EXHIBIT /7
`WIT. kR -ov
`DATE 3.tya-
`KRAMM COURT REPORTING
`
`Therapeutics, Targets, and Chemical Biology: Mather,,.,,-
`
`Increase of Plasma VEGF after Intravenous Administration
`of Bevacizumab Is Predicted by a Pharmacokinetic Model
`
`Marianne 0. Stefanini 1, Florence T. H. Wu', Feilirn Mac Gabhann2, and Aleksander S. PopeI 1
`
`(A) Check for updates
`
`Cancer
`Research
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`Abstract
`Vascular endothelial growth factor (VEGF) is one of the most potent cytokines targeted in antiangiogenic
`therapies. Bevacizumab, a recombinant humanized monoclonal antibody to VEGF, is being used clinically in
`combination with chemotherapy for colorectal, non-small cell lung and breast cancers, and as a single agent for
`glioblastoma and is being tested for other types of cancer in numerous clinical trials. It has been reported that
`the intravenous injection of bevacizumab leads to an increase ofplasma VEGF concentration in cancer patients.
`The mechanism responsible for this counterintuitive increase has not been elucidated, although several
`hypotheses have been proposed. We use a multiscale systems biology approach to address this problem.
`We have constructed a whole-body pharmacokinetic model comprising three compartments: blood, normal
`tissue, and tumor tissue. Molecular interactions among VEGF-A family members, their major receptors, the
`extracellular matrix, and an anti-VEGF ligand are considered for each compartment Diffusible molecules
`extravasate, intravasate, are removed from the healthy tissue through the lymphatics, and are cleared from the
`blood. Cancer Res 7q'23); 9886-94. ©2010 zIACR.
`
`Major Findings
`
`Our model reproduces the experimentally observed
`increase of plasma VEGF following intravenous adminis-
`tration of bevacizumab and predicts this increase to be a
`consequence of intercompartmental exchange of VEGF,
`the anti-VEGF agent and the VEGF/anti-VEGF complex.
`Our results suggest that a fraction of the anti-VEGF drug
`extravasates, allowing the agent to bind the interstitial
`VEGF. When the complex intravasates (via a combination
`of lymphatic drainage and microvascular transport of
`macromolecules) and dissociates in the blood, VEGF is
`released and the VEGF concentration increases in the
`plasma. These results provide a new hypothesis on the
`kinetics of VEGF and on the VEGF distribution in the body
`caused by antiangiogenic therapies, as well as their
`mechanisms of action and could help in designing anti-
`angiogenic therapies.
`
`Authors' Affiliations: 'Department of Biomedical Engineering, and 2lnsti-
`tute for Computational Medicine and Department of Biomedical Engineer-
`ing, Johns Hopkins University, Baltimore, Maryland
`Note: Supplementary data for this article are available at cancer Research
`Online lhttp:/Jcancen'es.aacrjournals.org.
`Corresponding Author. Aleksander S. Popel, Department of Biomedical
`Engineering, Johns Hopkins University School of Medicine, 720 Rutland
`Avenue, 611 Traylor Research Building, Baltimore, MD 21205. Phone:
`410-955-6419; E-mail; apopel@jhu.edu.
`doi: 10.115B10008-5472.cAN-1O-1419
`C201 American Association for cancer Research.
`
`9886
`
`Cancer Res; 70(23) December 1, 2010
`
`Introduction
`
`VEGF is a key factor in tumor angiogenesis, and it has
`become a major target of antiangiogenic cancer therapy (1). A
`large body of evidence suggests that the free plasma VEGF
`concentration is elevated several fold in cancer patients
`compared to healthy subjects (2). Therapies targeting VEGF
`have shown promising results in cancer. Bevacizumab (Avas-
`tin, Genentech Inc.), a recombinant humanized monoclonal
`antibody to VEGF, has demonstrated efficacy in colorectal
`cancer, non-small cell lung cancer, breast cancer, renal cell
`carcinoma, and glioblastoma. The drug has been approved by
`the Food and Drug Administration (FDA) for these indications
`under certain conditions in combination with chemothera-
`peutic agents and is being tested for other types of cancer and
`other conditions in numerous clinical trials.
