DRAFT - Phase 1 Study of AME-133v in Patients with ... · Results of a Phase 1 Study of AME-133v (LY2469298) an Fc-Engineered Humanized Monoclonal Anti-CD20 Antibody, in FcγRIIIa-Genotyped

DRAFT - Phase 1 Study of AME-133v in Patients with Follicular Lymphoma

CONFIDENTIAL 1

Title:

Results of a Phase 1 Study of AME-133v (LY2469298) an Fc-Engineered Humanized Monoclonal Anti-

CD20 Antibody, in FcγRIIIa-Genotyped Patients with Previously Treated Follicular Lymphoma

Running Title: Phase 1 Study of AME-133v in Patients with Follicular Lymphoma Key Words: Anti-CD20 Previously treated Non-Hodgkin’s Lymphoma AME-133v Monoclonal Antibody FcγRIIIa Authors: Andres Forero-Torres1, Sven de Vos2, Brad L. Pohlman3, Maksim Pashkevich4, ,Damien M. Cronier5, Nam H. Dang6, Susan P. Carpenter7, Barrett W. Allan7, James G. Nelson7, Christopher A. Slapak4, Mitchell R. Smith8, Brian K. Link9, James E. Wooldridge4, Kristen N. Ganjoo10

Authors Affiliations: 1University of Alabama at Birmingham, Birmingham, Alabama 2David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 3Cleveland Clinic Taussig Cancer Institute, Cleveland, Ohio 4Eli Lilly and Company, Indianapolis, Indiana 5Eli Lilly and Company, Windlesham, United Kingdom 6University of Florida, Gainseville, Florida 7Applied Molecular Evolution, San Diego, California 8Fox Chase Cancer Center, Philadelphia, Pennsylvania 9Holden Comprehensive Cancer Center at University of Iowa, Iowa City, Iowa 10Stanford University Cancer Center, Palo Alto, California Reprint Requests: 7Susan Carpenter, PhD Applied Molecular Evolution Lilly Biotechnology Center-San Diego 10300 Campus Point Drive, Suite 200 San Diego, CA 92121 [email protected]

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CONFIDENTIAL 2

Statement of Translational Relevance

A single nucleotide polymorphism (SNP) in the FcγRIIIa gene resulting in a valine at amino acid

position-158 in both alleles (158-VV), rather than a phenylalanine in at least one allele (F-carriers), leads

to a high affinity CD16a on immune effector cells such as NK cells and macrophages. This results in a

greater ability to mediate ADCC and is associated with better outcomes to rituximab therapy in patients

with follicular lymphoma (FL). As the FcγRIIIa F-carrier population comprises approximately 80% of

those patients diagnosed with FL; an opportunity exists to enhance the effectiveness of antibody treatment

by improving the interaction between the antibody, CD16a, and CD20. AME-133v, a humanized

monoclonal antibody to CD20, was engineered to have greater affinity for CD20 and greater potency in

mediating ADCC than rituximab. This provides a strong rationale for the development of AME-133v

therapy in FL patients, and in particular, those patients who are FcγRIIIa F-carriers.





CONFIDENTIAL 3

Abstract

Purpose: AME-133v is a humanized monoclonal antibody engineered to have increased affinity

to CD20 and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) better than rituximab.

Safety, pharmacokinetics (PK), and efficacy were assessed in a Phase 1/2 trial in patients with previously

treated follicular lymphoma (FL).

Patients and Methods: AME-133v was characterized in vitro using ADCC and cell binding

assays. A Phase 1 study was conducted in which 23 previously treated patients with FL were assigned

sequentially to one of 5 dose-escalation cohorts of AME-133v at 2, 7.5, 30, 100 or 375 mg/m2 weekly × 4

doses.

Results: AME-133v demonstrated a 13- to 20-fold greater binding affinity for CD20 and was 5-

to 7-fold more potent than rituximab in ADCC assays. Cell binding assays showed AME-133v and

rituximab competed for an overlapping epitope on the CD20 antigen, and AME-133v inhibited binding of

biotinylated rituximab to CD20 in a concentration-dependent manner. AME-133v was well tolerated by

patients and common related adverse events included chills and fatigue. One patient experienced a dose-

limiting toxicity (DLT) of neutropenia. AME-133v demonstrated non-linear PK with properties similar

to rituximab. Selective reduction of B-cells during and after AME-133v treatment was demonstrated by

flow cytometry of peripheral blood. A partial or complete response was observed in 5 of 23 (22%)

patients and the median progression-free survival (PFS) was 25.4 weeks.

