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`SCIENCES
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`APPLIEDBIOLOGICAL
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`VEGF Trap complex formation measures production
`rates of VEGF, providing a biomarker for predicting
`efficacious angiogenic blockade
`
`John S. Rudge*, Jocelyn Holash†, Donna Hylton, Michelle Russell, Shelly Jiang, Raymond Leidich,
`Nicholas Papadopoulos, Erica A. Pyles, Al Torri, Stanley J. Wiegand, Gavin Thurston, Neil Stahl,
`and George D. Yancopoulos*
`
`Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591
`
`This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 20, 2004.
`
`Contributed by George D. Yancopoulos, September 21, 2007 (sent for review July 16, 2007)
`
`VEGF is the best characterized mediator of tumor angiogenesis.
`Anti-VEGF agents have recently demonstrated impressive efficacy
`in human cancer trials, but the optimal dosing of such agents must
`still be determined empirically, because biomarkers to guide dos-
`ing have yet to be established. The widely accepted (but unveri-
`fied) assumption that VEGF production is quite low in normal
`adults led to the notion that increased systemic VEGF levels might
`quantitatively reflect tumor mass and angiogenic activity. We
`describe an approach to determine host and tumor production of
`VEGF, using a high-affinity and long-lived VEGF antagonist now in
`clinical trials, the VEGF Trap. Unlike antibody complexes that are
`usually rapidly cleared, the VEGF Trap forms inert complexes with
`tissue- and tumor-derived VEGF that remain stably in the systemic
`circulation, where they are readily assayable, providing unprece-
`dented capability to accurately measure VEGF production. We
`report that VEGF production is surprisingly high in non-tumor-
`bearing rodents and humans, challenging the notion that systemic
`VEGF levels can serve as a sensitive surrogate for tumor load; tumor
`VEGF contribution becomes significant only with very large tumor
`loads. These findings have the important corollary that anti-VEGF
`therapies must be sufficiently dosed to avoid diversion by host-
`derived VEGF. We further show that our assay can indicate when
`VEGF is optimally blocked; such biomarkers to guide dosing do not
`exist for other anti-VEGF agents. Based on this assay, VEGF Trap
`doses currently being assessed in clinical trials are in the efficacious
`range.
`
`aflibercept 兩 angiogenesis 兩 tumor 兩 endothelial cell
`
`VEGF is critical in many settings of physiological and patho-
`
`logical angiogenesis (1). In particular, high VEGF expres-
`sion is characteristic of many types of cancers (1), suggesting that
`it might be an attractive target for therapeutic intervention
`aimed at preventing tumors from recruiting the blood supply that
`they need to survive (2). The first attempts at validating this
`particular approach were taken by Ferrara and colleagues (3),
`who demonstrated that a murine anti-human VEGF antibody
`suppressed the growth of human tumor cell lines implanted in
`nude mice. This led to the generation of a humanized mono-
`clonal antibody, bevacizumab (Avastin; Genentech, South San
`Francisco, CA), which yielded impressive results in a controlled
`clinical trial in patients with metastatic renal cell cancer (4, 5).
`At doses of 3 and 10 mg/kg, bevacizumab treatment resulted in
`a significant prolongation in time to tumor progression com-
`pared with placebo, although the increased efficacy of the higher
`dose in this study suggested that the maximally efficacious dose
`may not yet have been attained (4, 5). Bevacizumab was subse-
`quently granted FDA approval based on the demonstration that
`it significantly improved the progression-free and overall sur-
`vival in patients with metastatic colorectal cancer when given in
`combination with irinotecan 5-FU/LV chemotherapy (6). Sev-
`
`eral other drugs designed to block VEGF signaling have since
`been developed and recently approved [BAY 43–9006 (sor-
`afenib) and SU11248 (sunitinib)] or are proceeding through
`clinical trials [PTK787 (vatalanib), ZD6474 (zactima), ZD6126,
`SU5416 (semaxanib), and AG-013736] (7–9).