`Despite the growing clinical applications of bevacizumab,
`the mechanism of action of this anti-VEGF agent and that of
`other anti-VEGF large molecules is not sufficiently understood
`(3). Specifically, two important questions remain: whether the
`drug acts by sequestering VEGF in the blood, tumor inter-
`stitium or both; and whether, as a result, the VEGF concen-
`tration in these compartments is reduced to 'normal' levels.
`Answering these questions would significantly contribute to
`understanding the mechanism of action not only at the
`molecular level, but also at the levels of tissue, organ, and
`whole body and would help in the design of anti-VEGF agents.
`Gordon and colleagues reported that the intravenous injection
`of bevacizumab led to an increase in serum total VEGF in
`clinical trials whereas free VEGF concentration was reduced
`(4). Since then, other groups have reported counterintuitive
`increases in the plasma VEGF level following bevacizumab
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`Increased Plasma VEGF Predicted by a Phanmacokinetic Model
`
`Quick Guide to Equations and Assumptions
`
`Key Equations
`
`The molecular detailed compartmental model is described by nonlinear ordinary differential equations on the basis of the
`principles of chemical kinetics andbiological transport (summarized in Supplement l).The following example equation describes
`the changeover time of the concentration of vascular endotlielial growth factor VEGF 121 isoform in the interstitial space of the
`normal tissue, denoted by the subscript N. The blood compartment is denoted by the subscript B.
`
`
`
`dt
`
`- N
`Will - krn,v121,Rl [VI2flv[RI] -+k011 y121R1 [VI2IRI]N
`
`- k00vi2ie2IVi2i],.IR2]+k0ijvi2iit2[Vi2iR2]
`- k0n,vl2,,,jNI [V12d [R 1 .WI] v+kofLvl2rnm-1 [V111 R1 A'i1
`
`-
`
`(kL + k9SNs\ Vi2iJç
`UN
`)CAr..v +kvUu[l2I)B
`
`The right-hand side terms represent: secretion of VEGF 121 isoform (qv)2,); binding to VEGF 12 , to its receptors (VEGFRI and
`VEGFR2) and to the complex VEGFRl/NRPt; binding of VEGI? i21 to the anti-VEGE agent A; and the intercompartmental
`transport of VEGF, 2, by lymphatics (1<,) and microvascular permeability to macromolecules (kr). S 8 and KAvNrepresent the
`total surface of microvessels at the normal tissue/blood interface and the available volume fraction for VEGF III in the total
`volume U, respectively. The total volumes are denoted by U. The subscript p in tJ denotes plasma as distinct from blood.
`Note that, with this nomenclature, the ratio (4,/UB represents the available fluid volume fraction for VEGF, 21 in the blood.
`The injection ofthe anti-VEGI'agent occurs after establishment of a physiologic steady state (t<0).At t = 0, the anti-VEGF agent is
`administered intravenously at a rate q,, for a duration at
`00 (typically in minutes). The subscript T represents the tumor. The
`equation governing the change of the antiVEGF agent concentration in the blood over time reads:
`
`-
`
`'lA cA[A]n_kPV &iA]n+(j) .
`
`-
`
`B STD [Al T
`A
`pv L/ [ lR + k pv
`p
`LiE "MIT
`
`- km,v121,A[VIIIID[A]a+koff,vIIIA[VIIIA]a
`
`-
`
`and q,, = 0 for all other times in = number of
`where q,, = total dose/(n x a• nr,aon) during the duration of each treatment
`injections). The first two terms on the right-hand side are the intravenous infusion of ar,ti-VEGF at rate q4 and the clearance of anti-
`VEGF from the blood at a rate CA. The next terms represent. drug extravasation; removal of anti-VEGF agent bylymphatics; and drug
`intravasation (when the intercompartnient transports are included). The last two terms describe the binding of the anti-VEGF agent
`to both VEGE isoforms.