Conclusions: AME-133v was safe and well tolerated at the doses tested. AME-133v

demonstrated encouraging results as an anti-CD20 therapy in heavily pretreated FL patients with the less

favorable FcγRIIIa F-carrier genotype.





CONFIDENTIAL 4

Introduction

Rituximab is effective as monotherapy or in combination with chemotherapy in patients with FL

(1). Several studies have demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) is a

key mechanism of action for rituximab (2). In a murine lymphoma model, a single dose of rituximab

prevented lymphoma growth in wild-type FcR animals while rituximab had minimal activity in animals

lacking activating FcR (3). A single nucleotide polymorphism (SNP) in the FcγRIIIa gene that results in

a valine at amino acid position-158 in both alleles (158-VV), rather than a phenylalanine in at least one

allele (158-VF or –FF; F-carrier), leads to a CD16a on immune effector cells with higher affinity for IgG1

mAbs, including rituximab (4).

In pre-clinical studies, an optimized CD20 antibody was more effective at activating NK cells, a

surrogate for ADCC, than rituximab or CD20 antibodies modified to have only higher affinity for CD20

(5, 6). The greatest improvement in NK-cell activation was noted in the presence of effector cells from

FcγRIIIa F-carrier donors; however, the fully optimized CD20 antibody also activated NK cells from 158-

homozygous-VV donors better than the other two antibodies. This suggests that an antibody with

enhanced ability to mediate ADCC may be more effective clinically, particularly in patients who are

FcγRIIIa F-carriers.

Although rituximab monotherapy is effective in patients with FL, several studies suggested that

rituximab is less effective in F-carriers. In a clinical trial of rituximab in 49 previously-untreated patients

with FL, patients with the 158-VV FcγRIIIa genotype had a significantly higher response rate at 2 months

(100% vs. 67%) and 12 months (90% vs. 51%) compared to F-carriers (7). A retrospective assessment of

87 patients with FL treated with rituximab demonstrated that patients with the 158-VV genotype had

significantly higher response rates compared to F-carriers, as well as a significantly longer time to

progression (534 days vs. 174 days, respectively) (8). The Swiss Clinical Cancer Research (SAKK)

group analyzed the effect of FcγRIIIa genotype on outcome to rituximab monotherapy in patients with

either newly diagnosed or previously treated FL, again finding a significant and substantial improvement

in event free survival in patients with the 158-VV genotype (9). Based on these data, an opportunity

exists to improve antibody therapy in patients with FL.

AME-133v (LY2469298) is a humanized immunoglobulin G1 (IgG1) monoclonal antibody

against CD20 that was optimized through protein engineering both for increased affinity to CD20 and

enhanced effector function in ADCC assays. We hypothesized that optimization of AME-133v would

result in an anti-CD20 therapy with greater potency and efficacy in all patients, but particularly in the

FcγRIIIa 158-F-carriers. This Phase 1 study was designed to provide evidence of the safety and a

preliminary understanding of the efficacy of AME-133v.





CONFIDENTIAL 5

Methods

AME-133v engineering. AME-133v is a humanized IgG1-variant monoclonal antibody

engineered to have greater affinity for CD20 and greater ADCC activity compared with rituximab in

vitro. The parent murine IgG2a anti-CD20 antibody was humanized using codon-substituted variations of

the antibody complementarity determining regions (CDRs) inserted into human antibody germline

frameworks. CDR variants were screened for binding to CD20 antigen present on human Ramos B

lymphoma cells in an ELISA format. Beneficial CDR mutations were combined and selected Fab clones

were expressed as whole IgG1 molecules, purified and characterized further. To directly compare cell

binding kinetics in solution, rituximab and AME-133 were tested for their binding to both SKW6.4 B

lymphoma cells (intermediate CD20 antigen expression) (10) and primary human CD19-positive

peripheral blood B cells (high CD20 antigen expression) using a Sapidyne (Boise, ID) KinExA

instrument.

Starting from a humanized high affinity anti-CD20 IgG1, 2 amino acid changes were introduced

into its Fc-region and tested for ADCC activity using a single-point ADCC assay format. Variants

demonstrating superior activity were confirmed by full titrations using purified Mabs.