`As new anti-VEGF agents proceed through the clinic, it would
`be very useful to have biomarkers that could either identify
`patients whose tumors depend most on VEGF or that could
`guide dosing by indicating when optimal VEGF blockade has
`been achieved. Unfortunately, accepted biomarkers do not
`currently exist for VEGF blockade and are few and far between
`for other targeted agents, such as epidermal growth factor
`receptor for colon cancer, Kit for gastrointestinal stromal tumor,
`and HER2/NEU for breast cancer (10). VEGF itself has been
`suggested as a candidate biomarker for guiding the application
`of anti-VEGF therapies. It is widely assumed that VEGF
`production is quite low in healthy adults in the absence of active
`angiogenesis. Were that the case, blood levels of VEGF in cancer
`patients might provide a useful index of tumor VEGF production
`(11, 12). However, because VEGF is rapidly cleared from the
`systemic circulation (having a half-life of only minutes), the
`sensitivity of assays measuring VEGF in the peripheral blood
`leads to a wide variability for blood levels of VEGF in published
`reports. Furthermore, VEGF is present at substantial levels
`within platelets and released upon their lysis such that prepa-
`ration of peripheral blood samples that avoid contamination
`from platelet-derived VEGF becomes difficult. These limita-
`tions are reflected in the disparate values reported for circulating
`VEGF levels in cancer patients, which range from 0.04 to 1
`ng/ml, calling into question the utility of plasma VEGF levels as
`a useful biomarker for guiding anti-angiogenic therapy (11,
`13–19).
`VEGF Trap is a fully human soluble decoy receptor protein
`that consists of a fusion of the second Ig domain of human VEGF
`receptor (VEGFR) 1 and the third Ig domain of human
`VEGFR2 with the constant region (Fc) of human Ig IgG1 (20).
`
`Author contributions: J.S.R., J.H., and G.D.Y. designed research; J.S.R., S.J.W., G.T., and N.S.
`analyzed data; J.H., D.H., M.R., S.J., R.L., N.P., E.A.P., A.T., and G.T. performed research; N.P.
`contributed new reagents/analytic tools; and J.S.R. and G.D.Y. wrote the paper.
`
`The authors declare no conflict of interest.
`
`Freely available online through the PNAS open access option.
`
`Abbreviations: AMD, age-related macular degeneration; MALLS, multiangled laser light
`scattering; SEC, size exclusion chromatography; VEGFR, VEGF receptor.
`
`*To whom correspondence may be addressed. E-mail: john.rudge@regeneron.com or
`george@regeneron.com.
`†Present address: Novartis, 1400 53rd Street, Emeryville, CA 94608.
`
`This article contains supporting information online at www.pnas.org/cgi/content/full/
`0708865104/DC1.
`
`© 2007 by The National Academy of Sciences of the USA
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.0708865104
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`PNAS 兩 November 20, 2007 兩 vol. 104 兩 no. 47 兩 18363–18370
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`s.c. injection of VEGF Trap into SCID mice at different doses reveals different levels of circulating free VEGF Trap but similar levels of circulating mouse
`Fig. 1.
`VEGF–VEGF Trap complex. At all doses ranging from 1 mg/kg (A) to 25 mg/kg (D), a steady-state level of VEGF–VEGF Trap complex is achieved, which plateaus
`at ⬇1 ␮g/ml. Dose-dependent levels of free VEGF Trap are observed as follows: 1 mg/kg to 10 ␮g/ml Cmax falling below complex levels at 4 days (A); 2.5 mg/kg
`to 20 ␮g/ml Cmax falling below complex levels at 7 days (B); 10 mg/kg to 80 ␮g/ml Cmax falling below complex levels at 9 days (C); and 25 mg/kg to 200 ␮g/ml Cmax
`falling below complex levels at 17 days (D). The half-life of VEGF Trap is ⬇2 days at doses ⬎2.5 mg/kg. (n ⫽ 6 for each dose.)
