WO2014006113A1

WO2014006113A1 - Method of treating cancer by effective amounts of aflibercept

Info

Publication number: WO2014006113A1
Application number: PCT/EP2013/064079
Authority: WO
Inventors: Emmanuelle BOELLE; Pankaj Bhargava; Marielle Chiron; Nathalie FAGNIEZ
Original assignee: Sanofi SA
Current assignee: Sanofi SA
Priority date: 2012-07-03
Filing date: 2013-07-03
Publication date: 2014-01-09
Anticipated expiration: 2015-01-03

Description

Method of treating cancer by effective amounts of aflibercept

The present invention relates to a method of treating cancer by effective amounts of aflibercept

Colorectal cancers are among the most frequent tumor types in the western countries, second to breast in women and third to lung and prostate in males. The end prognosis is dependent upon the extent of the disease. The five year survival rate in early localized stage of about 90% decreased to approximately 60-65% after spread to adjacent organ(s) or lymph nodes and is of less than 10% after spread to distant sites.

When diagnosed before nodal involvement treatment is usually limited to surgical resection (and radiotherapy for patients with rectal cancer) and potential participation to clinical trials for adjuvant therapy. Patients with nodal involvement are candidates for adjuvant chemotherapy following initial surgery in the attempt to prevent metastatic recurrence of the disease. Once spread to distant sites treatment essentially consists of palliative chemotherapy.

About 75 to 80% of all the patients with colorectal carcinoma will present at a stage when all gross carcinoma can be surgically removed. However, almost half of these patients will ultimately die from metastatic disease. Furthermore 20 to 25 % of the patients present with metastatic disease at diagnosis. Once metastases are present median overall survival with available combination therapy is around 20 months.

Over the past decades 5-Fluorouracil (5-FU) has remained the mainstay of the chemotherapy in colorectal cancer. During years the major determinant in the treatment of colorectal cancer patients has been the improvement in the schedules of 5-FU administration.

Among these, the bimonthly regimen (LV5FU2) of 5-FU given as bolus/infusion over 2 days has been shown to be superior to the monthly 5 day bolus regimen (Mayo regimen) in terms of response rate (RR) (32.6% vs 14.4%), in terms of progression free survival (PFS) (27.6 vs 22.0 weeks), and safety (de Gramont et al, Journal of Clinical Oncology 1997;15(2):808-815).

However, no statistically significant improvement in the overall survival (OS) was seen until development, starting in the beginning of nineties, of two novel cytotoxic agents, oxaliplatin, a DACH platinum, and the topoisomerase I inhibitor, irinotecan. With each of these two new agents median overall survival in the first line metastatic setting reached 15 to 19 months in multiple Phase III trials.

In a study, published in 2004 by Tournigand et al. (Journal of Clinical Oncology 2004;22(2):229-237), where these two drugs were administered in sequence in the same protocol, as first then second line treatment in metastatic colorectal cancer patients, the threshold of 20 months median overall survival was crossed whatever was the order of the treatment sequence.

Aflibercept (ziv-aflibercept in the US) is the INN of a fusion protein comprising the signal sequence of VEGFR1 fused to the D2 Ig domain of the VEGFR1 receptor, itself fused to the D3 Ig domain of the VEGFR2 receptor, in turn fused to the Fc domain of lgG1 Aflibercept. It is also referred to as VEGF trap, VEGFR1 R2-Fc.DELTA.C1 or Flt1 D2.Flk1 D3.Fc.DELTA.C1 . Aflibercept binds to VEGF-A, VEGF-B and PIGF ligands with high affinity in vitro. The amino acid sequence (SEQ ID N°1 ) of Aflibercept is illustrated in Figure 1 and is also shown inter alia in FIG. 24 of patent application WO 00/75319.

Aflibercept is marketed in the EU and in the US under the trade name ZALTRAP®.

It has been shown that free aflibercept molecules bind to VEGF-A in the circulation to form aflibercept-ligand complexes (also called bound aflibercept) that have no biological activity; the binding or 'trapping' of these VEGF ligands by aflibercept results in the anti-angiogenic effect in tumors.

The complex formed by aflibercept and VGEF-A is referred below as "bound aflibercept".

Rudge et al (PNAS 2007,104, 18363) performed pre-clinical experiments on mice bearing tumors. They increased doses of aflibercept from 0.5mg/kg to 25mg/kg and concluded that assays of free and bound aflibercept can serve as useful indicators of the proportion of bioavailable VEGF that is bound and neutralized at a given dose.

In patients, Maximum plasma concentration of free VEGF Trap increased proportionally with dose. Maximal VEGF-bound VEGF Trap complex levels were reached at doses≥ 2.0 mg/kg (Lockhart et al, J Clin Oncol. 2010 January 10; 28(2): 207-214).

5-fluorouracil (5-FU or f5U) is a drug that is a pyrimidine analog which is used in the treatment of cancer. It is a suicide inhibitor and works through irreversible inhibition of thymidylate synthase. It belongs to the family of drugs called antimetabolites.

Folinic acid or leucovorin is an adjuvant to cancer chemotherapy used in combination with 5-fluorouracil.

Irinotecan is a drug used for the treatment of cancer. Irinotecan is a topoisomerase 1 inhibitor, which prevents DNA from unwinding. FOLFIRI is the combination of folinic acid, 5-fluorouracil (5-FU) and irinotecan and will be used throughout the document.

In a phase I study (TCD61 18) aflibercept was administered IV in combination with irinotecan (180 mg/m² on day 1 ), leucovorin (200 mg/m² on day 1 and day 2), and 5- FU (bolus/infusional 400/600 mg/m² on day 1 and day 2), every 2 weeks in patients with advanced solid malignancies. The aflibercept 4 mg/kg dose every 2 weeks was considered to be the optimum dose.

In a phase II trial (NCI7498) aflibercept was administered in previously treated patients with metastatic colorectal cancer. This trial showed that aflibercept is well tolerated in pre-treated patients with MCRC. The conclusions are that based on the study results, studies of aflibercept as single agent or in combination should be explored (Tang et al, J Clin Oncol 26: 2008 (May 20 suppl; abstr 4027).

But the results provided in these two studies provided no insight as to efficacy.

Furthermore a phase III trial of aflibercept in metastatic pancreatic cancer was discontinued in 2009 and in 201 1 the data of a phase III trial evaluating aflibercept for the second-line treatment of non-small cell lung cancer (NSCLC) showed that adding aflibercept to the chemotherapy drug docetaxel did not meet the pre-specified criteria for the primary endpoint of improvement in overall survival compared with a regimen of docetaxel plus placebo. However, in 201 1 , a phase III study evaluating the combination of aflibercept and FOLFIRI for the second line treatment of metastatic colorectal cancer (MCRC) showed improvement in overall survival (OS), progression free survival (PFS) and response rate (ORR). This study formed the basis of regulatory filings and subsequent marketing approvals of aflibercept (ZALTRAP®) in the United States and European Union.

It has now been found, and this is an object of the present invention, that the effectiveness of aflibercept on Overall Survival (OS) in patients with cancer can be significantly improved by adapting the treatment of the patient in function of the ratio free/bound aflibercept;

The invention relates to methods, compositions and articles as disclosed herein.

In one aspect the invention provides for a method of treating cancer or cancer symptom in a patient in need thereof, said method comprising the following steps:

^■ measuring the ratio free/bound aflibercept in a biological sample of a patient, and

^■ adapting the treatment of the patient in function of the ratio free/bound

aflibercept.

In another aspect the invention provides for a method of treating cancer or cancer symptom in a patient in need thereof, said method comprising administering to said patient therapeutically effective amounts of aflibercept to patient displaying a ratio free/bound aflibercept comprised between 1 and 4, as measured by ELISA as described in the example 2 below.

This method comprises the following steps:

^■ administering therapeutically effective amounts of aflibercept to patient

displaying a ratio free/bound aflibercept comprised between 1 and 4. In another embodiment ratio free/bound aflibercept is around 1.6.

In another embodiment ratio free/bound aflibercept is comprised between 1 .6 and 3.2.

In another embodiment ratio free/bound aflibercept is comprised between 1 .5 and 1.8.

The free/bound ratio may be measured any time during the treatment of a patient. In a particular embodiment of the invention the free/bound ratio is determined after the first cycle of treatment of the patient, or after the second cycle of treatment of the patient.

Free aflibercept concentrations and VEGF:aflibercept complex concentrations are measured in plasma by the methods known by the man skilled in the art.

Such a method can be an enzyme linked immunosorbent assay (ELISA) method. In such a case the free and bound aflibercept concentrations are measured with the help of specific antibodies.

However the man skilled in the art may use other method to measure the free aflibercept concentrations and VEGF:aflibercept complex concentrations such as chromatographic methods, and for instance mass spectrometry methods.

In a particular embodiment of the invention the cancer is Colorectal Cancer (CRC).

In another embodiment the method comprising administering to said patient therapeutically effective amounts of aflibercept and FOLFIRI. This method is safe and effective. In another feature the invention provides a method wherein said patient has already been treated for the CRC or CRC symptom (second-line treatment).

In a specific embodiment CRC is a Metastatic Colorectal Cancer (MCRC).

However the said method can be used for treating any other cancer such as lung cancer, gastric cancer, ovarian cancer and any other cancer susceptible to be treated with aflibercept. The said method can be used for treating cancer with aflibercept as single agent or with aflibercept in combination with other chemical molecule or biologic molecule of the cancer. Such biologic molecule can be for instance an anti-Ang2 antibody.