Antibody-Dependent Cell-Mediated Cytotoxicity Assays. Standard cytolytic assays were used to

measure the ADCC activity of AME-133 Fc-region variants. Briefly, immune effector cells were isolated

from the blood of healthy donors using Histopaque-1077 (Sigma) separation and plastic adherence.

Effector cells were added to CD20 opsonized target cells at a ratio of 20 to 1 and incubated for 3 h at

37°C. Assay plates were centrifuged and supernatants analyzed for lactate dehydrogenase released from

the cytosol of damaged cells using a cytotoxicity detection kit (Roche Applied Science). The plates were

read at 490 nm and raw absorbance (A) values were converted to % maximal response using the

following equation: % maximal response = (experimental A − basal A)/(maximal A − basal A) × 100,

with maximal A determined by adding 2% Triton X-100 to the target cells and basal-release measured for

a mixture of effector and target cells in the absence of sensitizing IgG.

Cell Binding Assays. For competition experiments involving pre-bound rituximab, titrations of

biotinylated rituximab were incubated with SKW6.4 B-cells overnight at 37°C. After incubation, non-

biotinylated competitor antibody (AME-133v or a non-specific IgG1) or PBS was added directly without

prior washing. The time-dependent displacement of rituximab from the cells was followed at 37°C by

detecting the residual bound biotinylated rituximab using neutravidin alkaline phosphatase conjugate and

development with AMP/PMP substrate. Absorbance was read at 560 nm using a Molecular Devices

(Sunnyvale, CA) plate reader. For direct competition experiments titrations of biotinylated rituximab and





CONFIDENTIAL 6

AME-133v were prepared individually, mixed together and added to SKW6.4 cells followed by

incubation for 2 hours at 37°C. Following incubation, the plates were processed as previously described.

Phase 1 patient selection. Patients were eligible for the study if they were at least 18 years old

with diagnosis of CD20+ follicular B-cell NHL; had the FcγRIIIa (with SNP coding for F/F or F/V at

position 158) as determined by FcR genotyping (Beckmann Coulter Genomics, Beverly, MA) and had

measurable disease. Prior treatment with chemotherapy, rituximab, or both for FL was required. Prior

rituximab was allowed if the patient had not relapsed or progressed within 120 days of the last infusion of

rituximab. In addition, patients had no evidence of hepatitis B or C infection and had discontinued all

previous cancer and high-dose corticosteroid therapies at least 30 days prior to study entry. All eligible

patients had adequate hematopoietic, renal, and hepatic function, and women of childbearing potential

were required to use a medically acceptable contraceptive regimen.

Phase 1 trial design and dose escalation. The trial was an open-label, multicenter, Phase 1,

dose-escalation study of AME-133v (LY2469298), administered intravenously in four weekly doses, in

patients with previously treated CD20+ FL. The primary objective of the Phase 1 study was to determine

the safety and tolerability of repeat administration of AME-133v at 5 dose levels: 2 mg/m², 7.5 mg/m²,

30 mg/m², 100 mg/m², and 375 mg/m². A standard “3+3” enrollment design was employed. After the

last patient in a cohort received their fourth dose, a 2-week safety period was observed. The study was

conducted in accordance with the principles of the Declaration of Helsinki, Good Clinical Practice ICH

Tripartite Guideline (January 1997), and basic principles of Good Clinical Practice as outlined in the

current version of 21 Code of Federal Regulations (CFR). The protocol and informed consent for this

study was approved by institutional review boards at each participating institution and all patients were

required to sign informed consent prior to study entry (ClinTrials registry number NCT00354926).

AME-133v was diluted in normal saline (NS) and administered using an infusion or syringe

pump. The 2-, 7.5-, and 30-mg/m2 doses were delivered over fixed infusion times of 30, 60, and 180

minutes, respectively. For the 100- and 375-mg/m2 doses, the first dose was delivered at a rate up to 25

mg/hr for the first 30 minutes, which was increased by up to 50 mg/hr every 30 minutes. Subsequent

infusions were administered at an initial rate of up to 100 mg/hr and increased by up to 100 mg/hr

increments every 30 minutes until a maximum rate of 300 mg/hr was reached. All patients were pre-

medicated with acetaminophen 650 mg and diphenhydramine 50 mg . Pre-medication with steroids was

prohibited.