`
`The VEGF Trap was engineered to have optimized pharmaco-
`kinetic properties and a very high affinity for all isoforms of
`VEGF-A (⬍1 pM), as well as placental growth factor, a closely
`related angiogenic factor (20). VEGF Trap has shown robust
`antitumor effects in numerous mouse models of cancer and is
`now in clinical trials (21, ‡, §, ¶, 储). Here, we show that—unlike
`VEGF antibodies that tend to form multimeric immune com-
`plexes that are rapidly cleared from the circulation and can form
`immune complex deposits in tissues—the VEGF Trap forms a
`stable and inert 1:1 complex with VEGF. This VEGF–VEGF
`Trap complex has a long plasma half-life and can readily be
`measured in the systemic circulation, thus affording a reliable
`way to measure the rates of VEGF production in both tumor-
`bearing and non-tumor-bearing adult animals and humans. This
`unique ability to capture and thus precisely measure total VEGF
`levels, regardless of whether the VEGF comes from tumor or
`normal host tissues, allows for the unprecedented opportunity to
`accurately determine tumor and host VEGF production rates.
`Surprisingly, we find that total body VEGF production rates are
`quite high in normal adult rodents and humans, with the
`fractional contribution made by tumors being comparatively
`small. This finding has the important implication that therapies
`directed toward neutralizing VEGF produced by tumors must be
`provided in sufficient amounts so as to avoid being largely
`consumed by the significant levels of VEGF produced by the rest
`of the body. Toward this end, measurement of VEGF Trap
`complex allows the identification of VEGF Trap doses required
`to completely capture and block tumor-derived VEGF, provid-
`ing a useful guide for optimizing angiogenic blockade; such
`assays do not exist for other anti-VEGF agents. Based on this
`
`‡Rixe, O., Verslype, C., Meric, J. B., Tejpar, S., Bloch, J., Crabbe, M., Khayat, D., Furfine, E. S.,
`Assadourian, S., Van Cutsem, E. (2006) J. Clin. Oncol. 24:13161 (abstr.).
`§Mulay, M., Limentani, S. A., Carroll, M., Furfine, E. S., Cohen, D. P., Rosen, L. S. (2006) J. Clin.
`Oncol. 24:13061 (abstr.).
`¶Tew, W. P., Colombo, N., Ray-Coquard, I., Oza, A., del Campo, J., Scambia, G., Spriggs, D.
`(2007) J. Clin. Oncol. 25:5508 (abstr.).
`储Massarelli, E., Miller, V.A., Leighl, N., Rosen, P., Albain, K., Hart, L., Melnyk, O., Sternas, L.,
`Akerman, J., Herbst, R. S. (2007) J. Clin. Oncol. 25:7627 (abstr.).
`
`assay, we report that VEGF Trap doses currently being assessed
`in clinical trials appear to be in the efficacious range.
`
`Results
`VEGF Trap Forms an Inert Complex with VEGF That Remains Stably in
`the Circulation. Initial studies to determine the clearance rate of
`VEGF Trap revealed that it could form stable detectable
`complexes with endogenous VEGF in normal adult mice. After
`single injections of increasing amounts of VEGF Trap, we
`measured total VEGF Trap, uncomplexed/unbound or ‘‘free’’
`VEGF Trap, and VEGF Trap–mouse VEGF ‘‘complex’’ at
`various times after injection (Fig. 1 A–D represent increasing
`amounts of injected VEGF Trap). Because no exogenous VEGF
`was provided, complexes represent the association of VEGF Trap
`with endogenous murine VEGF. As expected, total VEGF Trap
`levels increased proportional to dose (determined by combining
`free VEGF Trap levels with complex levels) (Fig. 1, see green
`curves). Somewhat unexpectedly, substantial levels of VEGF
`Trap complexed with mouse VEGF accumulated rapidly (Fig. 1,
`see blue curves). At all doses of VEGF Trap tested, maximal
`levels of complex (⬇1–2 ␮g/ml) were attained within 24–48 h of
`injection and sustained at this level for at least several days.
`Consistent with conversion of free VEGF Trap into complexed
`VEGF Trap, most of the injected VEGF Trap is initially found
`in the free, unbound form, but after reaching peak levels (⬇24
`h after injection) free VEGF Trap in the circulation declines
`progressively (Fig. 1, note that red curves, corresponding to free
`VEGF Trap, initially overlap at early time points with green
`curves, representing total VEGF Trap, but then drop, as is most
`obvious at the lowest dose). Levels of free VEGF Trap decline
`because of a ‘‘consumption’’ (binding VEGF, thus being con-
`verted to complex) and clearance, which occurs at an identical
`rate for free and bound Trap. Thus, as long as free VEGF Trap
`remains in excess of bound, maximal steady-state levels of
`complex are maintained in the circulation. VEGF Trap is also
`able to bind placental growth factor with high affinity and is
`capable of forming stable circulating placental growth factor–
`VEGF Trap complexes in vivo with the same profile as VEGF–
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`VEGF Trap complex, albeit at ⬇10-fold lower levels (data not
`shown).
