a low-toxicity il-2 based immunocytokine retains anti-tumor activity … · 2011. 4. 29. ·...

A low-toxicity IL-2 based immunocytokine retains anti-tumor activity

despite its high degree of IL-2 receptor selectivity

Stephen D. Gillies*, Yan Lan**, Thore Hettmann#, Beatrice Brunkhorst, Yaping Sun,

Stefan O. Mueller1,2 and Kin-Ming Lo

EMD Serono Research Institute, 45A Middlesex Turnpike, Billerica, MA 01821-3936,

1 Merck Serono, 64297 Darmstadt, Germany and 2 Karlsruhe Institute of Technology

(KIT), Institut für Angewandte Biowissenschaften, 76128 Karlsruhe, Germany

* Present address: Provenance Biopharmaceuticals, 830 Winter Street, Waltham, MA 02451 # Present address: U3 Pharma AG Bunsenstr. 1, 81243 Munchen, Germany

** Corresponding author: Yan Lan, e-mail, [email protected]

Key Words: immunocytokines, interleukin-2, immune therapy, toxicity, bioactivity

on July 9, 2021. © 2011 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 29, 2011; DOI: 10.1158/1078-0432.CCR-10-2921

http://clincancerres.aacrjournals.org/

NHS-IL2LT low toxicity immunocytokine

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STATEMENT OF TRANSLATIONAL RELEVANCE

The approach of selective IL-2 receptor activation such as the use of low-dose IL-

2 in the clinic and the screening for IL-2 mutants that selectively activate the high affinity

receptor is aimed at immune activation with less side effects. We have been using

antibody targeting of cytokines to deliver immune stimulators to the tumor

microenvironment in order to minimize systemic toxicity. In the current study we

describe the characterization of such an immunocytokine called Selectikine, which

contains an IL-2 (D20T) variant that is selective for both the human and mouse high

affinity IL-2 receptors, and hence translational research can be performed in mouse

models to evaluate anti-tumor efficacy and toxicity. Selectikine is currently in Phase I

clinical trial, and this potent tumor-targeting immune stimulator with a low side effect

profile is a good candidate for combination with traditional chemo and radiotherapies that

by themselves have immune potentiating activity.





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ABSTRACT

Purpose The goal of the study was to engineer a form of IL-2 that, when delivered as a

tumor-specific antibody fusion protein, retains the ability to stimulate an anti-tumor

immune response via interaction with the high affinity IL-2 receptor, but has lower

toxicity due to the reduced activation of the intermediate affinity IL-2 receptor.

Experimental Design We investigated changes in the proposed toxin motif of IL-2 by

introducing a D20T mutation that has little effect on the activity of free IL-2. We

expressed this IL-2 variant as a fusion protein with an antibody (NHS76) that targets the

necrotic core of tumors, and characterized this molecule (NHS-IL2LT) in vitro and in

vivo.

Results NHS-IL2LT was shown to have near normal biological activity in vitro using T

cell lines expressing the high affinity IL-2 receptor, but little or no activity using lines

expressing only the intermediate IL-2 receptor. Relative to the control antibody fusion

protein containing wild-type IL-2, NHS-IL2LT retained anti-tumor activity against

established neuroblastoma and non-small cell lung cancer metastases in syngeneic mouse

tumor models, but was much better tolerated in immune competent mice as well as in

cynomolgus monkeys.

Conclusions The demonstrated qualities of low toxicity and single agent efficacy

suggest that NHS-IL2LT is a good candidate for therapeutic approaches combining

standard cytotoxic and immune therapies. In fact, this molecule (also known as

Selectikine or EMD 521873) is currently in Phase I clinical trial.





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INTRODUCTION

Despite many years of clinical use, the mechanisms of IL-2 toxicity and

therapeutic efficacy for cancer are not well understood. Clinical dose schedules showing

the highest objective response rates in renal carcinoma and melanoma are associated with

severe toxicities, especially those of the vascular compartment such as vascular leak

syndrome (1). Many mechanisms have been proposed for this vascular toxicity. In one

case, toxicity has been attributed to direct binding of IL-2 to endothelial cells via a motif

resembling a component of bacterial toxins (2) and centered around aspartic acid residue

20 (D20). Another group has reported a vasopermeability enhancing fragment of IL-2

extending from residues 22-58 that increases vascular permeability independent of IL-2

bioactivity (3). A third group of investigators has proposed that activation of cells bearing

the intermediate affinity IL-2 receptor in the vascular compartment leads to inflammatory

cytokine release by NK and other cells (4). In the latter case, it was proposed that a

receptor-selective form of IL-2, i.e. one that effectively binds the high affinity and not the

intermediate affinity IL-2 receptor, would avoid this activation in the vascular

compartment, but provide IL-2 to activated T-cells expressing the high affinity receptor.

This approach of selective receptor activation is not unlike the use of low-dose

IL-2 in the clinic that has been shown to result in immune activation with far less side

effects (5). Surprisingly, this treatment regimen appears to result in the expansion of a

subset of CD56bright NK cells rather than T cells, at least in the circulation, so it is not

clear whether this approach can effectively stimulate an anti-tumor T cell response. It is

generally accepted that this treatment approach results in fewer long term clinical

responses (1).