Side effects were assessed according to the National Cancer Institute (NCI) Adult Common

Toxicity Criteria (CTCAEv.3.0). A dose-limiting toxicity (DLT) was defined as the occurrence of any

Grade 3 or higher drug-related adverse event (AE), with the following modifications: Grade 3

hematopoietic toxicity was defined as an absolute neutrophil count (ANC) nadir of 500 to 1000/uL, or a





CONFIDENTIAL 7

decrease in platelets or hemoglobin of 50% to 74% from the lower limit of normal or the pre-treatment

value, whichever is less (11). Grade 3 infusion reactions (e.g., fever, chills, rigors, bronchospasm,

urticaria, and hypotension) and tumor lysis syndrome that were transient and resolved without sequelae

were not considered DLTs in this study. Any patient who experienced a DLT did not receive additional

study medication and was followed until the toxicity resolved to a CTCAE v.3.0 Grade 1 severity.

All patients who received at least 1 dose of AME-133v were evaluated for safety,

pharmacokinetic (PK), and lymphoma response. SAS procedures (Version 8 or higher) were employed

for the generation of all tables, graphs, and statistical analyses, except where otherwise specified.

Pharmacokinetic and pharmacodynamic studies. AME 133v concentration and time data were

collected from a total of 23 patients enrolled in the 2, 7.5, 30, 100 and 375 mg/m2 dose groups. All PK

samples were drawn pre-dose before infusion 1; 3 to 5 days after infusion 1, pre-dose before infusions 2,

3, and 4; and 1, 5, and 9 weeks after infusion 4.

The PK data collected from these patients were analyzed by means of nonlinear mixed effect

modeling using NONMEM software (version VI.2) with NM-TRAN and PREDPP. The PK model

describing the PK profile of AME-133v was parameterized in terms of CL, V1, Q and V2, which

correspond to the clearance, volume of the central compartment, intercompartmental clearance, and

volume of the peripheral compartment, respectively. Peripheral blood B-cells were quantitated by

fluorescence activated cell sorting (FACS) on samples collected at baseline and at all subsequent study

visits.

Response criteria. Response to treatment was assessed according to the 1999 NCI Working

Group criteria, 9 weeks after the last dose (12) and subsequently every 3 months until disease progression,

death, or another therapy was initiated. Additional endpoints included duration of response and

progression-free survival (PFS).





CONFIDENTIAL 8

Results

AME-133v characterization. Relative to rituximab, AME-133v demonstrated a 13- to 20-fold

increase in binding affinity for CD20 with a Kd of ~100 pM when tested on SKW6.4 cells and primary B

lymphocytes (Supplemental Table 1). The increased binding was due to a large improvement in the

antibody off-rate, although a modest improvement in on-rate was also observed. AME-133v was also

approximately 6-fold more potent than rituximab in ADCC assays and demonstrated improved binding

affinity to the V/V as well as V/F and F/F forms of the FcR (Supplemental Figure 1a, 1b). Competitive

binding experiments were performed to determine whether AME-133v and rituximab bound to the same

epitope on the CD20 antigen expressed on SKW6.4 B-cells. Direct competition assays, where AME-

133v and rituximab were pre-mixed prior to the addition of the mixture to the cells, showed that AME-

133v inhibited the binding of biotinylated rituximab to CD20 in a concentration-dependent manner

(Supplemental Figure 1c, 1d). The ability of AME-133v to displace pre-bound rituximab from the

surface of SKW6.4 cells was assessed as a function of time (Supplemental Figure 1e). The non-specific

competitor IgG1at 150 µg/mL had no effect upon the binding of rituximab even after 32 hour incubation;

however, in the presence of AME-133v, a time-dependent reduction in rituximab binding signal was

observed, with most of the biotinylated rituximab being competed from the cells within 32 hours.

Additional prebound rituximab experiments assessing concentrations of 75 and 150 µg/ml of AME-133v

yielded similar results to the 25 µg/ml concentration (data not shown). These results further underscore

the time rather than dose dependent nature of rituximab displacement by AME-133v.