In another feature the invention provides for a method wherein said patient has previously been treated with chemotherapy, radiotherapy or surgery. In an embodiment said patient has failed chemotherapy, radiotherapy or surgery.

In another feature the invention provides a method wherein said patient has previously been treated with therapy based on oxaliplatin with or without bevacizumab. In an embodiment said patient has failed therapy based on oxaliplatin with or without bevacizumab.

In another feature the invention provides a method wherein folinic acid at a dosage comprised between about 200 mg/m² and about 600 mg/m², 5-fluorouracil (5-FU) at a dosage comprised between about 2000 mg/m² and about 4000 mg/m², irinotecan at a dosage comprised between about 100 mg/m² and about 300 mg/m² and aflibercept at a dosage comprised between about 1 mg/kg and about 10 mg/kg are administered to patient.

In the present application the dosage of folinic acid indicated should be understood as the dosage of the racemate of folinic acid, i.e. comprising the D and L forms. Should only the L form be used the dosage should be half of the dosage indicated for the racemate.

In other words a dosage of folinic acid of about 200 mg/m² as indicated in the present application corresponds to about 200 mg/m² of racemate and about 100 mg/m² of L form. In another feature the invention provides a method wherein folinic acid at a dosage of about 400 mg/m², 5-fluorouracil (5-FU) at a dosage of about 2800 mg/m², irinotecan at a dosage of about 180 mg/m² and aflibercept at a dosage of about 4 mg/kg are administered to patient. In another feature the invention provides a method wherein said patient receives intravenous folinic acid at a dosage comprised of about 400 mg/m², intravenous 5- fluorouracil (5-FU) at a dosage of about 2800 mg/m², intravenous irinotecan at a dosage comprised of about 180 mg/m² and intravenous aflibercept at a dosage of about 4 mg/kg every two weeks.

In another feature the invention provides a method wherein said patient receives intravenous folinic acid, intravenous 5-fluorouracil (5-FU), intravenous irinotecan and intravenous aflibercept every two weeks for a period comprised between about 9 and about 18 weeks. In another feature the invention provides a method wherein said patient receives intravenous folinic acid immediately after aflibercept administration.

In another feature the invention provides a method wherein said patient receives intravenous irinotecan immediately after aflibercept administration.

In another feature the invention provides a method wherein said patient receives intravenous irinotecan immediately after aflibercept administration over almost 90 minutes.

In another feature the invention provides a method wherein said patient receives intravenous 5-fluorouracil (5-FU) immediately after aflibercept administration.

In another feature the invention provides a method wherein said patient receives a first quantity of intravenous 5-fluorouracil (5-FU) immediately after aflibercept administration and a second quantity in continous infusion.

In another feature the invention provides a method wherein said patient receives about 400 mg/m² of intravenous 5-fluorouracil (5-FU) over about 2 to 4 minutes after aflibercept administration and 2400 mg/m² over about 46 hours after aflibercept administration in continuous infusion.

In another aspect the invention features a composition comprising therapeutically effective amounts of aflibercept in combination with folinic acid, 5-fluorouracil (5-FU) and irinotecan for treating patients with CRC for simultaneous administration. In another aspect the invention features a composition comprising therapeutically effective amounts of aflibercept in combination with folinic acid, 5-fluorouracil (5-FU) and irinotecan for treating patients with CRC for sequential administration.

In another aspect the invention features a composition comprising therapeutically effective amounts of aflibercept in combination with folinic acid, 5-fluorouracil (5-FU) and irinotecan for treating patients with CRC for administration that is spaced out over a period of time so as to obtain the maximum efficacy of the combination.

In another aspect the invention features a composition comprising therapeutically effective amounts of aflibercept in combination with folinic acid, 5-fluorouracil (5-FU) and irinotecan and comprising a pharmaceutically acceptable carrier for treating patients with CRC.

In on feature of any of these aspects the patient has liver metastases.

In another aspect the invention features an article of manufacture comprising:

a) a packaging material

b) aflibercept, and

c) a label or package insert contained within said packaging material which indicates that the patient should adapt his treatment in function of the ratio free/bound aflibercept.

In another aspect the invention features a kit for treating patients with CRC

comprising:

a) at least one compound chosen from the list consisting of aflibercept, folinic acid, 5-fluorouracil (5-FU) and irinotecan; and

b) a label or package insert contained within said kit indicating that aflibercept is to be used in combination with folinic acid, 5-fluorouracil (5-FU) and irinotecan (FOLFIRI) or folinic acid, 5-fluorouracil (5-FU) and irinotecan (FOLFIRI) is to be used in combination with Aflibercept

In another aspect the invention features a kit comprising in separate containers pharmaceutical compositions for combined use in treating CRC in a patient which comprises (1 ) a pharmaceutical composition comprising aflibercept, (2) a pharmaceutical composition comprising folinic acid, (3) a pharmaceutical composition comprising 5-fluorouracil (5-FU) and (4) a pharmaceutical composition comprising irinotecan.

In another aspect the invention relates to aflibercept for use in treating patients with cancer or cancer symptom wherein the ratio free/bound aflibercept in a biological sample of said patient is measured and the treatment of said patient is adapted based on the ratio free/bound aflibercept.

In still another aspect the invention relates to aflibercept for use in treating patients with cancer or cancer symptom wherein the ratio free/bound aflibercept in a biological sample of said patient is measured and a therapeutically effective amount of aflibercept is administered to patient displaying a ratio free/bound aflibercept comprised between 1 and 4.

The production process of aflibercept is typical for a recombinant-Fc fusion protein. The upstream process includes expansion of the CHO host cells and expression of recombinant aflibercept. The downstream process involves clarification and purification of the protein from the culture medium.

The manufacturing process is initiated with the thawing and inoculation of one working cell bank (WCB) vial. The cell culture is expanded until reaching sufficient density for inoculation into the production bioreactor.

The downstream process consists of several chromatography steps (protein A affinity chromatography, Cation exchange chromatography, Anion exchange chromatography and Hydrophobic Interaction chromatography), and includes viral inactivation and filtration steps to clear potential adventitious viral agents. After processing through the step of concentration/diafiltration, the drug substance is filled into containers and stored frozen. Aflibercept can be formulated as described in WO2006/104852.

In particular Zaltrap is formulated in vials of 4 ml containing 100 mg of aflibercept (25 mg/ml) or in vials of 8 ml containing 200 mg of aflibercept (25 mg/ml).

Zaltrap formulation also contains sucrose, sodium chloride, sodium citrate dihydrate, citric acid monohydrate, polysorbate 20, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate, sodium hydroxide and/or hydrochloric acid and water for injections.

However aflibercept can be formulated differently if it is intended to be administered by other ways for instance by sub cutaneous administration. If the sub cutaneous administration route is used aflibercept can be formulated as described in US 8,404,638.

The man skilled in the art may refer in particular to WO2006/104852 or to WO 00/75319 to carry out the present invention.

Figures Figure 1 : Aflibercept amino acid sequence (SEQ ID NO:1 )

Figure 2 : Overall survival (months) - Kaplan-Meier curves by treatment group- ITT population

Figure 3 : Overall survival (months) - Subgroup analyses (forest plot) - By stratification factors as per IVRS - ITT population

Figure 4: Overall survival (months) - Subgroup analyses (forest plot) - By patient demographics - ITT population

Figure 5 : Overall survival (months) - Subgroup analyses (forest plot) - By baseline characteristics - ITT population

Figure 6 : PFS based on tumor assessment by the IRC (months) - Subgroup analysis (forest plot) - By stratification factors as per IVRS - ITT population

Figure 7: VELOUR - OS Kaplan-Meier curves by free aflibercept Cmax

Figure 8: VELOUR - OS Kaplan-Meier curves by average free aflibercept AUC

Figure 9: VELOUR - OS Kaplan-Meier curves by free/bound aflibercept median ratio (threshold=2)

Figure 10: Velour - OS KM curves by free/bound aflibercept median ratio

(thresholds .6)

Figure 1 1 : Velour - OS KM curves by free/bound aflibercept median ratio

(threshold=3.2) Figure 12: Velour - OS KM curves by free/bound aflibercept ratio at end of Cycle 2 (thresholds .6)

Figure 13: Velour - OS KM curves by free/bound aflibercept ratio at end of Cycle 2 (threshold=2)

Figure 14: Velour - OS KM curves by free/bound aflibercept ratio at end of Cycle 2 (threshold=3.2)

Figure 15: VELOUR - OS Kaplan-Meier estimates according to Free/Bound aflibercept median ratio (looking at various thresholds)

The following examples illustrate the invention. EXAMPLE 1 : EFC10262 (VELOUR)/ A Multinational. Randomized. Double-blind Study. Comparing the Efficacy of Aflibercept Once Every 2 Weeks versus Placebo in Patients with Metastatic Colorectal Cancer (MCRC) Treated with Irinotecan / 5-FU Combination (FOLFIRI) after failure of an oxaliplatin based regimen EFC10262 was designed as a randomized, double-blind, multi-centre study comparing aflibercept at 4 mg/kg to placebo, in combination with Irinotecan and 5 Fluorouracil combination (FOLFIRI) given intravenously every 2 weeks as second line treatment for patients with metastatic colorectal cancer (MCRC) after failure of an oxaliplatin based regimen. Each randomized patient was to be treated until disease progression, death, or unacceptable toxicity.