`In separate experiments, the bioavailability of VEGF Trap
`and the efficiency of VEGF capture were determined by inject-
`ing s.c. [supporting information (SI) Fig. 7 A] or i.v. (SI Fig. 7B)
`preformed complexes of the Trap and its VEGF target, or both
`agents separately. The results show that the bioavailability of s.c.
`(SQ) injected complex was essentially identical to that of i.v.
`injected complex, indicating that negligible complex was depos-
`iting within tissues. Moreover, whether the VEGF Trap was
`injected as a preformed complex with VEGF (single bolus) or
`the Trap and its target were injected separately, similar levels of
`complex were rapidly noted in the circulation, indicating that the
`Trap efficiently captures its target and brings it into the systemic
`circulation. In addition, VEGF Trap is also capable of seques-
`tering VEGF already bound in target tissues as shown by
`injecting VEGF before VEGF Trap (SI Fig. 7). Thus, VEGF
`Trap efficiently captures and forms inert complexes with VEGF
`that enter and remain stably in the circulation, readily accessible
`for measurement.
`
`Although VEGF Trap Forms a 1:1 Complex with VEGF, VEGF Antibodies
`Form Heterogeneous, Multimeric Immune Complexes with VEGF. The
`above findings suggested that VEGF Trap might behave very
`differently than VEGF antibodies, because antibodies com-
`monly form multimeric immune complexes that rapidly deposit
`in tissues and thus are rapidly cleared from the circulation.
`Because immune complexes rapidly disappear, the amount of
`captured ligand cannot be determined from levels of bound or
`unbound antibodies remaining in the circulation. To demon-
`strate directly that the VEGF Trap behaves in a fundamentally
`different way than antibodies, we compared VEGF Trap com-
`plex formation and clearance with that of a well characterized
`VEGF antibody, bevacizumab (Avastin). As predicted, size
`exclusion chromatography (SEC) of a preformed VEGF Trap–
`VEGF165 complex revealed a single major homogenous peak,
`with an approximate molecular mass (as judged by comparison
`to molecular mass standards, data not shown) of ⬇150 kDa
`corresponding to that expected of a 1:1 complex between VEGF
`Trap (⬇110 kDa) and VEGF165 (⬇40 kDa) (Fig. 2A, solid red
`line); a minor peak of free excess VEGF165 was also seen, as was
`a small shoulder of higher molecular mass. The molecular masses
`of the peaks were confirmed by using coupled multiangled laser
`light scattering (MALLS) (dashed red lines in Fig. 2 A). In
`contrast, SEC of preformed bevacizumab–VEGF165 complexes
`revealed a heterogeneous mixture corresponding to very high
`molecular masses (Fig. 2 A, solid blue line) in addition to the
`small peak of free excess VEGF165. The purity of free VEGF
`Trap, bevacizumab, and VEGF was ⬎97%, as determined by
`SEC (data not shown). Coupled MALLS analysis revealed
`molecular masses of the heterogeneous mixture ranging from
`370 kDa (corresponding to a multimer consisting of two bev-
`acizumab molecules, each with a molecular mass of ⬇145 kDa,
`and two VEGF165 molecules, each with a molecular mass of
`⬇40kDa) to ⬎2,000 kDa (corresponding to much larger mul-
`timers) (Fig. 2 A, dashed blue line). Consistent with the apparent
`tendency of bevacizumab to form multimeric immune complexes
`with VEGF, preformed bevacizumab–VEGF165 complexes rap-
`idly disappeared from the circulation when injection intrave-
`nously, as would be expected for multimeric immune complexes
`(SI Fig. 8; note that the levels of Bevacizumab when complexed
`with VEGF rapidly drop compared with the levels of free
`Bevacizumab that remain much higher), and in contrast to what
`was described above with VEGF Trap complexes that remain
`stably in the circulation. Because immune complexes can often
`be cleared by depositing in the renal glomeruli, we further
`explored apparent differences in the clearance of bevacizumab–
`VEGF and VEGF Trap–VEGF complexes by performing im-
`
`The molar masses of VEGF Trap–VEGF and bevacizumab–VEGF com-
`Fig. 2.