Phase 1 dose escalation. Between August 2006 and November 2007, 23 patients with previously

treated CD20+ FL were enrolled to the Phase 1 portion of this study. The median age of the patients was

61 years (range, 37 to 77) with a slight male predominance (13/23). All patients were F-carriers in at

least one allele of the FcγRIIIa gene. Patient characteristics including genotypes for FcRs II and III are

shown in Table 1. At baseline, 16 patients had an Eastern Cooperative Oncology Group (ECOG)

performance status (PS) of 0, and 6 had an ECOG PS of 1. All patients received prior systemic therapy,

with a median number of 2 prior regimens (range, 1 to 6). Only one patient was rituximab naïve.

Safety. All 23 patients reported one or more AEs (Supplemental Table 2). The most common

AEs were chills (13 patients; 57%), nausea (7 patients; 30%), fatigue and dizziness (each 6 patients;

26%), vomiting (5 patients; 22%), and arthralgia, headache, hypotension, and upper respiratory tract

infection (each 4 patients; 17%). Nine patients reported infections (including influenza, nasopharyngitis,

tinea pedis, upper respiratory tract infection, urinary tract infection, and viral infection) and 6 of these

patients were in the two highest dose groups. The only Grade 3/4 AE that occurred in more than one

patient was neutropenia (two patients). The Grade 3 and 4 AEs, regardless of attribution are listed in

Table 2A. The only Grade 3 AE attributed to study therapy was bronchospasm, which was an infusion





CONFIDENTIAL 9

reaction and was not considered a DLT. Other AEs, including potential infusion reaction-related AEs

were of Grade 1 or 2 (Table 2B). The majority of patients experienced infusion-related reactions, which

were –most commonly Grade 1. There were more events reported at the first infusion (91% of patients)

compared with subsequent infusions (14% to 23% of patients), as depicted in Figure 1. Two patients

reported treatment-emergent serious AEs: one with neutropenia as previously described, and one with a

small intestinal obstruction due to progression of lymphoma. The latter patient received only one dose of

study drug, withdrew consent, and received no further study drug.

One dose limiting toxicity (DLT) was observed during the dose escalation period. In cohort 4

(100 mg/m2), a patient experienced Grade 4 neutropenia one week after the fourth dose of AME-133v.

The event met the criteria for a serious AE and was considered possibly related to the study drug. The

patient recovered completely in 11 days. As a result of this DLT, three more patients were added to the

cohort, and no additional DLTs were observed. The dose was escalated to the highest planned dose level

of 375 mg/m2, 6 patients were enrolled according to the protocol, and no DLTs were observed. In the 2

mg/m2 cohort, one additional patient was enrolled to replace a patient who withdrew consent after receipt

of one dose of study drug. In the 30 mg/m2 dose cohort, 2 eligible patients were consented concurrently

and both were allowed to enroll. Thus, a maximum tolerated dose was not reached during the dose-

escalation period.

Multiple gated acquisition (MUGA) cardiac scans were evaluated for patient eligibility, at

baseline and at 1 week and 9 weeks post last infusion. No clinically significant findings were observed

for any patient at any visit.

Pharmacokinetics. Due to values below the limit of quantitation, the data from the 4 patients

enrolled in the 2 mg/m2 dose group were excluded from the analysis. In addition, 1 patient from the

375 mg/m2 dose group was deemed an outlier and was excluded from the analysis. Therefore the analysis

was conducted on a total of 216 serum AME-133v observations from 18 patients. Due to nonlinearity in

the pharmacokinetics of AME-133v across the dose range tested and the small number of observations,

the best fit was obtained with a 2-compartment model with a dose-dependent clearance term.

The population pharmacokinetic parameters of AME-133v are summarized in Supplemental

Table 3 and are similar to those previously reported for rituximab (13). The typical clearance (CL)

estimates range from 0.75 to 0.24 L/day from 7.5 to 375 mg/m2, and the typical volume of the central and

peripheral compartments are 2.98 and 2.70 L, respectively. A visual predictive check (VPC) of the AME-

133v concentration-time profile is illustrated in Figure 2a-d. Inter-individual variability was moderate to

large with 50 and 36% for CL and V1, respectively (Supplemental Table 3).

Response and time to event assessments. All 23 patients were included in the investigator

reported response assessment. Twenty-two patients received all 4 doses of study drug and one patient





CONFIDENTIAL 10

discontinued the study after receiving 1 dose of study drug. The majority of patients experienced a

reduction in lymphoma volume (Supplemental Figure 2). Two patients achieved a CR and 3 patients

achieved a PR (Supplemental Table 4). Four of the 5 responses were observed in patients treated at 100

mg/m2 and 375 mg/m2 doses. The duration of response ranged from 21 weeks to 145.1 weeks.