`As a final example, the change over time of the corresponding VEGF/anti-VEGF concentration in the normal tissue when the
`extravasation of the anti-VEGE agent is governed by
`
`d[V]IIA]N
`dt
`
`IN
`
`(/c. + k?'V2SNfl) 1Vi214]N + kEN SND U3
`
`-
`
`UN
`
`KAVN
`
`pV
`
`[V 2 A] 8
`
`
`and is dependent on: VEGF, 21 binding to the anti'VEGF agent and transport of the VEGF/anti-VEGF complex between the
`compartments.
`
`Major Assumptions of the Model
`
`Our model does not represent a particular stage or type of cancer to keep the model general in light of the fact that
`bevacixumab is administered in primary and metastatic diseases and in adjuvant or neaadjuvant settings. Therefore, our tumor
`compartment can either be a primary tumor or the aggregate of metastases in tissue.
`
`www.aacrjoumals.org
`
`Cancer Res; 70(23) December 1, 2010
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`Stefanini at al.
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`Because the simulation results for a smaller tumor (half the diameter of the tumor considered in this study) were not
`significantly different (both qualitatively and quantitatively--data not shown), our model does not consider the possible change
`in tumor volume that may result from the injection of the anti-VEGF agent for the duration of our simulations.
`The degradation of VEGF by proteases is not currently included in the model. Effects of platelets and leukocytes as potential
`sites for sequestering VEGF, antiVEGF, and their products are not considered and should also be added in the future. We assume
`that only endothelial cells express VEGF receptors. Our model does not include the presence of receptors on the luminal surface
`of endothelial cells and the quantification of abluminal receptors has been estimated from previous studies.
`The model does not include multimeric binding of the anti-VEGF or the ability of the anti-VEGF to bind to matrix-bound
`VEGF. We assume that the anti-VEGF has a half-life of 21 days. Its complexes formed by the binding of VEGF,,, or VEGF, are
`assumed to have the same half-lives because hound and free bevacizumab exhibit the same pharmacokinetic profile. The binding
`and unbinding rates of the anti-VEGF to VEGF are taken from the literature to be 9.2 x iO4 (mol/L)'-C' and 2.0 x 10 s'
`respectively leading to a dissociation constant Kdof 2.2 nM. The above assumptions can be relaxed, if warranted by experimental
`data, within the framework of the model that is generally suitable for simulating anti-VEGF therapeutics.
`
`aded from htp:!/aactjournaIsorg/canrresJartide-pdfI7OI23I9886a64471I9886.pdf by guest on 03 M
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`isoforms and placental growth factor (PIGF). This fusion
`protein serves as a soluble decoy receptor and is currently in
`clinical trials.
`Our model includes two VEGF-A isoforms (VEGF 121 and
`VEGF 165), as well as VEGF receptors (VEGFI11 and VEGFR2)
`and the coreceptor neuropilin-1 (NRPI). In this study, we
`assume that VEGFRI, VEGFR2, and NEPI are present only on
`the abluminal surface of the endothelial cells. The transcap-
`illary microvascular permeability for the diffusible molecules
`(VEGF, anti-VEGF, and the VEGF/anti-VEGF complex) is
`included, as well as lymphatic drainage from the interstitial
`space into the blood compartment. The model equatiOns are
`presented in the Supplemental Information (Supplement I).
`
`Materials and Methods
`
`Most of the parameters for the anti-VEGF agent were taken
`from published data on hevacizumab. We assume a half-life of
`21 days (4) for the anti-VEGF whether unbound or bound to
`VEGF J2, or VEGF 165, as bound and free bevacizumab exhibit
`the same pharmacokinetic profile (9). Kinetic parameters (k0 ,
`k0ff) for the binding and unbinding of the anti-VEGF to the
`vascular endothelial growth factor were taken to be 9.2 >< io
`(molfL)'-s' and 2.0 x iO 4 s- ' respectively, leading to a
`dissociation constant K,, of 2.2 nM (17).