The primary objective of EFC10262 was to demonstrate improvement in overall survival (OS) for aflibercept + FOLFIRI compared to placebo + FOLFIRI. The predefined statistical significance level for this final analysis was 0.0466 after adjusting the type I error spent for the two interim analyses using the O'Brien-Fleming spending function.

The study included one formal interim analysis, planned for the purpose of efficacy, when 561 death events (65% information time) had occurred. Upon request of the independent Data Monitoring Committee (DMC), an additional interim analysis of OS was performed to provide an early evaluation of the benefit-risk ratio, when 315 death events (36.5% information fraction) had occurred.

A total of approximately 863 deaths were required to detect 20% hazard rate reduction in OS with 90% power using the two-sided log rank test at an overall 0.0499 alpha level. The median survival times was expected to be 1 1 months for the control group. The overall alpha level was split between overall survival (0.0499) and progression-free survival as a secondary efficacy endpoint (0.0001 ).

Approximately 1200 patients (i.e. 600 patients per treatment group) were planned to be randomized. Treatment assignment was stratified according to prior therapy with bevacizumab (yes or no), and ECOG performance status (PS) (0 vs 1 vs 2).

The enrolment started in November 2007 and was completed in March 2010. A total of 1226 patients were randomized. The efficacy analysis was based on all randomized patients (Intent-to-Treat (ITT) population: 614 in the placebo arm and 612 patients in the aflibercept arm). The safety analysis was based on all treated patients (safety population: 605 and 61 1 patients in the placebo and aflibercept arms, respectively). Treatment arms were evenly balanced for demographics, disease characteristics and prior anti-cancer treatments, including prior exposition to bevacizumab.

Dosage and schedule of administration

Patients were administered either aflibercept or placebo, depending on arm assigned. Immediately after, patients received irinotecan, 5-FU and leucovorin (FOLFIRI regimen).

This treatment was repeated every 2 weeks. Aflibercept placebo

Arm A, aflibercept: 4 mg/kg was administered IV over 1 hour on Day 1 , every 2 weeks,

OR

Arm B, placebo: 4 mg/kg was administered IV over 1 hour on Day 1 , every 2 weeks. FOLFIRI regimen

Immediately after aflibercept/placebo administration, all the patients received:

Irinotecan 180 mg/m² IV infusion in 500 mL in 5% dextrose solution in water

(D5W) over 90 minutes and dextro-levogyre (dl) leucovorin 400 mg/m² IV infusion over 2 hours, at the same time, in bags using a Y-line, followed by:

5-FU 400 mg/m² IV bolus given over 2-4 minutes, followed by:

5-FU 2400 mg/m² continuous IV infusion in 500 mL D5W (recommended) over 46-hours. Results of EFC10262

Demographics and baseline characteristics

Patient demographics and characteristics at baseline were similar the 2 treatment arms (Table 1 ). Table 1 - Summary of patient demographics and patient characteristics at baseline - ITT population

Placebo/Folfiri Aflibercept Folfiri All (N=614) (N=612) (N=1226)

Gender [n(%)]

Number 614 612 1226

Male 353 (57.5%) 365 (59.6%) 718 (58.6%)

Female 261 (42.5%) 247 (40.4%) 508 (41.4%)

Age (Years)

Number 614 612 1226

Median 61 .0 61 .0 61 .0

Mean (SD) 60.2 (10.8) 59.5 (10.5) 59.8 (10.7)

Min : Max 19 : 86 21 : 82 19 : 86

Age class [n(%)]

Number 614 612 1226

<65 376 (61 .2%) 407 (66.5%) 783 (63.9%)

≥65 but <75 199 (32.4%) 172 (28.1 %) 371 (30.3%)

≥75 39 (6.4%) 33 (5.4%) 72 (5.9%)

Race [n(%)]

Number 614 612 1226

Caucasian/White 523 (85.2%) 548 (89.5%) 1071 (87.4%)

Black 27 (4.4%) 16 (2.6%) 43 (3.5%)

Asian/Oriental 51 (8.3%) 35 (5.7%) 86 (7.0%)

Other 13 (2.1 %) 13 (2.1 %) 26 (2.1 %) Placebo/Folfiri Aflibercept Folfiri All

(N=614) (N=612) (N=1226)

Region

Number 614 612 1226

Western Europe 217 (35.3%) 208 (34.0%) 425 (34.7%)

Eastern Europe 136 (22.1 %) 161 (26.3%) 297 (24.2%)

North America 75 (12.2%) 63 (10.3%) 138 (1 1.3%)

South America 56 (9.1 %) 62 (10.1 %) 1 18 (9.6%)

Other countries 130 (21 .2%) 1 18 (19.3%) 248 (20.2%)

Note: Other countries = Australia, New Zeland, South Africa and Korea

Disease characteristics at initial diagnosis and time from diagnosis to randomization were similar in the 2 treatment arms (Table 2).

Table 2 - Disease characteristics at initial diagnosis - ITT population

Placebo/Folfiri Aflibercept Folfiri All (N=614) (N=612) (N=1226)

Primary site [n(%)]

Number 614 612 1226

591

Colon 302 (49.2%) 289 (47.2%) (48.2%)

259

Recto sigmoid 136 (22.1 %) 123 (20.1 %) (21.1 %)

371

Rectum 174 (28.3%) 197 (32.2%) (30.3%)

Other 2 (0.3%) 3 (0.5%) 5 (0.4%)

- cea & ck20 postive - presumed colorectal 1 primary 1 (0.2%) 0 (<0.1 %)

1

- Appendix 0 1 (0.2%) (<0.1 %)

1

- Colon plus appendix 0 1 (0.2%) (<0.1 %)

- Presumed

colorectal, cea positive

and history of colon 1 cancer>20 years ago 0 1 (0.2%) (<0.1 %)

- Synchronous primary, 1 cecum and rectum 1 (0.2%) 0 (<0.1 %)

Patient accountability

Overall, 30.4% of the randomized patients were allocated in the prior bevacizumab stratum (Table 3).

Table 3 - Summary of randomized patients by stratification factor (as per IVRS) - ITT population

Placebo/Folfir

Aflibercept Folfiri All

Stratification factors (N=614) (N=612) (N=1226)

ECOG PS [n(%)]

350

0 (57.0%) 349 (57.0%) 699 (57.0%)

250

1 (40.7%) 250 (40.8%) 500 (40.8%)

2 14 (2.3%) 13 (2.1 %) 27 (2.2%)

Prior Bevacizumab

[n(%)]

187

Yes (30.5%) 186 (30.4%) 373 (30.4%)

427

No (69.5%) 426 (69.6%) 853 (69.6%)

Note: ECOG: Eastern Cooperative Oncology Group, PS: Performance Status, IVRS: Interactive Voice response System Dosage and duration

The median overall study treatment exposure (i.e. either both study drugs aflibercept/placebo and FOLFIRI, or one of them alone) was 8.0 and 9.0 cycles in the placebo and aflibercept treatment arms, respectively (Table 4). Table 4 - Summary of overall study treatment exposure - Safety population

The median number of aflibercept/placebo infusions was 8.0 and 7.0 in the placebo and aflibercept treatment arms, respectively (Table 5). The median relative dose intensity was 83% with aflibercept as compared to 92% with placebo.

Table 5 - Exposure to Aflibercept Placebo - Safety population

Placebo/Folfiri Aflibercept Folfiri (N=605) (N=611)

Number of cycles received by patient

Sum 6035 5632

Mean (SD) 10.0 (8.0) 9.2 (7.2)

Median 8.0 7.0

Min : Max 1 : 67 1 : 35

Placebo/Folfiri AfliberceptFolfiri

(N=605) (N=611)

1 24 (4.0%) 43 (7.0%)

2 32 (5.3%) 52 (8.5%)

3 85(14.0%) 70 (11.5%)

4 31 (5.1%) 45 (7.4%)

5 32 (5.3%) 43 (7.0%)

6 45 (7.4%) 29 (4.7%)

7 29 (4.8%) 28 (4.6%)

8 34 (5.6%) 29 (4.7%)

9 45 (7.4%) 29 (4.7%)

10 21 (3.5%) 28 (4.6%)

11-15 112 (18.5%) 94 (15.4%)

16-20 57 (9.4%) 68 (11.1%)

21-25 28 (4.6%) 34 (5.6%)

>25 30 (5.0%) 19 (3.1%)

Duration of exposure to

aflibercept/placebo (weeks)

Number 605 611

Mean (SD) 22.3 (17.5) 21.7 (16.7)

Median 18.0 17.9

Min : Max 2 : 135 2 : 85

Total cumulative dose received (mg/kg)

Number 605 611

Mean (SD) 39.63 (31.65) 35.69 (27.96)

Median 32.00 28.00

Min : Max 0.6 : 266.4 3.8 : 140.0

Actual dose intensity (mg/kg/week)

Number 605 611

Mean (SD) 1.78 (0.25) 1.55 (0.44)

Median 1.84 1.66

Min : Max 0.3 : 2.1 0.1 : 2.1 Placebo/Folfiri Aflibercept Folfiri

(N=605) (N=611)

Relative dose intensity

Number 605 61 1

Mean (SD) 0.89 (0.12) 0.78 (0.22)

Median 0.92 0.83

Min : Max 0.2 : 1 .1 0.1 : 1 .1

Number of cycles received: Number of cycles with at least one dose infusion of aflibercept placebo.