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One potential reason for this lack of efficacy with low-dose protocols may be due

to the inability to deliver sufficient levels of IL-2 to the tumor microenvironment where it

is needed to activate anti-tumor T cells. We and others have been using antibody

targeting of cytokines (immunocytokines) such as IL-2 to deliver these immune

stimulators to the tumor microenvironment with the hope of increasing efficacy without

associated toxicity (reviewed in ref (6)). Numerous studies have documented that it is

possible to generate (or enhance) potent T cell responses to tumors in mice and that these

responses are mostly MHC class I restricted CD8 T cell responses, and are dependent on

specific tumor targeting. Unlike many studies with IL-2, our treatment is based on short-

term dosing (e.g. 3 to 5 successive days followed by at least two weeks without dosing)

rather than repeated dosing over extended periods. The consequence is that we do not

provide IL-2 to cells after their initial activation that includes up-regulation of high

affinity IL-2 receptor and enhanced responsiveness. In this way toxicity is minimized, but

apparently the targeting aspect results in efficacy that is not possible with short term

dosing with non-targeted IL-2. In fact, many of our studies have included the use of IL-2

(at equal or higher doses) that was found to be completely ineffective in situations where

our targeted molecules were completely effective in eradicating tumor cells (7). In the

current study, we used an antibody NHS76 that was selected from a phage display library

using Raji Burkitt’s lymphoma nuclear extracts as the binding ligand (8), to target the

DNA/histone complex in the necrotic core of tumors. Such Tumor Necrosis Treatment

(TNT) antibodies have been shown to be effective in targeting tumors in animal models

and cancer patients (9, 10).





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Despite the ability to target IL-2 to tumors, systemic administration of

immunocytokines still exposes the cells in the vascular compartment to IL-2 prior to their

delivery to the tumor. Therefore we have investigated ways of reducing their potential

toxicity in order to increase the doses that can be administered. We first investigated

changes in the proposed toxin motif (discussed above) by testing mutations of D20 of IL-

2 that were reported to maintain normal IL-2 bioactivity. One such mutation to threonine

(D20T) has little or no effect on activity of free IL-2, but would be predicted to eliminate

the toxin motif responsible for endothelial cell binding (2). Surprisingly, when this form

of IL-2 was expressed as a whole antibody immunocytokine, we found it to be highly

specific for activating the high affinity IL-2 receptor. The resulting molecule would be

expected to have two beneficial properties including selectivity for activated T cells, and

reduced binding to endothelial cells. In the current study we describe the characterization

of this molecule in vitro and in vivo, and demonstrated for the first time that a form of IL-

2 with receptor specificity for the mouse high affinity IL-2 receptor can demonstrate anti-

tumor responses without the normal levels of IL-2 toxicity. An immune stimulator with

such a low side effect profile is a good candidate for combination with traditional chemo

and radiotherapies that by themselves have immune potentiating activity (11).

MATERIALS AND METHODS

Cell lines and animals. The mouse myeloma NS/0 cell line was obtained from

the European Collection of Animal Cell Cultures (Salisbury, UK). The murine T cell line

CTLL-2 and the human astrocytoma U-87 MG were obtained from American Type

Culture Collection (Rockville, MD). The murine neuroblastoma NXS2 was a gift from





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Dr. Ralph Reisfeld at Scripps Research Institute. The murine Lewis lung carcinoma

(LLC) was a gift from the late Dr. Judah Folkman at Children’s Hospital, Boston. The

human T cell line Kit-225 (K6) was provided by Dr. Angus Grant, EMD

Pharmaceuticals, Durham, NC. The human TF-1β cell line was provided by Dr. Paul

Sondel, University of Wisconsin at Madison, WI. All cell culture media were purchased

from Invitrogen (Carlsbad, CA) and all cytokines from R&D Systems (Minneapolis,

MN). C57Bl/6 (female), A/J (female), SCID CB17 mice (male), and BALB/c (female)

mice (8-9 weeks old) were purchased from Taconic Farms (Germantown, NY) and

Jackson Lab (Bar Harbor, ME).

Expression and purification of NHS-IL2 immunocytokines. The pdHL vector

used for the expression of NHS-IL2 immunocytokines was described previously (12).

The genes encoding the V regions of the NHS antibody were based on the protein

sequences of NHS76, a single-chain Fv derived from screening a human scFv phage

library against Raji Burkitt’s lymphoma cell nuclear extracts (8). The VH and the VL

were chemically synthesized, using optimized codons for mammalian expression. The

VH was joined to the human IgG2 constant regions, which was in turn fused in frame to

the sequence encoding the mature IL-2 in the vector. At the fusion junction, the C-

terminal lysine residue of the CH3 was changed to alanine to increase serum half life

(13). The sequence of the VL revealed that it was derived from a human lambda chain

and contained the J3 region, hence it was joined to the constant region of human lambda

3, which was used to replace the human kappa chain in the pdHL vector. The D20T

mutation in the IL-2 moiety and the N297 mutation in the CH2 domain were introduced

by overlapping polymerase chain reaction. The antibody-IL2 fusion proteins were





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expressed in NS/0 cells by transfection and selection of producer cell clones as described

(13). Proteins were purified from conditioned cell culture media by binding to and

elution from protein A Sepharose, followed by diafiltration into PBS.