`Experiments have shown that bevacizumab may have mul-
`timeric binding to VEGF (9, 18) and can bind to extracellular
`matrix-sequestered VEGF (19). For simplicity purposes, we
`limit our model to monomeric binding to VEGF and neglect
`binding to VEGF sequestered by the extracellular matrix; these
`can be included when quantification of binding sites and the
`kinetics become available. Bevacizumab has also been
`reported to alter the VEGF-dependent microvascular perme-
`ability to soluble molecules (20). As a first approximation, we
`assume that the geometry of each tissue and the capillary
`density remains constant in the course of our simulations, that
`is, we do not include tissue remodeling after the injection of
`the anti-VEGF agent. Although it may be important, the
`inclusion of tissue remodeling would take the model beyond
`the scope of this study but could be of interest for further
`studies. This model does not include VEGF receptors on the
`luminal side of endothelial cells that have not been experi-
`
`administration (5-7). In the ocular setting, Campo and col-
`leagues reported that intravitreal bevacizumab injection
`increased the VEGF concentration in the aqueous humor
`(8). Several hypotheses have been formulated to explain this
`phenomenon. l-lsei and colleagues have suggested that the
`clearance of complexed VEGF is lower than that of free VEGF
`in rats and hypothesized that this lower clearance could
`explain the accumulation of total VEGF in serum (9). Other
`groups have suggested alternate pathways activated by the
`injection of bevacizumab, such as: accumulation of hypoxia-
`inducible factor leading to an increase of VEGF in serum, or
`secondary macular edema for the eye (8, 10, 11). Loupakis and
`colleagues immunodepleted plasma to remove bevacizumab
`and bevacizumab-VEGF complexes and found that plasma-
`free VEGF was significantly reduced after bevacizumab
`administration (12); this methodology helps to circumvent
`the problem that the ELISA method used in a number of
`studies cannot distinguish between free and total (including
`bevacizurnah-bound) VEGF. The results of the study corro-
`borate an earlier proposal by Christofanilli and colleagues (13)
`that free VEGF can serve as a surrogate marker.
`Systems biology approaches, specifically computational and
`mathematical modeling, are emerging as powerful tools in
`fundamental studies of cancer and design of therapeutics (14,
`15). To better understand VEGF distribution in the body, we
`have built a three-compartment model composed of normal
`(healthy) tissue, blood, and tumor (16). In this study, we have
`extended our computational model by including an anti-VEGF
`agent delivered by the intravenous infusion (i.e., into the blood
`compartment). The model describes the effect of such admin-
`istration on the VEGF distribution in the blood, normal, and
`diseased tissues. Our goal is to understand how the distribu-
`tion of VEGF, anti-VEGF agent, and their products changes
`following the agent administration: in particular, we will
`investigate whether the plasma VEGF level increases or
`decreases following an intravenous injection of the anti-VEGF
`agent.
`Even though the results are presented using the para-
`meters for bevacizumab, the model can be applied to other
`anti-VEGF agents. One such agent is aflibercept or VEGF
`Trap (Regeneron Pharmaceuticals Inc.), a soluble huma-
`nized VEGF receptor protein designed to bind all VEGF-A
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`Increased Plasma VEGF Predicted by a Pharrnacokinetic Model
`
`If the anti-VEGF agent is confined to the blood compart-
`ment, a single injection causes the concentration of free VEGF
`(i.e., not bound to anti-VEGF) in plasma to decrease precipi-
`tously by 98.4% (Fig. IA, dashed line; minimum as the infusion
`ends), as the anti-VEGF agent binds to VEGF available in
`plasma. However, this is not predicted to significantly affect
`the free VEGF level in the healthy tissue (maximum 0.1% drop
`at 9 hours (solid line)] or the free VEGF level in the tumor
`compartment [maximum 0.2% drop at 30 hours (dotted line)].