`plexes were determined by MALLS coupled to SEC. (A) Using a 1:2 molar ratio
`of VEGF Trap to VEGF165, discrete peaks were observed at ⬇17 ml for VEGF (41
`kDa) and ⬇14.5 ml for VEGF Trap–VEGF complex (148 kDa) with SEC (red line)
`and MALLS (dashed red line). In contrast, a 1:2 molar ratio of bevacizumab to
`VEGF165 revealed a heterogeneous multimeric complex that ranged in molar
`mass from ⬇370 kDa to ⬎2,000 kDa (SEC, solid blue line; MALLS, dashed blue
`line). (B–E) One milligram of a preformed complex of VEGF Trap and VEGF165
`(B and C) or bevacizumab and VEGF165 (D and E) were injected into the left
`ventricle of 2- to 3-month-old C57bl6 mice. After 10 min, mice were killed, and
`their kidneys were processed for immunocytochemistry, using an anti human
`Fc reporter antibody to the human Fc moiety present on both VEGF Trap and
`bevacizumab. Significant staining was observed in the glomeruli of bevaci-
`zumab/VEGF treated mice but not in the glomeruli of VEGF Trap/VEGF treated
`mice (white arrows).
`
`munostaining in the kidney. After i.v. administration, renal
`glomeruli stained strongly for bevacizumab–VEGF complexes
`(Fig. 2 D and E) but not for VEGF Trap–VEGF complexes (Fig.
`2 B and C). Current evidence indicates that, as a class, pharmaco-
`logical agents that block VEGF signaling may produce mechanism-
`based effects on kidney function. Deposition of immune com-
`plexes as noted for bevacizumab/VEGF in the renal glomeruli
`could further accentuate renal toxicity in a nonspecific and
`non-class-dependent manner.
`
`VEGF Trap Complex Formation Reveals Unexpectedly High Production
`of Endogenous VEGF in Normal Adult Mice. As shown above, VEGF
`antibodies form immune complexes that rapidly deposit in
`tissues and thus do not allow for easy ascertainment of the
`amount of complex formed. In contrast, VEGF Trap forms inert
`complexes with VEGF that remain stably in the circulation and
`are thus readily accessible for measurement. In fact, the above
`findings demonstrate that, if VEGF Trap is present at sufficient
`levels so as to be in excess of Trap bound in complexes, the
`steady-state levels of VEGF Trap complex in the circulation
`reflect the total amount of VEGF produced. Daily production
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`rates of VEGF can be calculated by assuming that steady-state
`levels of VEGF Trap–VEGF complex reflect a balance between
`production of VEGF leading to formation of complex, and
`clearance of the resulting complex. Based on experimentally
`determined values for the steady-state levels of complex and its
`clearance (see Materials and Methods), we estimate that mice
`produce ⬇0.065 ␮g of VEGF per day per ml of the volume of
`distribution, or ⬇0.006 ␮g per gram of tissue per day. Because
`VEGF is active at picomolar levels, this at first seems to be a
`surprisingly high level of production for a normal adult animal
`(see below for comparison to tumor production rates). However,
`it should be noted that in the absence of VEGF Trap, any VEGF
`that enters the systemic circulation is rapidly cleared. For this
`reason, among others noted above, it has not proven possible to
`consistently and reliably measure systemic VEGF levels, pre-
`venting accurate estimation of VEGF production rates in normal
`adult animals.