IL-2 bioactivity assays. The IL-2 activity of IL-2 containing immunocytokines

were assayed in standard T-cell proliferation assays using the mouse CTLL-2 cell line

(14), human T cell line Kit-225 (K6) (15) or human TF-1β cell line (16), which are all

dependent on IL-2 for growth. The CTLL-2 cell line expresses the high affinity mouse

IL-2 receptor, the Kit-225 (K6) cell line expresses the high affinity human IL-2 receptor

(17), and the TF-1β cell line expresses only the intermediate affinity human IL-2

receptor. For the Kit-225 assay proliferation was measured by the reduction of Alamar

Blue instead of 3H thymidine uptake (18). Briefly, washed Kit-225 cells that had been

starved for 4 days in AIM-V serum free media (12,500 cells/well) were incubated with

IL-2 or IL-2 containing immunocytokines for 36 hours. Alamar Blue (Trek Diagnostics

Cleveland, OH) was then added (30 μl/well) and the incubation continued for an

additional 34 hours. The fluorescence was then read using a fluorescence plate reader

(excitation 530 nm, emission 590 nm). The maximum response used for ED50 calculation

was attained using an internal reference standard in each assay. The ED50 concentration

was calculated using least squares analysis (TREND analysis from Excel).

Mouse NK cell isolation and bioassay. Cells were harvested from spleens taken

from C57Bl6 mice and NK cells enriched using a kit from Stem Cell Technologies

(Vancouver Canada). NK cells were separated from the rest of the cells through negative

selection using a depletion cocktail tailored to highly enrich NK cells from suspensions

of murine spleen cells. NK cell purity was measured using cell staining for NK1.1 and





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DX5, known mouse NK cell markers. Cells were incubated with IL-2 or IL-2 containing

immunocytokines for a total of 96 hours with 3H thymidine added during the last 16

hours. The cells were harvested from the wells with water onto glass microfiber filter

plates and radioactivity was measured by liquid scintillation counting.

PBMC proliferation and FACS analyses. PBMC from normal donors were

prepared by Ficoll gradient centrifugation and labeled with carboxyfluorescein diacetate

succidimidyl ester (CFSE, Molecular Probes Inc., Eugene OR) for 15 min at 37o C. After

washing with culture medium (RPMI containing 10% FBS), 2 x 106 cells were cultured

per ml, alone or with added IL-2 or immunocytokine. In some cases, cultures included

OKT3 anti-CD3 antibody at a final concentration of 0.1 μg/ml. Cell samples were

collected at the indicated time points and stained with the following antibodies: anti-

CD56PE, anti-CD4PE, anti-CD8PE and anti-CD25FITC according to the instructions of

the manufacturer (BD Pharmingen, San Diego, CA). After incubation on ice for 30 min

and washing, labeled cells were analyzed using a Coulter Epics XL-MCL flow cytometer.

Pharmacokinetic analysis. BALB/c mice were injected with 25 μg of an

immunocytokine in a volume of 0.2 ml in the tail vein using a slow push. At various time

points, small blood samples were taken by retro-orbital bleeding and collected in tubes

coated with heparin to prevent clotting. After centrifugation to remove the cells, the

plasma was assayed by capture with anti-human IgG H&L antisera and detection with an

anti-human IL2 antibody (12). Results were normalized to the initial concentration in the

serum of each mouse taken immediately after injection.

Pilot toxicity study in cynomolgus monkey. The animal experiment was

approved by the local authorities and was conducted in compliance with the principles of





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Good Laboratory Practice (GLP) as well as the local animal welfare and health

regulations. In this pilot monkey toxicity study (performed by MDS Pharma Services,

France), a group of 2 male and 2 female cynomolgus monkeys (Macaca fascicularis) with

an age of 25 to 27 months at the beginning of treatment were treated in an 3-week cyclic

regimen that reflected the intended clinical dosing scheme of NHS-IL2LT. Animals were

treated by a 1-hour intravenous (i.v.) infusion on 3 consecutive days (day 0 – day 2)

followed by an 18-day treatment-free period (21-day cycle) for 3 cycles. The vehicle

control was 0.9% (v/v) NaCl solution. NHS-IL2LT was administered at 1, 3 and 10

mg/kg/d in 0.9% (v/v) NaCl solution. Animals were analyzed for standard toxicity

parameters, i.e. clinical observations, body weight determinations, vital functions (e.g.,

blood pressure measurements, electrocardiogram (ECG)), clinical pathology (hematology

including lymphocyte subset analysis, serum chemistry, urinalysis), toxicokinetic and

immunogenicity investigations, and histopathology examinations.

Blood samples for measurement of the α-subunit of the soluble IL-2 receptor

(sIL-2Rα) (2ml samples collected in tubes containing EDTA) and lymphocyte subtyping

(1 ml samples collected without anticoagulant) were taken at day 0, 3, 5, 7 and 14 in each

cycle. Blood samples were analysed by flow cytometry using a FACScan (Becton

Dickinson, France) with anti-CD4 (FITC-labeled; #556615), anti-CD8 (PerCP - Cy5.5-

labeled; #341050 and #341051) and anti-CD25 antibodies (PE-labeled; #557138, BD

Pharmingen, France) according to the instruction provided by the manufacturer. SIL-2Rα

was measured using a commercially available ELISA kit (#DR2A00, R&D Systems,

France) according to the method described by the supplier.





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Tumor models. Experimental liver or lung metastases were induced by tail vein

injection of viable single cells of NXS2 (0.4 x 106) or Lewis Lung Carcinoma (0.5 x 106)

in 0.2 ml PBS into A/J mice or C57Bl/6 mice (n=7), respectively, on Day 0. Mice

received 5 daily intravenous injections of PBS, fusion proteins, or free IL-2 with or

without the naked NHS antibody, on days 4 to 8. Metastases were scored and organ

weights were measured on day 28 as previously described (12). For depletion studies,

mice received intraperitoneal injections of 50 μg of rat IgG2b anti-murine CD4 mAb

(clone 2.43, TIB-210, ATCC), 100 μg of rat IgG2b anti-murine CD8 mAb (clone GK1.5,

TIB-207, ATCC), or 20 μl of NK-cell-specific rabbit anti-asialo GM1 antiserum

(WAKO, Richmond, VA) on day 3 and once weekly thereafter for 3 weeks, which had

been shown previously to result in about 95% depletion of the respective T cell subsets or

NK cells by indirect immunofluorescence staining and cytofluorometric analysis of

lymph nodes and spleens.