`The free anti-VEGF agent saturated the blood (Supplementary
`Figure SM) and reached a maximum of -4.7 Ismol/L in
`plasma (-8S ig/mL plasma) at the end of the infusion, which
`corresponds to the total injected amount of the 150-kDa agent
`distributed in the volume of plasma for a 70-kg patient. The
`VEGF/anti-VEGF complex reached its maximum concentra-
`tion in the blood (--'2.1 nmol/L) alter about 12 days (Supple-
`mentary Figure S2A). The total (free and bound to the anti-
`VEGF agent) VEGF concentration is typically what is mea-
`sured by VEGF ELISA methods (see Supplement 2 for a
`compilation of experimental data on free/total VEGF changes
`following bevacizuniab administration). Our results show a
`100- to a 1,000-fold difference between free VEGF concentra-
`tion (Fig. I) and the concentration of VEGF bound to the anti-
`VEGF (Supplementary Figure 82). Because of this difference in
`magnitude, the unbound VEGF concentration represents only
`a small percentage of the total VEGF concentration, and thus
`Supplementary Figure 52 also illustrates the total VEGF con-
`centration profile.
`For metronomic therapy (lower daily dose of 1 mg/kg over
`10 days), the free VEGF in plasma declines 86.8% following the
`first infusion, but is predicted to reach a pseudo-steady state
`after multiple infusions (Fig. IC, dashed line). The concentra-
`tion of free VEGF returned to its baseline level within 3 weeks
`once the treatment was stopped. Metronomic therapy showed
`delayed and lowered maximum levels of anti-VEGF compared
`to the single-dose regimen (Supplementary Figure SIC versus
`Supplementary Figure SIA); although the half-life of the anti-
`VEGF agent is relatively long, it is being cleared from plasma
`continuously. The VEGF/anti-VEGF complex (and therefore
`the total VEGF concentration) reached its maximum about a
`week later than for the single dose (Supplementary Figure S2C
`versus Supplementary Figure S2A).
`
`For an anti-VEGF agent that extravasates, plasma-free
`vIcl: is predicted to first decrease and then increase
`above the baseline level
`As for a nonextravasating anti-VEGF agent (Fig. IA),
`plasma-free VEGF decreased (97.0% drop in the first 45
`minutes) following administration of anti-VEGF that can
`extravasate (Fig, 18, dashed line), as the agent binds to the
`available free VEGF. In this case, however, VEGF concentra-
`tion then rebounded to 41.1 pmol/l. (a 9.1-fold increase over
`baseline) after about I week. Unlike the no-extravasation case
`where the concentration returned to baseline after 3 weeks,
`the free VEGF concentration in plasma was predicted to
`remain significantly elevated after 3 weeks (40.5 pmol/L, 9-
`fold the baseline level). The free VEGF concentration in the
`normal (solid line) and tumor (dotted line) tissues both also
`
`mentally characterized, but we have recently shown how such
`expression would alter the VEGF distribution (21); we do not
`expect that any qualitative conclusions of the study would be
`affected by the presence of luminal receptors.
`Note that the simulations are not aimed at representing a
`particular type or stage of cancer, recognizing that VEGF-
`neutralizing agents may be administered in cases of both
`metastatic and primary tumors. Thus, in the model, the tumor
`compartment can represent either an aggregate volume of
`metastases or a primary tumor. Due to the wide range of
`possibilities that could be represented for different types and
`stages of cancer, we adopt the parameters for this compart-
`ment from our previous study (16) and conduct a sensitivity
`study to ascertain that our qualitative conclusions are not
`dependent on the choice of parameters.
`For each simulation, the system was first equilibrated at a
`baseline for a cancer patient with tumor before the injection of
`the VEGF-neutralizing agent. At time zero, intravenous infu-
`sion of the anti-VEGF agent begins and delivery to the blood
`compartment continues as a slow infusion for 90 minutes. We
`considered two treatment regimens: a single-dose treatment
`of 10 mg/kg or 10 consecutive daily doses of I mg/kg (metro-
`nomic therapy).
`The parameters and their assigned numerical values are
`summarized in Supplement 3. The equations governing the 3-
`compartment VEGF transport system have been described in
`our previous papers (16,21) and can be found in Supplement I.
`We have also added equations to describe the interactions and
`intercompartmental transport of the anti-VEGF molecule
`(Equations S.30-S.38).