The median number of irinotecan infusions was 8.0 and 9.0 in the placebo and aflibercept treatment arms, respectively (table 6). The median relative dose intensity was 84% in the aflibercept arm as compared to 91 % in the placebo arm. Of note, two patients did not receive irinotecan; the dose was considered equal to 0 for the calculation of the cumulative dose, actual and relative dose intensity.

Table 6 - Exposure to irinotecan- Safety population

Placebo/Folfiri Aflibercept Folfiri (N=605) (N=611)

Number of cycles received by

patient

Sum 5992 6157

Mean (SD) 9.9 (7.8) 10.1 (7.4)

Median 8.0 9.0

Min : Max 1 : 67 1 : 50

1 23 (3.8%) 34 (5.6%)

2 29 (4.8%) 39 (6.4%)

3 87 (14.4%) 64 (10.5%)

4 33 (5.5%) 36 (5.9%)

5 29 (4.8%) 37 (6.1 %)

6 48 (7.9%) 31 (5.1 %)

7 27 (4.5%) 27 (4.4%)

8 32 (5.3%) 29 (4.8%)

9 47 (7.8%) 29 (4.8%)

10 21 (3.5%) 38 (6.2%)

1 1 -15 1 14 (18.9%) 1 1 1 (18.2%)

16-20 58 (9.6%) 78 (12.8%)

21 -25 31 (5.1 %) 35 (5.7%)

>25 25 (4.1 %) 22 (3.6%) Placebo/Folfiri Aflibercept Folfiri

(N=605) (N=611)

Duration of exposure to irinotecan

(weeks)

Number 604 610

Mean (SD) 22.2 (17.2) 23.5 (16.9)

Median 18.1 21 .0

Min : Max 2 : 135 2 : 105

Total cumulative dose received

(mg/m²)

Number 605 61 1

Mean (SD) 1736.30 (1355.52) 1730.37 (1273.76)

Median 1440.00 1472.50

Min : Max 0.0 : 1 1948.1 0.0 : 9046.1

Actual dose intensity

(mg/m²/week)

Number 605 61 1

Mean (SD) 78.82 (1 1.74) 73.59 (13.68)

Median 82.08 75.60

Min : Max 0.0 : 95.0 0.0 : 95.0

Relative dose intensity

Number 605 61 1

Mean (SD) 0.88 (0.13) 0.82 (0.15)

Median 0.91 0.84

Min : Max 0.0 : 1 .1 0.0 : 1 .1

Number of cycles received: Number of cycles with at least one dose infusion of irinotecan.

The median number of 5-FU infusions was 8.0 and 9.0 in the placebo and aflibercept treatment arms, respectively (Table 7). The median relative dose intensity was 83% in the aflibercept arm as compared to 91 % in the placebo arm. Of note, two patients did not receive 5-FU; the dose was considered equal to 0 for the calculation of the cumulative dose, actual and relative dose intensity. Table 7 - Exposure to 5-FU - Safety population

Placebo/Folfiri Aflibercept Folfiri (N=605) (N=611)

Number of cycles received by patient

Sum 6030 6155

Mean (SD) 10.0 (7.9) 10.1 (7.4)

Median 8.0 9.0

Min : Max 1 : 67 1 : 50

1 22 (3.6%) 35 (5.7%)

2 28 (4.6%) 39 (6.4%)

3 88 (14.6%) 63 (10.3%)

4 33 (5.5%) 35 (5.7%)

5 28 (4.6%) 37 (6.1 %)

6 48 (8.0%) 32 (5.2%)

7 27 (4.5%) 28 (4.6%)

8 33 (5.5%) 28 (4.6%)

9 47 (7.8%) 29 (4.7%)

10 20 (3.3%) 39 (6.4%)

1 1 -15 1 14 (18.9%) 1 13 (18.5%)

16-20 59 (9.8%) 77 (12.6%)

21 -25 28 (4.6%) 35 (5.7%)

>25 28 (4.6%) 21 (3.4%)

Duration of exposure to 5-FU

(weeks)

Number 603 61 1

Mean (SD) 22.4 (17.5) 23.5 (16.9)

Median 18.1 21 .0

Min : Max 2 : 135 2 : 105

Total cumulative dose received

(mg/m²)

Number 605 61 1

27142.02

Mean (SD) (21341 .89) 26644.81 (19245.24)

Median 22400.00 22702.44

Min : Max 0.0 : 185874.8 409.0 : 126701 .4 Placebo/Folfiri Aflibercept Folfiri

(N=605) (N=611)

Actual dose intensity (mg/m²/week)

Number 605 61 1

Mean (SD) 1227.42 (190.51 ) 1 140.36 (214.35)

Median 1276.38 1 165.56

Min : Max 0.0 : 1477.3 177.0 : 1491 .3

Relative dose intensity

Number 605 61 1

Mean (SD) 0.88 (0.14) 0.81 (0.15)

Median 0.91 0.83

Min : Max 0.0 : 1 .1 0.1 : 1 .1

Number of cycles received: Number of cycles with at least one dose infusion of 5- FU.

Results of EFC10262

1. Overall survival

The median follow-up time at the cutoff date (07 February 201 1 ) for the ITT population was 22.28 months (Figure 2 and Table 8). The study met its primary endpoint demonstrating a significant difference in overall survival in favor of aflibercept over placebo (stratified HR: 0.817, 95.34% CI: 0.713 to 0.937; p = 0.0032). The hazard ratio translates into a reduction of risk of death of 18.3% (95.34 CI: 6.3% to 28.7%) with aflibercept compared to placebo. After 12 and 18 months from randomization, the estimated probabilities of being alive were 50.3% in placebo arm and 56.1 % aflibercept arm, and 30.9% in placebo arm and 38.5% in aflibercept arm. Median overall survival was 13.50 months vs 12.06 months in aflibercept and placebo treatment arms, respectively. Sensitivity analyses and subgroup analyses showed a very consistent treatment effect confirming robustness of results on the primary endpoint.

Table 8 - Overall survival (months) - Kaplan-Meier survival estimates by treatment group- Primary analysis- Stratified according to stratification factors at randomization (IVRS) - ITT population Placebo/Folfiri Aflibercept Folfiri

Time to Event or Censoring (N=614) (N=612)

Overall

Number of death events,

n/N(%) 460/614 (74.9%) 403/612 (65.8%)

Median overall survival 13.50 (12.517 to (95.34% CI) (months) 12.06 (1 1.072 to 13.109) 14.949)

Number of patients at risk

3 months 573 566

6 months 485 498

9 months 401 416

12 months 286 31 1

18 months 131 148

24 months 51 75

Survival probability (95.34% CI)

0.931 (0.91 1 to

3 months 0.935 (0.915 to 0.955) 0.951 )

0.819 (0.788 to

6 months 0.791 (0.759 to 0.824) 0.850)

0.687 (0.650 to

9 months 0.654 (0.616 to 0.692) 0.725)

0.561 (0.521 to

12 months 0.503 (0.462 to 0.543) 0.602)

0.385 (0.343 to

18 months 0.309 (0.269 to 0.348) 0.427)

0.280 (0.237 to

24 months 0.187 (0.149 to 0.225) 0.324)

Stratified Log-Rank test p-value^a

vs Placebo/Folfiri - 0.0032

Stratified Hazard ratio (95.34%

Cl)^a

0.817 (0.713 to vs Placebo/Folfiri 0.937)

Cutoff date = 7 FEBRUARY 201 1

Median follow-up time = 22.28 in months

a : Stratified on ECOG Performance Status (0 vs 1 vs 2) and Prior Bevacizumab (yes vs no) according to IVRS . Significance threshold is set to 0.0466 using the O'Brien-Fleming alpha spending function. Subgroup analyses of Overall survival (OS)

Subgroup analyses did not show any significant interaction (at the 2-sided 10% level) between treatment arms and stratification factors, indicating that the treatment effect was consistent across subgroups. This is illustrated in Table 9 and in Figures 3, 4 and 5.