RESULTS

Bioactivity of a mutant IL-2 immunocytokine. We originally tested the D20T

mutation of IL-2 based on data suggesting that this change conferred no selectivity for

activating either the intermediate or high affinity forms of the IL-2 receptor. Such a

mutation would allow us to test the effect of removing this key residue in the proposed

LDL toxin motif (2) and its possible role in the vascular toxicity of IL-2, independent of

its receptor selectivity. While this mutation has little selectivity as free IL-2 (unpublished

data), we found that when it is fused to the carboxyl terminus of the human IgG H chain,

the selectivity for the high affinity receptor-mediated proliferation is increased





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dramatically. Biological activity as measured by cell proliferation was compared using

two human cell lines: the Kit-225 T cell line expressing both intermediate βγ and high

affinity αβγ, and TF-1β, an erythroleukemic line naturally expressing the common γ

chain and transfected to express the human β chain. The resulting cell line responds to

IL-2 through the heterodimeric βγ intermediate affinity receptor (16). Bioactivity of the

final antibody-IL2 fusion molecule, NHS-IL2LT (LT stands for low toxicity), containing

the D20T mutation, was compared by dose titrations to both free human rIL-2 as well as

the same immunocytokine bearing wild-type IL2, NHS-IL2. While the ability of NHS-

IL2LT to induce proliferation of Kit-225 cells was reduced 4-5 fold relative to free IL-2,

the ability to induce proliferation of TF-1β was reduced more than 6000 fold (Fig. 1A

and B). This resulted in selectivity for high affinity IL-2R of more than 1000-fold. Since

we would be using mouse models for in vivo testing of toxicity and anti-tumor efficacy,

we also examined the receptor selectivity on murine immune cells. In this case we used

the standard IL-2 responsive T cell line, CTLL-2, for the high affinity measurement and

purified mouse NK cells for measuring response through the intermediate IL-2R. We

found that the NHS-IL2LT mutant immunocytokine had an even more profound

selectivity for the high affinity IL-2R (Fig. 1 C and D). We figured that such biological

activity assays are more sensitive and meaningful than just measuring binding affinity to

the different receptors, because the selectivity for activating the high affinity receptor

may not be completely accounted for by the difference in binding. In fact, we found that

NHS-IL2LT retained binding affinities to both the high and intermediate affinity IL-2

receptors (Supplementary Fig. 1A and B), and its differential activities on the

proliferation of Kit225 and TF-1β cells seemed to be mainly due to impaired signaling, as





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measured by phosphorylated Stat5a (Supplementary Fig. 1C and D). NHS-IL2LT

attained substantial levels of pStat5a signaling on TF-1β cells only at very high

concentrations of about 100 nM to 1 μM (i.e., containing respectively 1800 and 18000

ng/ml of IL-2 equivalents, Supplementary Fig 1D), consistent with the proliferation data

of these cells in Fig. 1B.

These results are in contrast to earlier studies with the N88R mutant form of IL-2

that reported selectivity for high versus intermediate IL-2R, but was later found to

demonstrate this effect only for human and monkey cells but not for mouse immune cells

(19). Therefore, in the case of NHS-IL2LT, it is justified to use mouse models to

determine relative toxicity and efficacy and thereby establish any potential improvements

in the therapeutic index. Although not the original intention of our studies, we found it

possible to test for the first time in relevant tumor models, the hypothesis that a form of

IL-2 with selectivity for the high affinity IL-2R would be less toxic and still maintain

anti-tumor efficacy. We also found in these studies that immunocytokines with wild type

IL-2 also show a moderate degree of selectivity for the high affinity over the intermediate

form of IL-2R, relative to free IL-2 (Fig. 1). We have also confirmed this to be the case

with other immunocytokines utilizing different antibody V regions (data not shown).

IL-2 receptor selectivity in cultured PBMC. Since both mouse and human

immune cells responded with the same degree of IL-2 receptor selectivity, we used

human PBMC to study differential proliferative responses in CD56+ NK cells as well as

CD4+ and CD8+ T cell populations. We also monitored expression of the CD25

component of the high affinity receptor in the same experiment. For proliferation

analysis, resting PBMC were labeled with CFSE and cultured with rIL-2 or equimolar





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amounts of either NHS-IL2 or NHS-IL2LT immunocytokines. Significant proliferation of

NK cells were observed in cultures containing rIL-2 and NHS-IL2 as visualized by CFSE

dilution in the CD56 gated population on days 6 and 9 of culture (Fig. 2A). In contrast,

little or no proliferation was observed in the culture containing NHS-IL2LT as measured

by CFSE dilution. This result is consistent with the fact that little or no CD25 expression

was observed in this CD56+ cell population and that only cells expressing intermediate

receptor accounted for the observed proliferation. Despite this lack of proliferation, the

number of CD56+ cells in the culture supplemented with NHS-IL2LT was increased on

day 6 compared to the culture with no added IL-2, suggesting that NK cell survival was

prolonged by this molecule.