`
`Results
`
`Experiments demonstrate an inverse relationship between
`microvascular permeability and the size of a molecule (mole-
`cular weight or Stokes-Einstein radius; refs. 22-24). Therefore,
`in the absence of active transport, large proteins, such as anti-
`VEGF agents (150 kDa for bevacizumab and 110 kDa for
`ailibercept), should extravasate relatively slowly. In apparent
`agreement with this, the level of bevacizumab following an
`intravenous injection has been observed to be several times
`lower in normal tissues (25) and in tumors (19) than in the
`blood. However, little is known about what role, if any, the
`extravasation of an anti-VEGF agent may play in the ther-
`apeutic mechanism. To address this issue, we considered two
`computational scenarios: in the first, the anti-VEGF agent is
`constrained in the blood compartment (negligible extravasa-
`tion); in the second, the extravasation of the anti-VEGF agent
`is included.
`
`Plasma-free VEGF is predicted to decrease following
`intravenous injection of an anti-VEGF agent confined
`to the blood compartment (no extravasation)
`Changes in plasma- and tissue-free VEGF are summarized
`in Fig. 1A for a single-injection (10 mg/kg) and Fig. IC for
`metronomic therapy (1 mg/kg daily for 10 days), that is,
`repeated lower doses over a longer period of time (26). Total
`amount of drug injected is the same in both scenarios.
`
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`Cancer Res; 70(23) December 1, 2010
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`0
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`7
`
`14
`
`21
`
`Time (days)
`
`50
`
`- -n
`
`(V
`
`0
`
`0 -
`
`Stefanini at al.
`
`C 200
`
`150
`
`100
`E
`0. 50
`
`Single injection (10mg/kg)
`
`Metonomic therapy (1 mg/kg)
`
`No extravasation of the anti-VEGF agent
`
`axnvasation of the anti-VEGF agent included
`
`200
`
`150
`
`too
`
`50
`
`0
`
`0 1
`
`B 200
`
`150
`
`100
`
`50
`
`D 200
`
`ISO.
`
`Figure 1. Free VEGF concentration profiles following the intravenous injection of an anti-VEGF agent. A and B, single injection (10 mg/kg). C and D,
`daily injection of 1 mg/kg for 10 days (metronomic therapy). One pmot'L of VEGF equivalent to 24 pg/mL of total blood. Solid line, normal tissue; dashed line,
`blood; dotted line: tumor.
`
`showed an initial transient decrease (58.596 and 88.9%, respec-
`tively), followed by a slight rebound, reaching steady states
`6S% and 69.7% below baseline, respectively. This could be due
`to the long half-life of the anti-VEGF agent (21 days) as
`compared to the characteristic times of clearance, binding
`affinities, and internalization rates of VEGF receptors. This
`may suggest that an important whether not the primary action
`of the anti-VEGF agent is to deplete the tumor VEGF after the
`anti-VEGF extravasation. Interestingly, the increase of free
`VEGF in plasma is also predicted even in the absence of a
`tumor compartment (data not shown).
`Supplementary Figure SIB shows the dynamic response of
`the free anti-VEGF agent concentration. Upon injection, the
`free anti-VEGF concentration at first increases but then
`decreases rapidly within the next 12 hours as it travels to
`the normal and tumor tissues. Interestingly, the free anti-
`VEGF concentrations in the blood and in the tumor were
`almost identical. This was mainly due to the higher micro-
`vascular permeability and the absence of functioning lympha-
`tics in the tumor. The formation of the VEGF/anti-VEGF
`
`complex (and the total VEGF concentration) reached a max-
`imum after about 4 days and was significantly higher in the
`tumor than in the other compartments due to higher VEGF
`concentrations (Supplementary Figure S2B).
`In metronomic therapy (Fig. 1D), similar results were
`observed. The free VEGF concentration decreased in the
`plasma upon the anti-VEGF injection then rebounded and
`increased further after each injection (dashed line). In the
`healthy and tumor compartments (solid and dotted lines,
`respectively), a decrease in the free VEGF concentration
`was observed, followed by a rebound effect without exceeding
`their respective baseline levels. In all three compartments, the
`free VEGF concentrations are predicted to reach a steady state
`at the end of the 10 days of treatment and then remain almost
`constant (varying within a small range) over the duration of
`the experiment: the free VEGF level in the tumor was sig-
`nificantly decreased ('-.70.1%), whereas that in the plasma was
`significantly increased (by 8-fold) as compared to the baseline.
