0.2 61 Peptide 2 TNFMPMDDLEQRLYEQFILQQG 5.4 ND CVX-37 (DFB)TNFMPMDDLEK(0P)RLYEQFILQQG 0.4 26 CVX-51 (0P)TNFMPMDDLEQRLYEQFILQQG 1.8 28




24

FIGURE LEGENDS

Figure 1. Generation of CovX-Bodies. A pharmacophore comprising a linker and a bioactive

component was synthesized. A spontaneous selective fusion reaction occurs between the

azetidinone ring and the Lys99 of the heavy chain. This nucleophilic reaction transforms the

azetidinone ring into a stable beta alanine-based peptide bond creating CovX-Bodies.

Figure 2. Ang2 CovX-Bodies inhibited staged Colo-205 tumor growth. Colo-205 tumors were

staged to about 200 mm3, and then were treated weekly with vehicle, 10mg/kg of CVX-37, CVX-

060, or CVX-87 (A), CVX-32 and CVX-32T (0.4 mg/kg, B). CVX-32T, a peptide, is the

pharmacophore of CVX-32, a CovX-Body. C. Ang2 protein levels in homogenates of tumors

were determined by a human Ang2 ELISA. Data was expressed as ng/g tumor tissue. D.

Tumor microvessel density was assessed by CD31 staining in vehicle, CVX-37 or CVX-060

treated tumors. Quantification of CD31 staining was done as described in Materials and

Methods. Number of microvessels was presented per 15 HPF per tumor (high power field). ** P

< 0.01, by one-way Anova. SEMs are shown, N=10 per group. E. Representative CD31

stainings (20x) of tumors are shown. Brown for CD31 and blue for nuclear counter staining.

Scale bars represent 100 microns.

Figure 3. Ang2 CovX-Bodies induced necrosis and reduced Tie2+CD11b+ cells inside treated

tumors. Representative H/E stainings from the center of tumors from vehicle, CVX-37 and CVX-

060 treated groups are shown (A) and the pale grey area indicates tumor death in the treated

tumors. Scale bar represents 2 mm and images are in proportion to tumor sizes. Necrotic area

was assessed by H&E staining and quantified using the ImagePro software (C). Representative

immunofluorecence staining (40x) of tumors are shown (B, red for CD11b, green for Tie2 and

blue for the nuclear counter staining). Scale bars represent 50 microns. Arrows on the zoomed-

in images indicate Tie2+ CD11b+ cells and not all positives cells are marked with arrows.




25

Number of Tie2+ CD11b+ macrophages was presented per HPF (D). *. P < 0.05, *** P < 0.001,

by one-way Anova. SEM was shown, N=9-10 per group.

Figure 4. Ang2 CovX-Body CVX-060 inhibited tumor growth when combined with other anti-

angiogenic agents. Colo-205 tumors were staged to about 200 mm3. Tumors were then treated

with weekly intravenous CVX-060 (10 mg/kg), sunitinib (A, 20mg/kg), sorafenib (B, 20mg/kg) or

combination. Combining CVX-060 and anti-VEGF antibody resulted in significant tumor growth

inhibition with Colo-205 (C, 3 mg/kg; D, 10 mg/kg) and MDA-MB-435 xenografts (E). All CovX-

Body treatments were weekly intravenous injections. * P < 0.05 vs. vehicle, ** P < 0.01 vs.

vehicle, P < 0.05 vs monotherapy groups, two-way Anova with Bonferroni test. N=10 per group.

Figure 5. Ang2 CovX-Body CVX-060 inhibited tumor growth when combined with chemotherapy

agents, 5-FU (A, 50 mg/kg), irinotecan (B, 50 mg/kg) and docetaxel (C, 20 mg/kg). 10 mg/kg

CVX-060 was dosed by weekly intravenous injections while chemotherapy agents were dosed

by weekly intraperitoneal injections. Data up to day 69 was shown. Significant tumor growth

inhibition was observed in the combination groups (). * P < 0.05, ** P < 0.01 vs. vehicle at day

28, two-way Anova with Bonferroni test. N=10 per group.




Figure 1




Figure 2

A B

C D

E

Vehicle CVX-37 CVX-060




Figure 3

A Vehicle CVX-37 CVX-060A

Tie2 CD11b Merge Zoom-inB

Vehicle

CVX-37

CVX-060

C D




Figure 4

2000

3000A Vehicle

SunitinibCVX-060Suntinib + CVX-060

% TGI

40um

e (

mm

3)

2000

3000

B VehicleSorafenibCVX-060Sorafenib + CVX-060

% TGI

um

e (

mm

3 )

5 150

1000 **4460

Days

Tu

mo

r V

ol

5 15 250

1000

****

585980

Days

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ol

2000

3000

CVehicle

Bevacizumab + CVX-060Bevacizumab 3mg/kgCVX-060 3 mg/kg

% TGI

e (

mm

3)

2000

3000D

Vehicle

Bevacizumab + CVX-060Bevacizumab 10 mg/kgCVX-060 10 mg/kg

% TGI

me

(m

m3)

5 15 250

1000

**

*33.452.9

83.1

Tu

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r vo

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1000

**

* 576382

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*

EVehicleBevacizumab 3 mg/kgCVX-060, 3 mg/kgBevacizumab + CVX-060

% TGI at day 69

31 43 71

mm

3 )

5 15 25Days Days

0

1000

2000

**

Tum

or

Volu

me (

m

15 40 65 90Days




Figure 5

3000

A Vehicle5-FUCVX-0605-FU + CVX-060

(mm

3)

0 20 40 600

1000

2000

*

*T

um

or

Vo

lum

e

0 20 40 60Days

3000

B VehicleIrinotecanCVX-060Irinotecan + CVX-060

(m

m3)

0 20 40 600

1000

2000

**

**

Tu

mo

r V

olu

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0 20 40 60Days

3000

C VehicleDocetaxelCVX-060Docetaxel + CVX-060

mm

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0

1000

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**

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olu

me

(m

0 20 40 600

Days




Published OnlineFirst January 13, 2011.Clin Cancer Res Hanhua Huang, Jing-Yu Lai, Janet Do, et al. AND TUMOR GROWTH

INFILTRATIONANGIOGENESIS, TIE2 EXPRESSING MONOCYTE SPECIFICALLY TARGETING ANGIOPOIETIN-2 INHIBIT

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SPECIFICALLY TARGETING ANGIOPOIETIN-2 INHIBITS ......Jan 12, 2011 · FBS in a 5% CO2 incubator with 98% humidity to sub-confluence. 5x10 6 Colo-205 cells alone or 5x106 MDA-MB-435