Analysis of proliferation and IL-2R expression in T cell populations was

performed in the same manner, but in the presence and absence of anti-CD3 antibody

stimulation. Again, PBMC were labeled with CFSE and cultured with rIL-2, ΝΗS-IL2 or

NHS-IL2LT. In this case, CD4+ and CD8+ cells were gated and analyzed separately for

proliferation and CD25 expression. In the absence of anti-CD3 stimulation, little or no

CD4+ T cell proliferation was observed with rIL2 or either immunocytokine (Fig. 2B),

since the IL-2Rβ chain is expressed constitutively in CD8 T cells, but not in un-

stimulated CD4 T cells (20). In cultures activated by anti-CD3 antibody, strong

proliferative responses were seen by day 6 in all cultures, including those with NHS-

IL2LT. Response to this molecule correlated with a strong up-regulation of CD25 in the

CD4 gated cell population as measured on day 6 of culture (Fig. 2B). Since anti-CD3

activation is known to induce CD25 expression (21), we assume that the CD4+CD25+

cells observed at this time have survived and proliferated in response to the selective





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NHS-IL2LT molecule, whereas the culture containing anti-CD3 antibody but no IL-2

would not have maintained expression of CD25 until day 6.

When CD8+ cells were examined in the same cultures, a somewhat different

pattern of response was observed. In this case, cultures without anti-CD3 activation

showed proliferative responses to the wild-type forms of IL-2, i.e. those capable of

triggering both intermediate and high affinity receptors, but not to the selective NHS-

IL2LT molecule (Fig. 2C). Also unlike CD4+ cells, some degree of CD8+ cell

proliferation was observed in response to anti-CD3 antibody alone (no added IL-2), but

that the addition of either the wild-type or mutant IL-2 molecules increased the number of

cell divisions dramatically and to roughly the same extent. Again, the ability of NHS-

IL2LT to stimulate cell proliferation was associated with strong induction of CD25

expression in the CD8 gated population in the anti-CD3 activated culture.

Effects on pharmacokinetic behavior in mice. Before comparing wild-type and

mutant immunocytokines, it was essential to eliminate other parameters that could effect

vascular toxicity and anti-tumor efficacy – especially changes in pharmacokinetics. We

reported earlier that the fusion of IL-2 to an antibody molecule has unpredictable

consequences to the pharmacokinetic behavior of immunocytokines in mice and man (22,

23). Generally, the distribution α phase in the blood is far more rapid than that of a whole

antibody and the t1/2 in the vascular compartment is shorter as well. These effects can be

partially abrogated by reducing FcR binding or by modifying the linker between the

antibody and IL-2 so that intracellular degradation is reduced (13). The NHS-IL2

immunocytokines described in this study have been optimized for both reduced FcR

binding (by using the γ2 isotype) and intracellular degradation (by mutating the C-





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terminal residue of the CH3 domain from K to A). Despite these changes, we found that

the mutation of D20 to T had a dramatic effect on clearance from the circulation,

primarily as a result of prolonging the distribution α phase (t1/2 α = 0.62 hr, t1/2 β = 4.6 hr,

AUC (area under the curve) = 18.7 μg-hr/ml), as compared to that of NHS-IL2 (t1/2 α =

0.93 hr, t1/2 β = 5.1 hr, AUC = 75.0 μg-hr/ml. Interestingly, this could be reversed by

removing the N-linked glycosylation at N297 of the CH2 domain, either by enzymatic

treatment (not shown) or through mutation to N297Q. This results in a longer exposure

time for the aglycosylated form in the circulation (AUC = 59.4 μg-hr/ml), despite

similarities in clearance rates (t1/2 α = 0.79 hr, t1/2 β = 4.9hr). The same phenomenon was

observed with other IgG isotypes indicating that this was not particular to the use of the

γ2 isotype (not shown). Since our antibody targets the necrotic portion of tumors through

binding of DNA released from dying cells, use of such a non-FcR binding antibody

should have little impact on anti-tumor efficacy.

Tolerability of NHS-IL2LT in mice. Our initial tolerability studies utilized

immune competent mice that make high-titered antibody responses to this human fusion

protein beginning around day 5. For this reason we limited intravenous (i.v.) dosing to

five consecutive days and increased dose amounts until significant body weight loss or

death were observed. For NHS-IL2 containing wild-type IL-2, a dosing regimen of 50

μg/mouse/day x 5 days resulted in animal deaths consistently in multiple experiments

(Fig. 3A). This is a lower tolerated dose than we had seen in earlier studies with other

immunocytokines, but was likely due to this molecule’s longer circulating t1/2 that led to

accumulation with daily administration. In contrast, using NHS-IL2LT, daily i.v. doses of

1 mg/mouse/day x 5 could be administered with no animal deaths, however at this point,





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mice were beginning to show signs of toxicity including transient weight loss (Fig. 3B).

These data demonstrate that NHS-IL2LT is greater than 20-fold less toxic than the same

molecule containing wild-type IL-2 when administered with this dosing schedule.

Since treatment with IL-2 is expected to modulate IL-2R on immune cells, we

also examined longer dosing schedules to see if there was IL-2R up-regulation or

expansion of cells with high affinity IL-2R. Due to anti-immunocytokine antibody

responses, we performed these studies in immune deficient SCID mice recognizing that

the lack of functional B and T cells would likely make any results difficult to interpret in

the context of immune competence. In the study shown in Fig. 3C, consecutive dosing for

5 days repeated weekly resulted in significant toxicity and death with doses as low as 20

μg per day, and with a delay of only about 5 days compared to the NHS-IL2 wild type

control molecule. Additional pilot experiments in SCID mice identified the shortest time

between 3-day repeated courses of treatment was 21 days (not shown), a schedule that is

quite similar to those used for standard chemotherapies. These data show that the benefit

of receptor selectivity is lost with continuous dosing, but that intermittent dosing

protocols may be able to maintain the low toxicity benefit of this immunocytokine and its

potential for use in combination therapies. In order to test this hypothesis in a more

relevant model, where biological responses are more similar to humans, and where

immunogenicity should be far less pronounced, we next studied repeated intermittent

dosing in non-human primates.