`Although the rebound in free VEGF in plasma occurred after
`45 minutes, the rebound still happens if we limit the duration
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`Increased Plasma VEGF Predicted by a Pharniacokinetic Model
`
`VEGF agent followed by subsequent binding to VEGF once in
`the normal or tumor tissues. For example, within 4 to S hours,
`58% of VEGF in the healthy tissue has become complexed with
`the anti-VEGF agent (Fig. 113). This is mainly due to the fact
`that the dose of the anti-VEGF agent injected is several orders
`of magnitude higher than the free VEGF concentration in the
`blood and, therefore, the anti-VEGF saturates the plasma
`upon injection. This concentration difference is counteracted
`by the microvascu1ar permeability of the high molecular
`weight (150 kDa) anti-VEGF being lower than that of VEGF
`(-'45 kDa).
`The net intercompartmental flow of the VEGF/anti-VEGF
`complex reveals that effectively, there is a net flow of complex
`intravasating from the normal tissue. This result suggests that
`the role of the anti-VEGF is to deplete VEGF from the
`interstitial spaces of the normal tissue and the tumor in
`the form of a complex.
`
`The VEGF distribution is modified by the injection of
`the anti-VEGF agent
`Relative VEGF distribution changes in normal tissue, blood,
`and tumor upon administration of the anti-VEGF agent
`(Fig. 3). In the blood (middle graph), there is no significant
`amount of free VEGF because most of it is complexed with the
`anti-VEGF agent. In the normal and tumor tissues (top and
`bottom graphs, respectively), most VEGF is complexed with
`the anti-VEGF agent (light gray region). The decrease in the
`relative amount of unbound VEGF in each compartment
`(black region), however, is mostly due to increase in total
`VEGF, due to the formation of VEGF/anti-VEGF complex
`(Supplementary Figure S3). In normal tissue, unbound VEGF
`declines only transiently; in tumors, there is a steady-state
`decline, where VEGF is much less bound to its receptors (drop
`by 48% - dark gray region) and less sequestered in the matrix
`(drop by 69% - white region) than before injection.
`
`The administration of the anti-VEGF agent significantly
`modifies the VEGFRI and Mfl'I occupancies in the
`tumor
`In keeping with the predicted effect on unbound VEGF
`(Fig. I), occupancy of the receptors in normal tissue was only
`transiently altered by the administration of the anti-VEGF
`agent (Fig. 4. solid line), whereas the VEGFRI and NRPI
`occupancies (top and bottom graphs) in the tumor were
`significantly decreased (from 31% to about 10%, and 35% to
`about 18% for VEGFR1 and NRPI, respectively) and remained
`fairly unchanged over the course of the experiment (dotted
`lines). Changes in VEGFR2 occupancy appeared less signifi-
`cant (middle graph), due to the saturation of tumor VEGFR2
`by cell-surface association of VEGF-bound NRPI; we assume
`10-fold higher NRPI expression on tumor endothelial cell
`surfaces than that of VEGFR2 in our model and 10-fold higher
`than on normal tissue endothelial cells. The effect of para-
`meters on the qualitative results of the study is discussed
`below. VEGFR2 occupancy after the anti-VEGF administration
`does not significantly change in the tumor compartment as
`compared to baseline (50% and 100% ligated in single and
`metronomic therapies, respectively).
`
`of the treatment to 45 minutes instead of 90 minutes (data not
`shown). The free anti-VEGF concentration peaked following
`each injection, reaching an overall maximum after 10 days of
`about half that for the single-dose treatment (Supplementary
`Figure S1D versus Supplementary Figure SIB). Interestingly,
`more VEGF/anti-VEGF complex was formed in the tumor
`(dotted line) than in the blood (dashed line) or in the normal
`tissue (solid line), regardless of the regimen (Supplementary
`Figures S2B and S2D) for single-injection and metronomic
`therapy, respectively). This also means that the total VEGF
`concentration was higher in the tumor than in the blood or the
`normal tissue.