Table 9 - Overall survival (months) - Summary of subgroup analyses

stratification factors as per IVRS - ITT population

Treatment effect for OS was consistent across subgroups with regards to baseline characteristics at study entry. Of note, the interaction between treatment arms and the presence of liver metastasis factor was significant at 10% level, indicating a higher treatment effect in 'liver metastasis only' group (HR (95.34% CI): 0.649 (0.492 to 0.855)) than in 'no liver metastasis, or other metastases' group (HR (95.34% CI): 0.868 (0.742 to 1 .015)) (quantitative interaction, p=0.0899) This is illustrated in Table 10. Table 10 - Overall survival (months) - Summary of subgroup analyses - By baseline characteristics - ITT population

Aflibercept Folfi

Placebo/Folfiri ri P-value

Median Median Hazard Ratio for

(Months) (Months) (95.34% CI) vs interact! o (95.34% CI) (95.34% CI) Placebo/Folfiri n^a

12.1 (1 1.07 to 13.5 (12.52 to 0.817 (0.713 to

All patients 13.1 1 ) 14.95) 0.937)

Prior

hypertension

1 1.7 (10.41 to 12.7 (1 1.17 to 0.883 (0.74 to

No 13.1 1 ) 14.39) 1 .054) 0.1309

12.7 (10.78 to 15.5 (12.91 to 0.714 (0.577 to

Yes 14.00) 18.56) 0.884)

Number of

metastatic

organs

involved

10.5 (9.72 to 12.1 (10.71 to 0.825 (0.692 to

> 1 12.06) 13.1 1 ) 0.982) 0.6992

13.7 (12.29 to 16.0 (14.42 to 0.767 (0.618 to

<= 1 16.30) 20.86) 0.953)

Liver

Metastasis

only

12.3 (1 1.07 to 13.2 (12.06 to 0.868 (0.742 to

No 13.73) 15.28) 1 .015) 0.0899

1 1 .4 (9.86 to 14.4 (12.68 to 0.649 (0.492 to

Yes 12.88) 18.04) 0.855)

Location of

primary tumor

10.6 (9.66 to 12.9 (1 1.50 to 0.739 (0.607 to

Colon 12.06) 16.16) 0.899) 0.1421

Recto 14.1 (12.71 to 14.3 (12.35 to 1.039 (0.772 to

sigmoid/Other 17.08) 16.39) 1 .4) Aflibercept Folfi

Placebo/Folfiri ri P-value Median Median Hazard Ratio for (Months) (Months) (95.34% CI) vs interactio (95.34% CI) (95.34% CI) Placebo/Folfiri n^a

12.6 (10.35 to 13.5 (1 1.93 to 0.806 (0.629 to

Rectum 14.55) 15.87) 1 .031 )

Median follow-up time = 22.28 in months

a : Interaction test from the Cox proportional hazard model including the factor, treatment effect and the treatment by factor interaction

2. Progression free survival based on tumor assessment by the IRC

The final analysis for PFS was performed at the time of the second interim analysis of OS (i.e. cut off date = 06 MAY 2010). Improvement in progression free survival (PFS) was demonstrated in patients of the aflibercept treatment arm compared to patients in the placebo treatment arm (stratified HR: 0.758, 99.99%CI: 0.578 to 0.995; p = 0.00007). Median PFS was 6.90 months in the aflibercept arm and 4.67 months in the placebo arm (Table 1 1 ).

Table 11 - PFS based on tumor assessment by the IRC (months) - Kaplan-Meier survival estimates by treatment group - Stratified according to stratification factors at randomization (IVRS) - ITT population

Placebo/Folfiri Aflibercept/Folfiri

Time to Event or Censoring (N=614) (N=612)

Overall

Number of events, n/N(%) 454/614 (73.9%) 393/612 (64.2%)

Median PFS (99.99% CI)

(months) 4.67 (4.074 to 5.552) 6.90 (5.881 to 7.852)

Number at risk

3 months 355 420

6 months 171 247

9 months 94 99

12 months 46 43

18 months 9 7 Placebo/Folfiri Aflibercept Folfiri

Time to Event or Censoring (N=614) (N=612)

Probability of surviving

(99.99% CI)

3 months 0.664 (0.587 to 0.741 ) 0.793 (0.727 to 0.859)

6 months 0.390 (0.306 to 0.475) 0.573 (0.488 to 0.659)

9 months 0.254 (0.174 to 0.334) 0.313 (0.222 to 0.404)

12 months 0.146 (0.076 to 0.216) 0.166 (0.085 to 0.246)

18 months 0.043 (0.000 to 0.091 ) 0.051 (0.000 to 0.108)

Stratified Log-Rank test p- value³

vs Placebo/Folfiri - 0.00007

Stratified Hazard ratio (99.99%

Cl)^a

vs Placebo/Folfiri - 0.758 (0.578 to 0.995)

Cutoff date = 06 MAY 2010

a : Stratified on ECOG Performance Status (0 vs 1 vs 2) and Prior Bevacizumab (yes vs no) according to IVRS

Significance threshold is set to 0.0001 .

Subgroup analyses of Progression free survival

Progression free survival (PFS) was analyzed in subgroups as illustrated in Table 12 and in Figure 6. No interaction between treatment arms and stratification factors was observed (Table 12).

Table 12 - PFS based on tumor assessment by the IRC (months) - Summary of subgroup analyses - By stratification factors as per IVRS - ITT population

Aflibercept/

Placebo/Folfiri Folfiri

Median Median Hazard Ratio

(Months) (Months) (99.99% CI) vs P-value for (99.99% CI) (99.99% CI) Placebo/Folfiri interaction³

4.7 (4.07 to 6.9 (5.88 to 0.758 (0.578 to

All patients 5.55) 7.85) 0.995)

Prior

bevacizumab Aflibercept

Placebo/Folfiri Folfiri

Median Median Hazard Ratio (Months) (Months) (99.99% CI) vs P-value for (99.99% CI) (99.99% CI) Placebo/Folfiri interaction³

5.4 (4.17 to 6.9 (5.82 to 0.797 (0.58 to

No 6.70) 8.15) 1 .096) 0.6954

3.9 (2.86 to 6.7 (4.76 to 0.661 (0.399 to

Yes 5.42) 8.74) 1 .095)

ECOG PS

5.4 (4.24 to 7.2 (6.37 to 0.761 (0.529 to

0 6.77) 8.87) 1 .094) 0.1958

4.1 (2.83 to 5.6 (4.60 to 0.749 (0.494 to

1 5.55) 7.46) 1 .135)

2.0 (1.18 to 2.7 (0.53 to 0.618 (0.1 1 to

2 5.75) 12.88) 3.476)

Cutoff date = 06 MAY 2010

For PFS, no significant interaction was shown between treatment arms and demographic variables or regions.

Treatment effect for PFS was consistent across subgroups with regards to baseline characteristics at study entry. Of note, the interaction between treatment arms and the presence of liver metastasis factor, that was noted on OS, was also significant at 10% level, indicating a higher treatment effect 'in liver metastasis only' group (HR

(99.99%CI): 0.547 (0.313 to 0.956)) than in 'no liver metastasis, or other metastases' group (HR (99.99%CI): 0.839 (0.617 to 1 .143)) (quantitative interaction, p=0.0076).

Results of the two sensitivity analyses for PFS were consistent with those of the primary PFS analysis. Moreover, adherence to the protocol-defined schedule for tumor assessment was assessed and showed no imbalance between treatment arms. 3. Overall Response rate

Overall response rate -IRC reviewed- was significantly higher in the aflibercept treatment arm when compared to the placebo treatment arm: 19.8% (95%CI: 16.4% to 23.2%) vs 1 1.1 % (95%CI: 8.5% to 13.8%) respectively (p=0.0001 ) (Table 13).

Table 13 - Summary of overall objective response rate by IRC - Evaluable patient population for response rate

4. Further anti-cancer therapy

Overall 60% of patients in both treatment groups received further antitumor therapies (Table 14). Table 14 - Summary of first further anti-cancer therapies - ITT population

About 32% of patients in each group receive further anticancer treatment that includes a "biologic (Table 15). Table 15 - Summary of all further anti-cancer therapies - ITT population

5. Safety

Adverse events

Treatment emergent adverse events, all grades, were reported in nearly 100% of the patients in both treatment arms, whereas occurence of grade 3-4 events was greater in the aflibercept treatment arm (83.5% vs 62.5%).

The rate of permanent discontinuation of study treatment due to adverse events was higher in the aflibercept treatment arm (26.8% vs 12.1 %). A similar pattern was observed for premature treatment discontinuation due to adverse events (19.5% vs 2.8%). Premature treatment discontinuation corresponds to an earlier discontinuation of either FOLFIRI, aflibercept/placebo being continued, or aflibercept/placebo, FOLFIRI being continued.

Within 30 days of last dosing, respectively 37 (6.1 %) and 29 (4.8%) patients in the aflibercept and placebo arm, respectively, experienced, adverse events that eventually led to death within 30 days (28 vs 17 in the aflibercept and placebo arm, respectively) or after 30 days (9 vs 12 in the placebo and aflibercept arm,

respectively) of last dosing. These included death due to disease progression.

A summary of safety data is illustrated in Table 16 and Table 17.

Table 16 - Summary of the most frequent TEAEs: incidence > 20% in aflibercept arm or (incidence < 20% in aflibercept arm and Δ all grades >5 %) - Safety population

% of Placebo/Folfiri Aflibercept Fol Δ>10% 5<Δ<10 Δ>2% patients (in N = 605 firi all Gr % Gr 3/4 the safety N = 611 all Gr

population) All Gr Gr 3/4 All Gr Gr 3/4

Incidence > 20% (aflibercept arm)

Diarrhea 56.5 7.8 69.2 19.3 X X (PT)

Asthenic 50.2 10.6 60.4 16.9 X X condition

(HLT)

Stomatitis & 34.9 5.0 54.8 13.7 X X ulceration

(HLT)

Nausea (PT) 54.0 3.0 53.4 1 .8

Infections 32.7 6.9 46.2 12.3 X X (SOC)

Hypertensio 10.7 1 .5 41 .4 19.3 X X n (grouping)

Gl and 29.1 3.3 34.0 5.4

abdominal

pains (HLT)

Vomiting 33.4 3.5 32.9 2.8

(PT)

Decrease 23.8 1 .8 31 .9 3.4 X

appetite

(PT) % of Placebo/Folfiri Aflibercept Fol Δ>10% 5<Δ<10 Δ>2% patients (in N = 605 firi all Gr % Gr 3/4 the safety N = 611 all Gr population) All Gr Gr 3/4 All Gr Gr 3/4

Incidence > 20% (aflibercept arm)

Weight 14.4 0.8 31 .9 2.6 X

decrease

(PT)

Epistaxis 7.4 0 27.7 0.2 X

(PT)

Alopecia 30.1 NA 26.8 NA

(PT)

Dysphonia 3.3 0 25.4 0.5 X

(PT)

Musculoskel 21 .2 2.3 23.1 1.3

etal &

connective

pain &

discomfort

(HLT)

Constipation 24.6 1 .0 22.4 0.8

(PT)

Headache 8.8 0.3 22.3 1.6 X

(PT)

Incidence < 20% (aflibercept arm) and Δ all grades > 5 %

Palmar 4.3 0.5 1 1.0 2.8 X

plantar

erythrodysa

esthesia

(PT)

Dehydration 3.0 1 .3 9.0 4.3 X

(PT)

Skin 2.8 0 8.2 0 X

hyperpigme

ntation (PT)

Medra classification: SOC (system organ class), HLT (high level term), PT (Preferred term).