`
`Tumor-Derived VEGF Represents a Minority of Total Body VEGF Under
`Conditions of Minimal Tumor Burden. Next, we compared the total
`body production rate of VEGF, as determined above, with tumor
`production rates of VEGF. Toward this end, we implanted mice
`with tumors, allowed these tumors to grow to 0.5–3% of total
`body weight (average mouse weight, ⬇25 g) and measured levels
`of VEGF Trap complex in these mice to compare them to
`complex levels found in healthy, non-tumor-bearing mice. Sur-
`prisingly, in mice bearing four different types of rodent tumors,
`the total levels of complex were not markedly different from
`those seen in non-tumor-bearing mice (1–2 ␮g/ml; see Fig. 3A
`and compare with Fig. 1). This finding implies that tumor-
`derived VEGF represented only a small proportion of total body
`VEGF or circulating bioavailable VEGF in these mice.
`To further validate this unanticipated finding, we analyzed
`VEGF Trap complex levels in mice bearing human tumors,
`where it is possible to distinguish complexes formed with en-
`dogenous mouse VEGF with those formed with human VEGF
`derived from the implanted tumors by analyzing human VEGF–
`VEGF Trap complex levels in mouse serum. The levels of
`mouse-derived complexes (Fig. 3B) in these animals were equiv-
`alent to those of non-tumor-bearing mice (Fig. 2, above) and
`mice bearing rodent-derived tumors (Fig. 3A). In contrast, the
`levels of VEGF Trap complexed with tumor-derived human
`VEGF were an order of magnitude lower (0.08–0.2 ␮g/ml) (Fig.
`3B). This result was seen in mice bearing tumors of three
`different human cell lines (SK-NEP, A673, and HT1080). To-
`gether, these studies demonstrate that normal total body pro-
`duction of VEGF eclipses the production from tumors that may
`weight as much as 3% of body weight (mouse weight ranges from
`23 to 29 g). Thus, it is unlikely that total levels of free VEGF in
`the systemic circulation would provide a sensitive index of tumor
`burden, even if accurate measurement of unbound VEGF in
`blood samples were readily achievable. Moreover, the above
`findings suggest that therapeutic compounds designed to bind
`and inactivate tumor-derived VEGF would have to be provided
`at sufficient levels to avoid being diverted by significant levels of
`VEGF normally produced by the rest of the body.
`
`VEGF Trap Complex Levels Provide Guidance on When Efficacious VEGF
`Blockade Is Achieved. Based on the results above, it is evident that
`drugs that bind and neutralize VEGF must engage significant
`levels of VEGF derived from normal tissues, in addition to that
`originating from tumors. Therefore, we reasoned that measure-
`ments of VEGF Trap complex might provide a useful guide to
`when the dose of VEGF Trap sufficient to substantially neu-
`tralize both host and tumor-derived VEGF had been achieved.
`Indeed, for three different tumors [B16F1 mouse melanoma
`(Fig. 4A); A673 human rhabdomyosarcoma (Fig. 4B); and MMT
`mouse mammary carcinoma (Fig. 4C)], increasing the VEGF
`
`In mice bearing tumors ⬍3% body weight, the tumor pool of VEGF
`Fig. 3.
`production is modest compared with endogenous mouse tissue VEGF produc-
`tion. (A and B) Mouse (A) or human (B) tumors were allowed to grow to ⬇100
`mm3, and then VEGF Trap was administered twice per week for 1–2 weeks at
`0.5, 1, 2.5, 10, and 25 mg/kg. At the termination of the experiment, free VEGF
`Trap, mouse, and human complex levels were measured in serum. In all cases,
`regardless of terminal tumor volume, levels of circulating mouse complex
`were ⬇1 ␮g/ml, whereas human complex levels in the mice bearing human
`tumors were ⬇0.1 ␮g/ml. Free Trap levels increased incrementally, with the
`dose levels rising above complex levels at the 2.5 mg/kg dose and reaching
`⬇100 ␮g/ml at the 25 mg/kg dose. (n ⫽ 6 for each dose). (C) Legend of mouse
`and human tumor types used.
`
`Trap dose resulted in progressive, marked improvements in
`anti-tumor efficacy until a dose at which free VEGF Trap
`substantially exceeded maximal steady-state levels of complex
`was reached (Fig. 4). For all three tumor types, this was achieved
`at a dose of 2.5 mg/kg VEGF Trap given twice weekly: at this
`dose, free VEGF Trap (blue curve) is severalfold the level of
`complex (green curve), and past this point further dose escala-
`tion yields only modest incremental increases in complex levels
`(green curve) and in anti-tumor efficacy (red curve). In other
`tumor types, such as U87 glioblastoma, higher levels of VEGF
`Trap are required to achieve maximal efficacy (22).