Toxicity and pharmacological activity of NHS-IL2LT in cynomolgus

monkey. We assessed NHS-IL2LT in a GLP-compliant pilot toxicology study in

cynomolgus monkeys, a relevant species for the assessment of IL-2 toxicities (24). The





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lowest dose administered (1 mg/kg) corresponded to the highest, but poorly tolerated,

dose we have administered to cynomolgus monkeys using other immunocytokines

containing un-mutated IL-2. The other doses were 3 and 10-fold higher. The maximum

tolerated dose (MTD) of immunocytokines in humans have been determined to be 6.4

and 7.5 mg/m2 for huKS-IL2 and hu14.18-IL2, respectively, when administersed as once-

daily 4-hour infusions in a 3-day cycle repeated every 4 weeks (23, 25). This MTD range

for humans corresponds to about 0.52 to 0.61 mg/kg for monkeys based on allometric

scaling (26). However, the actual MTD of IL-2 containing immunocytokines in monkeys

was determined empirically to be several-fold lower, about 0.1 mg/kg, and monkeys and

importantly, humans dosed at their respective MTD’s had very similar Cmax and AUC

(23, 25, and unpublished data).

Monkeys were treated using the intended clinical regimen, i.e., a 21-day cycle

consisting of treatment (1 h, i.v infusion) on 3 consecutive days (day 0-2) followed by an

18-day treatment-free period, which is a more aggressive schedule with the anticipated

lower toxicity of Selectikine. Toxicokinetic analysis showed a systemic and dose-

proportional exposure of all NHS-IL2LT-treated animals. The peak plasma levels were in

the range of 103 to 161 µg/ml in high dose treated male and female animals, which are

many times higher than the peak plasma levels of 2 to 5 µg/ml obtained at the MTD of

huKS-IL2 and hu14.18-IL2 in humans (23, 25), and in monkeys (unpublished data).

Although most animals developed low levels of antibodies against NHS-IL2LT, this fact

did not affect its kinetic properties and therefore drug exposure. NHS-IL2LT elicited no

overt clinical signs or changes in body weight. In addition, no treatment-related effects at

the injection sites or on cardiovascular parameters were observed. Treatment-related





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alterations with respect to hematology, biochemistry and pathology were mild to

moderate; the major target organs being the lymphatic system, liver, kidney and intestine

with lymphoid cell infiltrations. In summary, NHS-IL2LT induced moderate and typical

IL-2-derived toxicity (27) and was clinically well tolerated using the intermittent dosing

scheme, defined in our mouse models, up to the high-dose of 10 mg/kg tested.

In addition to the comprehensive toxicity assessment, we also evaluated some

pharmacological IL-2 response in the cynomolgus monkeys to prove the relevance of this

species and to show NHS-IL2LT’s pharmacologic activity in vivo. We first analyzed the

response of total lymphocytes, T-lymphocytes (CD3+) and NK (CD8+ CD16+) cells to

NHS-IL2LT treatment. The total lymphocytes (Fig. 4A) and T-cells (Fig. 4B) first

showed a very mild lymphopenia (considering these high doses) followed by a strong

lymphocytosis around days 5 and 7 that were increased (2-3 fold) over baseline,

compared to the vehicle control. Interestingly, all dose groups showed essentially the

same degree of total lymphocyte and T cell expansions indicating saturation of receptors

at the lowest dose level tested. NK cells showed a somewhat different pattern of response

(Fig. 4C). In this case, the degree of lymphopenia was much stronger as a percentage of

total NK cells, and lymphocytosis in the lowest dose group was clearly reduced compared

to the two higher dose groups. In fact, the peak level after recovery in the low dose group

was essentially a return to baseline. This level of response was not different from what

was observed in the vehicle control group.

We then looked at more specific IL-2 markers as the soluble IL-2 receptor α-

subunit (sIL2Rα) and CD25 positive (activated) CD4+ and CD8+ lymphocytes in serum.

The soluble form of the high affinity IL-2 receptor subunit α increases in serum





20

concomitant with its cellular expression. Although the function of sIL2Rα is unclear, it

correlates with increased T-cell and immune system activation (28). It has been shown

previously that mutated IL-2 molecules are able to induce activated (CD25+) T-Helper

(CD4+) as well as cytotoxic T-cells (CD8+) cells in non-human primate species (4).

Cynomolgus monkeys showed a typical and strong IL-2 response with a sharp and dose-

dependent increase in sIL2Rα one day after the last treatment in each cycle (day3, 24, 45)

(Fig. 4D), whereas CD25 positive CD4+ (Fig. 4E) and CD8+ (Fig. 4F) cells increased on

day 5 in each cycle. The peak expansion of activated CD4+ and CD8+ cells as well as the

increase of sIL2Rα was 20-40-fold compared to the vehicle control. Overall the effects

were reversible and returned to baseline values at the end of each cycle (day 20 and 41).