Grouping: grouping of selected PTs

Δ: difference in incidence in aflibercept arm compared to placebo

Table 17 - Overview of safety, number (%) of patients - Safety population Placebo/Folfiri Aflibercept Folfiri

(N=605) (N=611)

Patients with any TEAE 592 (97.9%) 606 (99.2%)

Patients with any grade 3-4 TEAE 378 (62.5%) 510 (83.5%)

Patients with any serious TEAE 198 (32.7%) 294 (48.1 %)

Patients with any TEAE leading to death 29 (4.8%) 37 (6.1 %)

Patients with any related TEAE leading

to death 3 (0.5%) 6 (1.0%)

Patients with any TEAE leading to

permanent treatment discontinuation 73 (12.1 %) 164 (26.8%)

Patients with any TEAE leading to

premature treatment discontinuation 17 (2.8%) 1 19 (19.5%)

Note : Adverse Events are reported using Med DRA version MEDDRA13.1 and graded using NCI CTC Version 3.0.

5. Conclusions

The study met its primary endpoint, with a significant improvement in overall survival in the aflibercept arm when compared to placebo.

In addition, a significant improvement was demonstrated on secondary efficacy endpoints (PFS and RR).

The safety profile was qualitatively consistent with that of anti VEGF treatment with enhancement of known toxicities of the background chemotherapy (such as diarrhea, stomatitis, infections, neutropenia/neutropenic complications).

EXAMPLE 2 : VELOUR exploratory PK-PD analyses on estimated free and bound aflibercept PK parameters

An exploratory PK-PD analysis was performed with the data obtained from VELOUR trial.

For the PK analysis, free aflibercept (not bound to VEGF) and the inert

"VEGF:aflibercept complex" (bound aflibercept) were quantified in plasma. Since VEGF:aflibercept complex contains one molecule of endogenous VEGF (MW=45390 g/mol) and one molecule of aflibercept (MW=1 15000 g/mol), VEGF-bound aflibercept concentrations were expressed as free aflibercept equivalents for PK analyses using the ratio of molecular weights between free aflibercept and VEGF:aflibercept complex. From a theoretical point of view, based on the law of mass action (Kd = [free aflibercept] x [free VEGF] / [bound aflibercept] = 0.5 pM), when [free aflibercept] > [bound aflibercept], VEGF concentrations are lower than 0.5 pM (or 20 pg/mL, expressed as VEGF equivalents, MW of VEGF = - 40,000 daltons). Thus, maintaining free aflibercept concentrations higher than bound concentrations over the dosing interval is expected to maintain endogenous VEGF levels at concentrations lower than 20 pg/mL (the median value reported in healthy subjects was 17 pg/mL).

Free and bound aflibercept trough concentrations were measured and maintaining free/bound ratio at least >1 in the dosing interval was used as a guide for dose selection in different clinical trials.

In the phase I study of aflibercept administered intravenously once every 14 days to patients with solid tumors (TED 61 15), PK studies demonstrated that at doses≥2 mg/kg, concentrations of free aflibercept exceeded bound aflibercept, and therapeutic concentrations of free aflibercept were maintained for the entire 14-day dosing period.

Measure of free and bound aflibercept

Free aflibercept and VEGF:aflibercept complex concentrations were measured in plasma by validated enzyme-linked immunosorbent assay (ELISA) methods with a LOQ of 0.0156 g/ml_ and 0.0439Mg/ml_, respectively. Plasma bound aflibercept concentrations were expressed as free aflibercept equivalents by adjusting the measured complex levels for the proportion of the molecular weight of the complex using the formula: [Bound aflibercept] = 0.717

[measured VEGF:aflibercept complex], with 0.717, the ratio of molecular weights between free and bound aflibercept. 1. Quantitation of Free Aflibercept in Human Plasma

Free aflibercept (unbound aflibercept) concentrations in human plasma were quantified using a validated, direct enzyme-linked immunosorbent assay. In this assay, hVEGF165 (aflibercept capture agent) is initially adsorbed to the polystyrene surface of the wells of microtiter plates and non-specific binding sites on the surface of the wells blocked with 5% BSA in PBS (blocking buffer). Calibration standards, quality control (QC) samples and subject samples, all in a 10% human CTAD plasma matrix (human CTAD plasma diluted with 0.5% bovine serum albumin [BSA] and 0.05% polysorbate 20 [Tween 20] in 1 x Dulbecco's phosphate-buffered saline [PBS]), are subsequently added and incubated with surface-bound hVEGF165, resulting in the binding of unbound (free) aflibercept to immobilized hVEGF165.

Following removal of excess reactants, a mouse monoclonal antibody specific to an epitope on the VEGFR1 domain of aflibercept report is bound to the immobilized hVEGF165:aflibercept complex. Following a wash to remove excess reagent, peroxidase-conjugated Affinipure goat anti-mouse IgG Fey report is bound to the immobilized mouse monoclonal antibody-hVEGF165:aflibercept complex. Following another wash, a luminol-based substrate specific for peroxidase is added to generate a chemiluminescent signal the intensity of which is directly proportional to the concentration of free aflibercept. 2. Quantitation of Bound Aflibercept in Human Plasma

Human VEGF:aflibercept complex was quantified using a validated direct ELISA. In this assay, a non-blocking goat anti-hVEGF antibody report is adsorbed to the polystyrene surface of the wells of a microtiter plate. Following adsorption of the capture antibody, non-specific protein binding sites on the surface of the wells are blocked with 5% BSA in PBS (blocking buffer) that contained 10 μg/mL goat IgG. Calibration standards, QC samples and subject samples all in a 20% human CTAD plasma matrix (human CTAD plasma diluted with 0.5% BSA, 10 μg mL goat IgG and 0.05% polysorbate 20 [Tween 20] in 1 x Dulbecco's phosphate-buffered saline [PBS]), are subsequently added and incubated with the surface bound anti-VEGF antibody resulting in the binding of hVEGF:aflibercept complex to the immobilized anti-human VEGF antibody. Following a wash to remove excess reactants, a mouse monoclonal antibody specific to an epitope on the VEGFR1 domain of aflibercept is bound to the immobilized complex. Following a wash to remove excess reactants, a mouse monoclonal antibody, peroxidase-conjugated Affinipure goat anti-mouse IgG Fey is bound to the immobilized mouse monoclonal antibody complex. Following an additional wash, a luminolbased substrate specific for peroxidase is added to generate a chemiluminescent signal the intensity of which is directly proportional to the concentration of the hVEGF:aflibercept complex. Population PK analysis

A population pharmacokinetic analysis of free aflibercept concentrations was performed using data from Phase 1 and Phase 2 patients where aflibercept was administered intravenously every two or three weeks as single agent or in

combination with various chemotherapy drugs at dose levels ranging from 2 to 9 mg/kg. The tested covariates were the following: age, gender, weight, race such as Caucasian, Black and Asian, albumin, serum alkaline phosphatase, total bilirubin, aspartate amino transferase, alanine amino transferase, total protein and creatinine clearance. Significant covariates were identified during the univariate analysis based on nonlinear mixed effect modeling using the likelihood ratio test to discriminate among alternative models, then incorporated into the model and final model building was accomplished by a backward elimination procedure (POH0253). Then, the PK of free aflibercept was further investigated with the successive addition of Phase 3 studies ie, EFC10547/VANILLA (POH0262), EFC10261A ITAL (POH0274) and EFC10262/VELOUR (POH0265).

In addition, based on its mechanism of action, aflibercept undergoes a target- mediated drug disposition (TMDD); a term used to describe the phenomenon in which drug is bound with high affinity to its pharmacologic target such that the interaction is reflected in the PK properties of the drug. Therefore, a mechanism-based PK model for aflibercept was developed in order to characterize its binding to VEGF and its pharmacokinetic properties in healthy subjects and then applied to the Phase 2 & 3 patients' database. No covariates were tested for inclusion in the model.