`
`Human VEGF/VEGF Trap Complex Levels Are Directly Related to Tumor
`Size. The finding that conventionally sized s.c. tumors in mice
`produced ⬍10% the amount of total body VEGF prompted us
`to determine whether there is a consistent relationship between
`tumor size and VEGF production levels. Human tumors (A673
`rhabdomyosarcoma) were implanted into mice and allowed to
`grow to various sizes before injecting VEGF Trap. In this case,
`we could define a clear linear relationship between tumor size
`(Fig. 5A) and complex levels (Fig. 5B, note that the assay reflects
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`readily detectable contribution to total body VEGF production.
`To determine whether or not this was indeed the case, we studied
`VEGF Trap complex formation in non-cancer patients [patients
`suffering from age-related macular degeneration (AMD)] and
`then compared these results with complex formation in cancer
`patients. In the AMD patients, the lowest dose of VEGF Trap
`tested (0.3 mg/kg, i.v.) was insufficient to neutralize all VEGF,
`as evidenced by the levels of free Trap quickly falling below those
`of bound VEGF Trap, and bound VEGF Trap did not approach
`the maximal steady-state levels seen with higher doses (Fig. 6 A
`and B). However, doses of 1.0 and 3.0 mg/kg (i.v.) maintained
`substantial free Trap levels throughout the dosing period (Fig.
`6A), and maximal complex levels were attained, as evidenced by
`equivalent levels of complex being generated at the two higher
`doses (⬇1–2 ␮g/ml, see Fig. 6B). In cancer patients with ad-
`vanced solid tumors or non-Hodgkin’s lymphoma, remarkably
`similar results were obtained. That is, similar doses of VEGF
`Trap were required to saturate VEGF binding and complex
`formation (Fig. 6 C–E). In addition, the maximal steady-state
`levels of VEGF–VEGF Trap complex were similar to those seen
`in non-cancer patients (Fig. 6 B, D, and E). These findings
`indicate that, consistent with our findings in mice, endogenous
`VEGF production in adult human subjects is quite high, whether
`or not the individuals harbor tumors (Fig. 6E).
`Using the same approach as was used for the mouse (see
`Materials and Methods), human production rates of VEGF in
`humans were found to be ⬇0.0025 ␮g per gram of tissue per day,
`which is remarkably similar to that calculated for mice (see
`above). If our findings in animal models continue to be predic-
`tive, these VEGF Trap levels achieved in ongoing clinical studies
`should be in the efficacious range.
`
`Discussion
`At present, there are a number of anti-angiogenic agents tar-
`geting the VEGF pathway that are proceeding through clinical
`trials or already approved for the treatment of cancer (9). One
`major challenge is the lack of objective measures to guide dosing
`to determine when sufficient blockade has been achieved or to
`inform pharmacological response to these drugs. VEGF itself
`has been suggested as a potential biomarker for the above
`purposes, based on the assumption that VEGF in the peripheral
`circulation was primarily derived from the tumor and therefore
`accurately reflected tumor burden (19). However, to date it has
`proven difficult to accurately measure systemic levels of VEGF,
`correlate these levels with tumor burden, or use them as a guide
`to dosing (11, 12). Here, we describe the use of the VEGF Trap,
`a potent VEGF antagonist that forms a stable, inert complex
`with VEGF, as an index that allows for the accurate assessment
`of VEGF production rates. In addition, this unique property of
`the VEGF Trap allows accurate assessment of the amounts of
`VEGF made by a resident tumor compared with the rest of the
`body. Furthermore, in animals, this approach has been shown to
`provide a useful guide to selecting dosing regimens that sub-
`stantially block available VEGF. This has not been possible with
`anti-VEGF antibodies, as VEGF–antibody complexes are rap-
`idly cleared.