These results confirmed our observations in vitro and show that NHS-IL2LT also has a

selective pharmacological activity on CD4/CD8 positive cells in non-human primates.

Anti-tumor activity in mouse models. Next we tested whether treatment with a

low-toxicity form of IL-2 could show efficacy in mouse tumor models. We were also

interested in testing whether tumor necrotic targeting could be effective in the setting of

minimal residual disease. Previous studies of the uptake of radiolabeled antibodies in

mouse tumor models had demonstrated that tumor necrosis can be used to target

micrometastases in well oxygenated organs when lesions are greater than 20 to 30 cells in

diameter (9). At this stage, cells undergoing apoptosis are known to express DNA or

DNA containing complexes on their surface (29, 30). Furthermore, for tumor cells with

high rates of mutation and daughter cell deaths, in vitro experiments using tumor

spheroids to model micrometastases had shown that 20 to 30% of the cells in the

proliferating rim are non-viable and necrotic (31).





21

To test anti-tumor activity, we induced experimental lung metastases by

intravenous injection of a lethal does of LLC cells in immune competent Bl/6 mice or

experimental liver metastases by intravenous injection of NXS2 neuroblastoma cells in

immune competent A/J mice. Treatment was initiated on day 4 when only small

metastases have been established. For these studies we limited dosing to 5 consecutive

days to avoid treatment after IL-2R up-regulation and thereby maintained receptor

selectivity. Animals were sacrificed when control mice showed signs of toxicity from

tumor burden (approximately day 28) and lungs or livers were removed for analysis of

tumor burden by two criteria. Surface metastases were apparent after staining, but most

surface lesions tended to fuse and could not be easily counted. Therefore, surface

metastases were semi-quantitated by estimation of percent surface coverage. For the LLC

model we first correlated this assessment method with tumor burden as measured by

histological examination after H&E staining, and found a strong correlation between %

surface coverage and overall tumor burden (Fig. 5A and B). In this experiment potent

antitumor activity was demonstrated with a daily dose of 80 μg and an intermediate level

of activity was observed using 20 μg per dose. The control groups treated with either the

same amounts of IL-2 or combinations of IL-2 and the NHS antibody showed no activity

at all in this model.

In the NXS2 neuroblastoma model we measured both % surface coverage by

metastases as well as overall organ weight as a measure of tumor burden. Again we

confirmed that diseased livers consisted of tumor mass by H&E staining (not shown).

Therefore, tumor-containing liver weight (approximately 5% of body weight) in excess of

normal liver weight was taken as a measure of tumor mass. Under these conditions we





22

found that equal doses of NHS-IL2 and NHS-IL2LT showed nearly the same level of

activity as assessed by the percent of surface coverage by tumor (Fig. 6A), and both

treatment groups were significantly different from the PBS control (P< 0.001). Treatment

was somewhat better with NHS-IL2, but this was not significant (P = 0.18). When data

were assessed by organ weights – a more objective measure of tumor burden - the

similarity between the treatment groups (shown as individual animal organ weights) were

even more striking. Recombinant IL-2 had no anti-tumor activity in the same model (not

shown).

In order to assess what effector cells were responsible for anti-tumor activity, we

tested the effect of antibody depletion of CD4, CD8 or NK cells in the LLC experimental

metastasis model. Depletion of the respective cell type was confirmed by FACS analysis

of peripheral blood for each antibody. The in vivo efficacy results showed that depletion

of CD4+ cells did not abrogate the anti-tumor effect of the two immunocytokines

containing either wild-type or mutated IL-2 (Fig. 6B) and may have improved their

activities, although the difference was not statistically significant (P = 0.16 for NHS-IL2

and 0.096 for NHS-IL2LT). This improvement in anti-tumor activity suggests that

elimination of CD4+CD25+ Treg cells might make this treatment approach more

effective.

In contrast to the results with CD4 depletion, treatment with anti-CD8 antibody

resulted in a complete loss of anti-tumor activity for both NHS-IL2 and NHS-IL2LT (Fig.

6B), indicating that CD8+ cells are the primary effectors in this model. This is in

agreement with most of our earlier results in other syngeneic tumor models (6). The

results with NK cell depletion were less dramatic and suggested some role for these cells





23

in anti-tumor activity (Fig. 6B), possibly providing a supporting function to the more

essential CD8+ effector cells. Interestingly, there was no difference between the groups

treated with NHS-IL2 and NHS-IL2LT with respect to NK cell depletion, despite the

dramatic selectivity differences in cytokine response in vitro and toxicity in vivo.

DISCUSSION

Several factors can play a role in the ability of a cytokine to effectively activate

the immune system against a foreign agent or a tumor cell. One of the first cytokines to

be approved for treatment of cancer is IL-2 despite the fact that its biology is one of the

most complex and, in some cases, contradictory to its proposed role in immune

stimulation. Not only does IL-2 potently stimulate NK and T cells to expand in numbers

and increase their cytolytic activity, but in the case of T cells, it sensitizes them to

activation-induced cell death (32) and is required for T regulatory (Treg) cells to suppress

ongoing immune responses (33). In addition, activation of many cell types in the

circulation through the intermediate IL-2R can lead to numerous side effects through the

production of secondary cytokines and over-activation of NK cells – both of which can

result in vascular injury (34).