Subsequently, the mechanistic model was used to simulate concentration-time courses of free and bound aflibercept for a typical patient who receives 1 -hour IV infusion of 1 , 2, 4 or 6 mg/kg aflibercept q2w and q3w. After q2w and q3w

administration, maximum bound aflibercept levels were reached at doses≥ 4 mg/kg with similar bound levels after 4 mg/kg q2w and 6 mg/kg q3w. According to these simulations, the free to bound aflibercept ratio exceeded 1 throughout all the dosing intervals for 89% of the population receiving 4 mg/kg q2w and 69 % of the population receiving 6 mg/kg q3w. Overall, these simulations suggest that the dose of 4 mg/kg every 2 weeks or 6 mg/kg every 3 weeks are adequate to maximize binding of the available endogenous VEGF in most patients, supporting the selection of these dosing regimens in clinical settings. Pharmacokinetic (PK) and Pharmacodynamic (PD) analysis

PK-PD analyses were performed to estimate the relationship between aflibercept PK parameters and various measures of efficacy and safety. Free aflibercept

concentrations and VEGF:aflibercept complex concentrations were measured in plasma by a validated enzyme linked immunosorbent assay (ELISA) method.

Since VEGF:aflibercept complex contains one molecule of endogenous VEGF (MW=45390 g/mol) and one molecule of aflibercept (MW=1 15000 g/mol), VEGF- bound aflibercept concentrations were expressed as free aflibercept equivalents using the ratio of molecular weights between free aflibercept and VEGF:aflibercept complex. For Free aflibercept, the following PK parameters were estimated:

• Clearance

• AUC of first cycle (i.e, AUCO-336 or AUCO-504 in the q2w or q3w schedule, respectively)

• AUC extrapolated (calculated from the model based on clearance and actual dose received at 1 st cycle)

• Cmax of first cycle

• Cumulative AUC up to last aflibercept administration + 90 days or date of death or cutoff date, whichever comes first, divided by the number of cycles (denoted as average AUCcumOS)

· Cumulative AUC up to last aflibercept administration + 90 days or date of progression (radiologically documented progression or death) or cutoff date, whichever comes first, divided by the number of cycles (denoted as average AUCcumPFS, not derived for EFC10547A ANILLA),

• Cumulative AUC over the first 2 cycles (not derived for E F C 10547 A/AN I L LA) For bound aflibercept, the following PK parameters were estimated:

• Clearance (apparent clearance estimated from the model, inversely

proportional to bound aflibercept exposure)

The safety pharmacodynamic parameters used for this analysis included: • Patients with safety events were defined as patients who experienced the events (any grade, unless otherwise specified) at least once during the first 2 cycles of treatment

• The following events were considered for the 3 studies: hypertension, venous thromboembolic event, arterial thromboembolic event, proteinuria, dysphonia, and hemorrhage. For proteinuria, in EFC10547/VANILLA, all grades were considered and only grades≥2 were considered in EFC10262/VELOUR and EFC10261 /VITAL

• In EFC 10261 /VITAL and EFC10262/VELOUR, renal events were also

assessed and defined as any renal failure event (grouped terms from the AE page) or grade 3 or 4 serum creatinine increase (from laboratory data) or clearance creatinine <30ml/min (calculated according to cokroft gault formula).

• For EFC10262/VELOUR only, the following additional safety endpoints were considered: diarrhoea grade 3, 4, headache grade 3, 4, stomatitis and ulceration (high level term [HLT]) and infections and infestations (SOC).

The efficacy pharmacodynamics parameters for this analysis included:

• Efficacy endpoints were overall survival (OS) for the 3 studies and

progression-free-survival (PFS) only for EFC10261 /VITAL and

EFC10262/VELOUR The following methodology was used for the PK-PD analysis:

• In each Phase 3 study, the relationship between aflibercept PK parameters as defined above and safety as well as efficacy (OS and PFS) endpoints were evaluated by conducting univariate analyses, using logistic (for safety) and proportional hazard Cox (for efficacy) regression models

· Multivariate analyses were also performed including covariates such as

demographics/baseline characteristics

• PK parameters were considered as continuous variables in the models

The PK-PD analysis of the VELOUR study in MCRC showed the following: High correlation between efficacy and all PK parameters (p<0.0001 for AUCext, AUCaverage, AUCI st cycle, p=0.0005 for Cmax, p=0.0147 for free clearance, p<0.0001 for bound clearance):

o The higher the AUC and Cmax (and the lower the free and bound aflibercept clearances), the longer the OS and PFS

Correlation between occurrence of hypertension during cycle 1 or 2 (and to a lesser extent of haemorrhage) and some PK parameters (p=0.0029 for AUCI st cycle)

o The higher the AUC and Cmax, the higher the incidence of hypertension

o The higher the AUC, the higher the incidence of haemorrhage

• Results were confirmed by multivariate analyses (adjusting for

demographic/baseline characteristics) or when adding baseline endogenous VEGF in the model Similar trends were observed in the PK-PD analysis for VITAL and VANILLA studies. Additional PK-PD analysis using Free to Bound Ratio

Additional PK-PD analyses were performed after submission of regulatory dossiers to FDA and EMA. The primary objective of these PK-PD analyses was:

• To get further insight on the high correlation between efficacy and PK

parameters (in PK-PD analyses included in the dossier), and evaluate impact on benefit-risk for some subpopulations

• To evaluate the use of free to bound ratio of aflibercept in the PK-PD analyses

The following methodology was used for these analyses:

Blood samples for free/bound ratio measurement were collected prior to the first administration of aflibercept, then prior to administration of aflibercept of each odd numbered cycle.

For each patient, the free/bound ratio for PK/PD analysis was calculated as the median of free/bound values from Cycle 3 to the end of treatment.

• Additional analyses were done using free/bound ratio at the end of Cycle 2 • For aflibercept-treated patients, PK parameters defined as categorical variables, dichotomizing patients according to the median PK parameter (< median,≥ median)

• In each subgroup:

o For efficacy: provide Kaplan-Meier OS and PFS estimates o For safety: provide incidences of AEs

o Assess the exposure to study treatment in each subgroup

• Identify a placebo group of patients that "mimicks" the aflibercept group of patients evaluable for PK; check to which extent the evaluable PK population is representative of the overall population

• Perform the same analyses on observed free to bound aflibercept trough concentration ratio (using same methodology)

The results of these analyses are illustrated by Figure 7- 15 and tables 18-22. They show that: Table 18: Estimated PK parameters in Velour - Aflibercept-treated patients evaluable for PK

Table 19: Anti-VEGF associated events by free aflibercept average AUC

Table 20: Subgroup analysis of overall survival according to laboratory parameters and weight (ITT population): Goal was to try to identify patients who benefit more from the treatment through their baseline characteristics (variables that explained PK inter-individual variability)

Table 21 : Safety (anti-VEGF associated events) by free/bond aflibercept median ration (threshold = 1.6)

Table 22: Safety (anti-VEGF associated events) by free/bond aflibercept median ration (threshold = 3.2)

Table 23: Velour - OS KM estimates according to free/bound aflibercept median ratio (cut-point ranging from 1 to 4)

Hazard Ratio (95% CI)

Median survival High vs Low

No. Of (95.34% CI) Free/Bound Aflibercept pts (months)³ ratio

Median ratio

Free/Bound <= 1 12 9.8 (5.06 to 21.32)

Median ratio

Free/Bound > 1 289 16.7 (14.78 to 18.66) 0.469 (0.237 to 0.929)

Median ratio

Free/Bound <= 1 .2 20 9.4 (7.56 to 1 1.53)

Median ratio

Free/Bound > 1 .2 281 16.7 (14.95 to 18.76) 0.372 (0.22 to 0.63)

Median ratio

Free/Bound <= 1 .4 33 9.8 (7.56 to 1 1.53)

Median ratio

Free/Bound > 1 .4 268 17.4 (15.44 to 18.96) 0.384 (0.25 to 0.589)

Median ratio

Free/Bound <= 1 .6 53 10.2 (8.18 to 1 1.53)

Median ratio

Free/Bound > 1 .6 248 17.6 (16.16 to 19.06) 0.41 (0.285 to 0.591 )

Median ratio

Free/Bound <= 1 .8 70 10.8 (9.40 to 13.37)

Median ratio

Free/Bound > 1 .8 231 17.9 (16.16 to 19.45) 0.482 (0.344 to 0.675)

Median ratio

Free/Bound <= 2 89 1 1 .5 (10.18 to 14.29)

Median ratio

Free/Bound > 2 212 18.0 (16.23 to 20.73) 0.539 (0.39 to 0.745)

Median ratio

Free/Bound <= 2.2 1 14 12.3 (10.41 to 15.64)

Median ratio

Free/Bound > 2.2 187 18.7 (16.23 to 21.26) 0.567 (0.416 to 0.774)

Median ratio

Free/Bound <= 2.4 138 13.4 (1 1 .79 to 16.85)

Median ratio

Free/Bound > 2.4 163 17.9 (15.47 to 19.45) 0.712 (0.524 to 0.969) Hazard Ratio (95% CI)

Median survival High vs Low

No. Of (95.34% CI) Free/Bound Aflibercept pts (months)³ ratio

Median ratio

Free/Bound <= 2.6 165 14.4 (12.29 to 17.35)

Median ratio

Free/Bound > 2.6 136 18.0 (15.47 to 19.45) 0.69 (0.505 to 0.943)

Median ratio

Free/Bound <= 2.8 179 14.4 (12.48 to 17.64)

Median ratio

Free/Bound > 2.8 122 18.0 (15.87 to 19.45) 0.713 (0.519 to 0.98)