In all patients the number of B-cells, identified using the B-cell antigen CD19, present in the

peripheral blood before and after AME-133v were analyzed. There was a dose-dependent, rapid and

specific depletion of the B-cells in all patients receiving at least 7.5 mg/m2 of AME 133v (Supplemental

Figure 3). In all dose groups except the 2 mg/m2 group, the depletion persisted for at least 3 months post

last dose before beginning to recover toward baseline levels.





CONFIDENTIAL 11

Discussion

We describe the pre-clinical and Phase 1 clinical trial results of AME-133v, an anti-CD20

monoclonal antibody rationally designed to improve upon the clinical activity of rituximab. Rituximab is

effective as a single-agent and, when combined with or sequenced after chemotherapy, it increases the

response rate and PFS in patients with treatment-naïve or previously-treated FL (14, 15, 16). Several

studies suggest that the survival of patients with FL has improved over time and at least some of this

improvement is likely due to rituximab (17, 18).

Despite the importance of rituximab, the impact of the FcγRIIIa genotype on outcome to

rituximab suggests that it is possible to improve outcomes even further by enhancing the activity of the

CD20 antibody. The preclinical studies by Bowles and colleagues demonstrated that a CD20 antibody

optimized for ADCC is more effective at activating NK cells in vitro than rituximab, and this

improvement was more profound in the FcγRIIIa F-carrier effector cells (5,6). As previously outlined,

patients with FL and the high affinity 158-VV FcγRIIIa genotypes have a higher response rate and time to

progression after rituximab treatment. Other studies in patients with FL have suggested that

chemotherapy negates this effect, although this has not been proven in a well-designed, prospective

clinical trial (19, 20, 21). To our knowledge, this is the first such trial to select (or stratify) patients on the

basis of their FcγRIIIa genotype. Two studies in patients with diffuse large B-cell lymphoma treated with

R-CHOP provide conflicting results, and the possibility persists that FcγRIIIa genotype may play a role

even in the face of chemotherapy (22, 23).

The impact of the FcγRIIIa genotype on outcome after cancer therapy is not limited to rituximab

or lymphoma. In patients with Her-2/neu+ breast cancer treated with trastuzumab and taxane, those with

158-VV FcγRIIIa genotype had a higher response rate and longer progression free survival than F-carriers

(24). Similarly, FcγRII and FcγRIIIa status combined was predictive of outcome to cetuximab combined

with irinotecan even after adjusting for k-ras mutational status in patients with metastatic colorectal

cancer (25). Furthermore, the hazard ratio was similar to the impact of k-ras mutational status.

AME-133v was developed to improve CD20 affinity and ADCC potency via protein engineering,

with the goal of achieving enhanced effectiveness in the entire FL patient population (which was not

tested in this clinical trial) and particularly in those patients expressing the 158 F/F or F/V FcγRIIIa

polymorphism. Pre-clinical studies of AME-133v showed that it has a higher affinity for CD20 and

mediates ADCC better than rituximab in vitro. Furthermore, competitive binding experiments confirmed

that AME-133v and rituximab bind to an overlapping epitope on the CD20 antigen expressed on SKW6.4

B-cells. The concentration of AME-133v competitor antibody used in the displacement assay (25 µg/mL)

was equivalent to the highest concentration used in the rituximab titration and approximately 5.2-fold

lower than the clinical levels achieved using 375 mg/m2. This amount of AME-133v displaced significant





CONFIDENTIAL 12

quantities of rituximab from the cell surface after 32 hours, even in the presence of saturating

concentrations of pre-bound rituximab. This is important because further development of this antibody

would occur in patients who had been treated with rituximab before, and may still have measurable levels

of serum rituximab.

In a small Phase I study of AME-133v (therein denoted LY2469298) in Japan (26) AME-133v

(100 or 375 mg/m2 weekly x4) was well tolerated and generated objective responses in 5/10 patients

(50%) who had previously treated FL and had received rituximab alone or rituximab-containing regimens.

Two of three complete responders were F-carriers and one complete responder was 158-VV FcγRIIIa

genotype. No DLTs were observed.