`
`Formation of the VEGF/anti-VEGF complex mediates
`the depletion of VEGF from the tumor
`The changes in VEGF concentrations induced by the anti-
`VEGF agent can be interpreted by a detailed study of the
`movement of VEGF and anti-VEGF between the three com-
`partments. We define the net flow for each molecule as the
`difference between the intercompartmental flows of that
`molecule (in moles per unit time) entering and leaving the
`compartment. For example, the net intercompartmental flow
`of VEGF in the normal tissue is the difference between VEGF
`influx (by extravasation) and VEGF leaving the compartment
`(by intravasation and lymphatic drainage). With this metric,
`any negative net flows represent flows of diffusible molecules
`traveling from the blood into the normal tissue or the tumor
`compartment, whereas positive net flow illustrateg the flow of
`molecules entering the blood compartment. To visualize the
`relative effects, Ave plotted the net intercompartmental flows
`on the same graph. Figure 2 illustrates the net flows for the
`free VEGF, free anti-VEGF agent, and VEGF/anti-VEGF com-
`plex for the few hours following the anti-VEGF agent intra-
`venous injection.
`The anti-VEGF net flows (light gray) increased significantly
`during the first 6 to 12 hours in the normal (solid line) and
`tumor tissues (dotted line), showing extravasation of the anti-
`
`VEGFIanTi.VEG ti0L VEGF(anti-VEGF(No,msl tissue)
`
`0
`
`-2 1'
`
`am;-VEGF
`(Tumurl
`
`/
`VEGF
`Both tissucal
`
`0
`S
`
`ii Aso
`
`anl,-VEGF (No,mal tissue
`
`0
`
`12
`
`24
`
`48
`
`Time
`
`Figure 2. Net intercompartmental flows of VEGF, anti-VEGF, and VEGF/
`anti-VEGF complex. Solid line, normal tissue; dotted line, tumor. Black,
`Free VEOF; light gray, anti-VEGF agent; dark gray, VEGF/anti-VEGF
`complex (also total VEGF).
`
`v,w.aacdoumals.org
`
`Cancer Res; 70(23) December 1, 2010
`
`9891
`
`Mylan Exhibit 1105
`Mylan v. Regeneron, IPR2021-00880
`Page 6
`
`

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`-pdfI7O23988&26447l9/9886.pdf by guest on 03 March 2022
`
`Stefanini et S.
`
`- srnØe irecton
`- Meeor,om,c therapy
`
`NormS issue
` Tunic,
`
`-
`
`
`
`7
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`10
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`e
`
`40%
`
`20%
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`0%
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`eo%
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`
`0%
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`80%
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`60%
`
`40%
`
`20%
`
`LI.
`'3
`
`0.
`
`Figure 3. Relative VEGF distribution profiles in normal (top), blood (middle),
`and tumor (bottom) tissues. Percentage of VEGF sequestered in the anti-
`VEGF agent, free, sequestered In the receptors, and sequestered in the
`extracellular matrix. From top to bottom: normal tissue, blood, and tumor.
`Light gray, VEGF sequestered in anti-VEGF (VEGF/anti-VEGF complex);
`white, WOE sequestered in the extracellular matrix; dark gray, VEGF
`sequestered in receptors; black, unbound VEGF.
`
`Discussion and Conclusion
`
`The mechanism of action of bevacizumab has been com-
`monly accepted as its binding to the VEGF protein resulted
`in inhibition of angiogenesis. However, recent analysis
`points out that "whereas the molecular targets for anti-VEGF
`therapy with large molecules are identified, the mechanism
`of action, that is, how an anti-VEGF approach can exert
`single-agent activity in some cancers and augment the
`efficacy of conventional chemotherapy, is not well under-
`
`Time (days)
`
`Figure 4. VEGF receptor occupancy profiles. Percentage of ligated
`and unligated receptors. P VEGFR1. B, VEGFR2. 0, NRPI. Solid
`line, normal tissue; dotted line, tumor, Black,