Median ratio

Free/Bound <= 3 197 15.4 (13.34 to 18.56)

Median ratio

Free/Bound > 3 104 17.6 (14.95 to 21.55) 0.787 (0.566 to 1 .093)

Median ratio

Free/Bound <= 3.2 216 16.4 (13.80 to 18.76)

Median ratio

Free/Bound > 3.2 85 16.2 (14.46 to 19.45) 0.912 (0.644 to 1 .29)

Median ratio

Free/Bound <= 3.4 233 16.4 (13.90 to 18.76)

Median ratio

Free/Bound > 3.4 68 16.2 (14.46 to 19.45) 0.929 (0.638 to 1 .351 )

Median ratio

Free/Bound <= 3.6 249 16.7 (14.39 to 18.76)

Median ratio

Free/Bound > 3.6 52 15.9 (14.29 to 19.45) 1 .094 (0.732 to 1 .634)

Median ratio

Free/Bound <= 3.8 260 16.7 (14.39 to 18.76)

Median ratio

Free/Bound > 3.8 41 15.9 (13.37 to 18.04) 1 .201 (0.78 to 1.85)

Median ratio

Free/Bound <= 4 273 16.7 (14.49 to 18.66)

Median ratio

Free/Bound > 4 28 15.9 (12.71 to 21.55) 1 .216 (0.731 to 2.024)

: Patients with at least a value of Free and Bound after cycle 3 Table 24: Velour - OS KM estimates according to free/bound aflibercept ratio at end of Cycle 2 (cut-point ranging from 1 to 4)

Hazard Ratio (95%

Median survival CI) High vs Low

(95.34% CI) Free/Bound

Number (months)³ Aflibercept ratio

Ratio Free/Bound

at C2 <= 1 99 12.7 (10.18 to 15.51 )

Ratio Free/Bound

at C2 > 1 366 15.2 (14.16 to 17.45) 0.807 (0.609 to 1 .071 )

Ratio Free/Bound

at C2 <= 1 .2 124 12.5 (10.18 to 14.49)

Ratio Free/Bound

at C2 > 1 .2 341 15.5 (14.29 to 17.94) 0.769 (0.593 to 0.996)

Ratio Free/Bound

at C2 <= 1 .4 147 12.5 (10.28 to 14.49)

Ratio Free/Bound

at C2 > 1 .4 318 16.2 (14.39 to 18.04) 0.753 (0.589 to 0.963)

Ratio Free/Bound

at C2 <= 1 .6 166 1 1 .4 (10.18 to 13.37)

Ratio Free/Bound

at C2 > 1 .6 299 16.7 (14.78 to 18.66) 0.679 (0.534 to 0.863)

Ratio Free/Bound

at C2 <= 1 .8 187 1 1 .5 (10.15 to 13.44)

Ratio Free/Bound

at C2 > 1 .8 278 17.3 (14.78 to 18.79) 0.674 (0.532 to 0.853)

Ratio Free/Bound

at C2 <= 2 217 1 1 .8 (10.41 to 13.44)

Ratio Free/Bound

at C2 > 2 248 17.4 (15.18 to 19.45) 0.655 (0.518 to 0.828)

Ratio Free/Bound

at C2 <= 2.2 237 12.5 (10.84 to 14.29)

Ratio Free/Bound

at C2 > 2.2 228 17.6 (14.85 to 19.02) 0.673 (0.532 to 0.852)

Ratio Free/Bound

at C2 <= 2.4 261 12.9 (1 1 .10 to 14.49)

Ratio Free/Bound

at C2 > 2.4 204 17.4 (14.78 to 19.02) 0.728 (0.573 to 0.924) Hazard Ratio (95%

Median survival CI) High vs Low (95.34% CI) Free/Bound

Number (months)³ Aflibercept ratio

Ratio Free/Bound

at C2 <= 2.6 278 12.9 (1 1 .10 to 14.49)

Ratio Free/Bound

at C2 > 2.6 187 17.6 (14.85 to 19.45) 0.725 (0.569 to 0.925)

Ratio Free/Bound

at C2 <= 2.8 299 12.9 (1 1 .40 to 14.49)

Ratio Free/Bound

at C2 > 2.8 166 17.6 (15.18 to 19.02) 0.721 (0.561 to 0.926)

Ratio Free/Bound

at C2 <= 3 323 13.1 (1 1 .96 to 14.82)

Ratio Free/Bound

at C2 > 3 142 17.4 (14.85 to 19.02) 0.756 (0.582 to 0.982)

Ratio Free/Bound

at C2 <= 3.2 345 13.5 (12.48 to 15.44)

Ratio Free/Bound

at C2 > 3.2 120 17.6 (14.46 to 19.02) 0.805 (0.61 to 1 .061 )

Ratio Free/Bound

at C2 <= 3.4 364 13.5 (12.48 to 15.28)

Ratio Free/Bound

at C2 > 3.4 101 17.9 (14.85 to 19.45) 0.789 (0.588 to 1.06)

Ratio Free/Bound

at C2 <= 3.6 383 13.8 (12.68 to 15.28)

Ratio Free/Bound

at C2 > 3.6 82 17.6 (15.18 to 19.02) 0.839 (0.609 to 1 .156)

Ratio Free/Bound

at C2 <= 3.8 396 14.2 (12.78 to 15.51 )

Ratio Free/Bound

at C2 > 3.8 69 17.6 (15.18 to 18.79) 0.852 (0.602 to 1 .206)

Ratio Free/Bound

at C2 <= 4 409 14.3 (12.88 to 15.87)

Ratio Free/Bound

at C2 > 4 56 17.4 (14.46 to 18.04) 0.871 (0.593 to 1.28)

• PK parameters and efficacy

• A difference of 5 months is observed between patients with Cmax > median vs patients with Cmax < median Cmax. • PK parameters and Safety:

• The incidence of grade 3/4 AE was similar in the 2 aflibercept subgroups (low or high PK parameter).

• A higher incidence of ATE, haemorhage, hypertension, VTE was

observed in the subgroup of patients with high PK parameter

• Free/bound aflibercet ratio and Efficacy:

• Higher OS in patients with free/bound ratio > 1 .6 (median OS: 17.4 months) than in patients with ratio<1.6 (median OS: 10.2 months).

• The difference in OS between subgroups decreased with threshold increasing from 1 .6 to 3.2.

• Safety:

• The incidence of grade 3-4 anti-VEGF associated AE was higher in patients with higher free/bound ratio.

• For a threshold of 3.2, efficacy was similar in both subgroups, however the incidence of grade 3-4 anti-VEGF associated AE was higher in patients with higher free/bound ratio (39.5% vs 26.7%).

Conclusions

The additional PK-PD analyses of the VELOUR data, suggest that free to bound ratio may be used to administer therapeutically effective amounts of aflibercept to patients with MCRC. The free to bound ratio may be measured at any time during the patient's treatment.

Similar trends were observed in other phase 3 trials (VITAL, VANILLA).

The optimal free to bound ratio that results in improved efficacy and acceptable safety appears to be > 2; however improvement in efficacy was observed at all cutoff points from 1 .6 to 3.2 with decreasing OS benefit seen with increasing cutoff value within this range.

Claims

1. A method of treating cancer or cancer symptom in a patient in need thereof, said method comprising the following steps:

^■ adapting the treatment of the patient based on the ratio free/bound

aflibercept.

2. A method of treating cancer or cancer symptom in a patient in need thereof, said method comprising: ■ measuring the ratio free/bound aflibercept in a biological sample of a patient, and

^■ administering therapeutically effective amounts of aflibercept to patient displaying a ratio free/bound aflibercept comprised between 1 and 4.

3. A method of increasing Overall Survival (OS) in a patient afflicted with cancer, said method comprising:

4. A method according to any one of claims 1 to 4 wherein said cancer is Colorectal Cancer (CRC).

5. A method according to any one of claims 1 to 4 comprising administering to said patient therapeutically effective amounts of aflibercept, folinic acid, 5-fluorouracil (5-FU) and irinotecan. 6. A method according to any one of claims 1 to 5 wherein the patient display a ratio free/bound comprised between 1.6 and 3.2. A method according to any one of claims 1 to 5 wherein the patient display a ratio free/bound comprised between 1.5 and 1.8.

A method according to any one of claims 1 to 6 wherein the patient display a ratio free/bound aflibercept around 1.6.

A method according to any one of claims 1 to 7 wherein the ratio free/bound aflibercept is determined after the first cycle of treatment of the patient.

A method according to any one of claims 1 to 7 wherein the ratio free/bound aflibercept is determined after the second cycle of treatment of the patient.

A method according to any one of claims 1 to 7 wherein the ratio free/bound aflibercept is determined any time during treatment of the patient.

An article of manufacture comprising:

a) a packaging material

b) aflibercept, and c) a label or package insert contained within said packaging material which indicates that the patient should adapt his treatment in function of the ratio free/bound aflibercept.

13. Aflibercept for use in treating patients with cancer or cancer symptom wherein the ratio free/bound aflibercept in a biological sample of said patient is measured and the treatment of said patient is adapted based on the ratio free/bound aflibercept.

14. Aflibercept for use in treating patients with cancer or cancer symptom wherein the ratio free/bound aflibercept in a biological sample of said patient is measured and a therapeutically effective amounts of aflibercept is administered to patient displaying a ratio free/bound aflibercept comprised between 1 and 4.