The safety and clinical impact of AME-133v were also evaluated in Phase 1 of this trial. Overall,

all doses of AME-133v evaluated were safe and well tolerated. There were no discontinuations due to

drug-related AEs and no deaths during the study. During Phase 1, there was no detection of human anti-

human antibodies (HAHA) in any of the patients in this dose escalation study either before or up to 5

weeks after the last infusion. Only one patient experienced a DLT, and a maximum tolerated dose was not

reached. Of the 4 serious AEs that occurred, only one was considered related to study drug, and two were

considered treatment-emergent. AME-133v displayed a safety profile similar to other anti-CD20

antibodies (27). AME-133v demonstrated a non-linear PK and moderate to large interpatient variability,

consistent with results observed for rituximab (13). Also, selective reduction in B-cell counts was

observed at all doses and particularly at higher doses. The clinical activity observed is encouraging but

difficult to interpret without a control arm or an historical control from similarly treated F-carrier patients

with relapsed FL. The median duration of response was not estimable, due to the large number of

censored responses; however median PFS was 25.4 weeks. Approximately 64% of patients showed at

least some decrease in tumor size. Further study will be necessary to establish the response rate not only

in FcγRIIIa F-carriers, but in patients expressing the homozygous valine receptor polymorphism as well.

Two anti-CD20 monoclonal antibodies are currently marketed, and more are in development (2).

Veltuzumab (hA20, IgG) has a 3-fold longer off rate than rituximab in Raji cells, while AME-133v had

an approximately 13-fold longer off rate than rituximab with SKW6.4 lymphoma cells in the current

study (27). In addition, AME-133v demonstrated a 13- to 20-fold increase in binding affinity for CD20

with a Kd of ~100 pM when tested on SKW6.4 cells and primary B lymphocytes. Veltuzumab has a Kd

reportedly similar to rituximab at around 8 nM (28, rituximab label), suggesting AME-133v has a nearly

80-fold increase in binding affinity in these cell binding assays compared to rituximab or veltuzumab.

Ofatumumab is an IgG1κ monoclonal antibody indicated for the treatment of patients with chronic

lymphocytic leukemia (CLL) refractory to fludarabine and alemtuzumab (29). The response rate after

treatment with ofatumumab was 43% in a Phase I/II study of patients with relapsed or refractory FL (30).





CONFIDENTIAL 13

Another second generation anti-CD20 mAb, GA101, has shown promise in refractory FL (31). This

glycoengineered, humanized mAb is primarily cytolytic toward CD20-expressing B-cells and binds a

different epitope than rituximab or AME-133v. Finally, ocrelizumab another humanized anti-CD20 mAb

with increased ADCC activity compared to rituximab has recently shown activity in relapsed/refractory

NHL patients (32, 33). The adverse events and infusion reactions appear to be similar among these

antibodies suggesting these effects may be relevant to the entire class of monoclonal antibodies to CD20

(2, 27-33).

In conclusion, AME-133v has a greater CD20 binding affinity and ability to mediate ADCC than

rituximab and is similar in these activities to several second-generation anti-CD20 antibodies in vitro. In

this Phase 1 study, AME 133v was safe and well tolerated across the dose range tested. The PK profile

was similar to rituximab, and B-cell depletion and objective clinical responses were observed.

Preliminary efficacy results in this small Phase I study in FcγRIIIa F-carriers demonstrated modest

activity, and further elucidation of the efficacy and safety profile of AME 133v from Phase II studies is

forthcoming.





CONFIDENTIAL 14

Disclosure of Potential Conflicts of Interest

Form attached

Acknowledgements

Greg Beuerlein, Julian Davies, Barbra Gaynor, Denise O’Neil, Jean Dang, Jill Woods, David

Ohannesian PhD





CONFIDENTIAL 15

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4. Koene HR, Kleijer M, Algra J, Roos D, von dem Borne AE, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood 1997;90: 1109-14.

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CONFIDENTIAL 16

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CONFIDENTIAL 17

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CONFIDENTIAL 18

Figure Legends

Figure 1: Patients reporting an AE on the day of infusion, stratified by infusion number. Ninety

nine percent of events were Grades 1 or 2.