`We find unexpectedly high levels of VEGF production in the
`normal adult setting, where it has long been assumed that, in the
`absence of ongoing angiogenesis, VEGF production rates would
`be quite low (11, 12). However, the unexpectedly high rates of
`VEGF production in non-tumor-bearing adult mice and humans
`is consistent with the recent realization that VEGF likely plays
`an ongoing role in the ‘‘quiescent’’ vasculature of normal adults
`(23). For example, treating normal adult mice and monkeys with
`VEGF antagonists can increase hematocrit (a measure of the
`proportion of the blood volume occupied by red blood cells)
`(24). Similarly, VEGF antagonists can also increase blood
`pressure (25), indicating that VEGF is involved in regulating
`
`VEGF Trap Complex provides guidance on when optimal VEGF
`Fig. 4.
`blockade is achieved for antitumor purposes. In mice bearing B16F1 mouse
`melanoma tumors (A), A673 human rhabdomyosarcoma (B), and MMT mouse
`mammary carcinoma tumors (C) grown to ⬇100 mm3 before treatment,
`increasing the dose of VEGF Trap from 0.5 mg/kg twice per week to 25 mg/kg
`twice per week results in a steady-state of mouse complex at ⬇1 ␮g/ml at 1–2.5
`mg/kg and free circulating VEGF Trap levels of ⬇10 ␮g/ml at the 2.5 mg/kg
`dose, rising to ⬇100 ␮g/ml at the 25 mg/kg dose. Tumors remain quite large
`at the 0.5 and 1 mg/kg doses but begin to show a significant lack of growth at
`the 2.5 mg/kg dose, where free Trap levels rise above steady-state complex
`levels (n ⫽ 6 for each dose). Tumors were treated with VEGF Trap from 6 –13
`(B16F1), 4 –13 (MMT), and 12–18 (A673) days after implantation.
`
`levels of complexes containing only human VEGF to specifically
`detect only tumor-derived complex). The amount of complex per
`unit weight of tumor was similar across different-sized tumors
`(Fig. 5C), indicating that tumors maintained their rates of VEGF
`production as they grew. Linear regression analysis confirmed
`that there was a very strong correlation between A673 tumor
`volume and circulating human VEGF complex (Fig. 5D).
`At these larger tumor sizes, the amount of complex (ranging
`from ⬇0.8 to 5 ␮g/ml) contributed by the tumor matched or even
`exceeded that contributed by the rest of the body, confirming
`that tumors do indeed make substantially more VEGF per cell
`than does the average cell in the normal adult host. For example,
`in the largest tumors (weighing ⬇10% of the total mass of the
`mouse, Fig. 5A), the tumor-derived human VEGF–VEGF Trap
`complex levels (⬇5 ␮g/ml, Fig. 5B) were ⬇3-fold above the levels
`of murine VEGF–VEGF Trap complex,
`indicating that the
`tumors made ⬇30 times the amount of VEGF per unit of weight
`compared with normal, adult tissues.
`
`VEGF Trap Complex Formation in Human Subjects With and Without
`Cancer. Very large tumors that substantially contribute to VEGF
`Trap complex formation in mice are generally not seen in the
`human patient. This in turn suggests that it is unlikely that most
`tumors in human patients become large enough to make a
`
`Rudge et al.
`
`PNAS 兩 November 20, 2007 兩 vol. 104 兩 no. 47 兩 18367
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`Downloaded from https://www.pnas.org by 74.105.34.50 on December 29, 2022 from IP address 74.105.34.50.
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`Fig. 5. Human VEGF–VEGF Trap complex levels are directly related to tumor size. Human A673 rhabdomyosarcoma tumors were grown in mice to ⬇100, ⬇300,
`⬇500, and ⬇750 mm3, at which point they were treated with a single bolus of 25 mg/kg. VEGF Trap, tumor volume, and human complex levels were measured
`after 2 weeks (n ⫽ 6). (A) Increasing tumor volume equates with an increase in tumor burden. (B) Increasing human tumor burden is reflected in an increase in
`circulating human VEGF–VEGF Trap complex. (C) The ratio of human VEGF–VEGF Trap complex to tumor volume remains steady at ⬇2-fold. (D) Linear regression
`analysis comparing systemic levels of human VEGF–VEGF Trap to tumor volume reveals that increasing tumor volume directly correlates with increasing complex
`levels. (P ⬍ 0.0001.)
`
`vascular tone in the adult (26). We m