Figure 2. Visual predictive check (VPC) of AME-133v concentration-time profile with 5th, 50th

and 95th percentiles shown (o, observed concentrations) at doses of 7.5 mg/m2 (A), 30 mg/m2 (B),

100 mg/m2 (C) and 375 mg/m2 (D). Population PK parameters are shown in (E).




Table 1. Patient Characteristics Patient Cohort

(mg/m2) Age

(Years) Stage FcγR2a

Genotype FcγR3a

Genotype Best

Response PFS

(Days) PFS

Censored Flag

Prior Therapy

0301 2 61 NR His/Arg Val/Phe SD 9 1 R, C 0401 2 63 IVA His/His Val/Phe PD 86 0 R, RM 0601 2 70 IVB His/His Val/Phe SD 178 0 R, C 0902 2 46 IIA His/His Val/Phe CR 1102 1 R+C 0302 7.5 58 IIB Arg/Arg Val/Phe PD 84 0 R+C 0906 7.5 66 IVA His/Arg Val/Phe PD 85 0 R+C,

RM 0907 7.5 54 NR Arg/Arg Phe/Phe ND ND ND R, C 0201 30 60 IVA His/Arg Phe/Phe SD 172 0 R, C 0403 30 45 IIIA His/His Phe/Phe SD 342 0 R+C 0707 30 64 NR Arg/Arg Val/Phe SD 178 0 R+C,

RIT 0908 30 67 NR Arg/Arg Val/Phe PD 85 0 R+C, C 0305 100 67 IA His/Arg Val/Phe PR 834 1 C 0404 100 49 IIIA Arg/Arg Phe/Phe PR 258 0 R, RM 0502 100 73 IVA Arg/Arg Phe/Phe SD 234 0 R, C,

RIT 0704 100 50 IIA His/Arg Phe/Phe SD 827 1 R+C, C 0708 100 66 NR His/Arg Val/Phe PD 52 0 R, R+C,

C 0909 100 37 IVA His/His Phe/Phe SD 274 0 R 0306 375 56 NR Arg/Arg Val/Phe PD 86 0 R+C, C 0307 375 69 IA His/Arg Val/Phe PD 63 0 R 0308 375 67 IA Arg/Arg Phe/Phe PR 364 0 R+C, C 0309 375 77 IA His/Arg Val/Phe CR 722 1 R, RM 0405 375 61 IVA His/Arg Phe/Phe SD 265 0 R+C, R,

RM 0703 375 49 NR His/Arg Phe/Phe SD 120 0 R, R+C,

C Abbreviations: R= rituximab, C= chemotherapy , RM= Rituximab maintenance, RIT= radioimmunotherapy




Table 2A: Adverse Events of Toxicity Grade 3 or Higher

Dose (mg/m2) 2 7.5 30 100 375 Total (N=4) (N=3) (N=4) (N=6) (N=6) (N=23)

Grade 3/4 AEs n n n n n n (%) Any Adverse Event 1 0 1 2 3 7 (30.4) Neutropenia 0 0 0 1*,# 1 2 (8.7) Alanine Aminotransferase Increased 1 0 0 0 0 1 (4.3) Anaemia 0 0 0 0 1 1 (4.3) Back Pain 0 0 1 0 0 1 (4.3) Bronchospasm 0 0 0 0 1# 1 (4.3) Dyspnoea 0 0 0 1 0 1 (4.3) Groin Pain 0 0 1 0 0 1 (4.3) Syncope 0 0 0 1 0 1 (4.3) * Grade 4 and a DLT; all others Grade 3 # Attributed to study therapy; all others considered not related to study therapy

Table 2B: AEs Reported at Infusion

Number of AEs Reported at Infusion

Dose Cohort

1st Infusion

2nd Infusion

3rd Infusion

4th Infusion Total

2 mg/m2

14 4 4 5 27

7.5 mg/m2

6 0 0 0 6

30 mg/m2

7 0 0 0 7

100 mg/m2

13 2 1 1 17

375 mg/m2

8 1 5 2 16

Total Events % pts reporting

48

22/23

7

6/22

10

5/22

8

5/22

73




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Published OnlineFirst January 5, 2012.Clin Cancer Res Andres Forero-Torres, Sven de Vos, Brad L Pohlman, et al. Patients with Previously Treated Follicular Lymphoma

RIIIa-GenotypedγHumanized Monoclonal Antibody in FcA Phase 1 Study of AME-133v (LY2469298) an Fc-Engineered

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