The initial goal of the present study was to engineer a form of IL-2 as an

immunocytokine with reduced potential for causing side effects while retaining the

ability to stimulate an anti-tumor immune response. An earlier report from another group

reported such a study with free IL-2 containing an N88R mutation (4), however it was

later found that the basis for the reduced toxicity (selectivity for high affinity over

intermediate IL-2R) was not maintained in the mouse tumor model used to show efficacy





24

(19). In our case, we tested the biological and anti-tumor activity of a different IL-2

mutation (D20T) that was found by others to be non-receptor-selective in the context of

free IL-2. This aspartic acid residue is the critical part of a toxin-like domain that is

believed to be responsible, in part, for direct vascular toxicity of IL-2. Surprisingly, we

found that the D20T mutation, in the context of a whole antibody immunocytokine, is

highly selective for the high affinity IL-2R (as measured by IL-2 induced proliferation)

for both human and mouse immune cells. Thus, for the first time, it was possible to test

the hypothesis of whether such a selective molecule could be both less toxic and retain

anti-tumor activity in a mouse model. Results indicate that the D20T form of IL-2,

contained in the NHS-IL2LT molecule, retained the majority of its anti-tumor activity

when tested in syngeneic mouse tumor models of experimental metastases. Even when it

was dosed at higher levels than the control NHS-IL2 molecule containing wild-type IL-2,

such doses represent a far greater therapeutic index. For example, we showed that for a

single dosing cycle, the NHS-IL2LT molecule is roughly 20-fold less toxic than NHS-

IL2 (containing normal IL2). In the tumor efficacy experiments we found no more than a

four-fold increase in dose was required to achieve the same level of efficacy. Thus, the

therapeutic index was improved a minimum of five-fold.

The tolerability of NHS-IL2 LT was also tested in cynomolgus monkeys and

shown to be dramatically improved over what we have observed with similar

immunocytokines containing normal IL-2. The lowest dose in these studies was 1 mg/kg

that resulted in sustained blood levels of NHS-IL2LT of several μg/ml - a level

approaching the ED50 of this molecule for the intermediate IL-2R. Interestingly, this level

showed a diminished ability to increase circulating levels of NK cells (following a brief





25

lymphopenia), compared to the two higher doses. T cells, on the other hand, appeared to

respond just as well at the low dose and proliferated to a much greater extent than NK

cells. This expansion correlated to the up-regulation of CD25 on both CD4 and CD8

positive T cells, as well as an increase in sIL-2 in the blood. We do not know yet whether

the cells responding at the lower dose were already expressing CD25 or whether the

initial response was stimulated through intermediate receptor-mediated induction of

CD25 on resting cells. In any case, the overall effect was to selectively expand both T

cell subsets, especially those expressing CD25, and to do this at increasingly high dose

levels in the apparent absence of typical IL-2 side effects.

How this selectivity for high affinity IL-2 receptor in NHS-IL2LT (Selectikine)

may translate to anti-tumor activity and lower toxicity remains to be tested clinically. In

fact, Selectikine (EMD 521873) is currently in Phase I clinical trial. The inherent

problem with immune therapy is that it works less well as tumor burden increases and, in

the case of NHS-IL2LT, we used a targeting approach (anti-DNA) that might be expected

to require large tumors as a source of antigen. The fact that even small metastases could

be treated with this approach, likely due to the appearance of DNA containing structures

on apoptotic cells, allowed us to demonstrate anti-tumor activity in this minimal disease

setting after a single dosing cycle and this activity was due mostly to CD8+ effector cells.

Only low titers of anti-human antibodies were seen in monkeys after an 8-week treatment

and these have not limited the number of cycles of NHS-IL2LT that can be administered.

Therefore, it is likely that multiple cycles of treatment in the clinic could be administered

safely and with the potential for improved efficacy. One challenge with IL-2 in general,

and with forms specific for the high affinity receptor in particular, is the possibility of





26

generating more T regulatory cells than anti-tumor effectors. In this regard it was

interesting to note that our anti-tumor activity in the Lewis Lung model was improved

when mice were treated with anti-CD4 antibody, although significant anti-tumor activity

was seen without CD4 depletion. Nonetheless, many approaches to reducing T regulatory

cells (11) could be combined with NHS-IL2LT, including many standard chemotherapies

and local radiation. The fact that its administration, even at high doses, is associated with

only mild side effects makes such an approach clinically attractive.





27

Figure 1 Relative IL-2 bioactivity of normal and mutant IL-2 based

immunocytokines (NHS-IL2 ( ), NHS-IL2LT( ), and control IL-2( )).

Proliferation responses of (A) the high affinity IL2 receptor expressing human T cell line

Kit-225 (K6), (B) the intermediate affinity IL2 receptor expressing human cell line TF-

1β, (C) the high affinity IL2 receptor-expressing mouse T cell line CTLL-2, and (D) the

intermediate IL2 receptor expressing mouse NK cells. Proliferation of Kit-225 (A) was

measured by the reduction of Alamar Blue, while that of the other cells (B, C and D) was

by 3H thymidine uptake. Mouse NK cells were freshly isolated from Bl/6 mice as

described in the Materials and Methods. Data are representative of at least 3 experiments.

Figure 2 CD45 positive immune cell proliferation in PBMC cultures containing

wild-type and mutated IL-2 immunocytokine as assessed by CFSE dilution. A. CD56

positive NK cells were gated and analyzed for CFSE staining intensity on days 6 and 9 of

culture. CD25 expression on CD56+ cells was assessed on day 6. A decrease in the

intensity of staining (shift to the left) was used as a measure of NK cell division. B.

CD4+ cells were gated and analyzed for CD25 expression and CFSE dilution on day 6 of

culture. One culture was activated with anti-CD3 antibody while a second was not. C.

CD8+ cells were gated and analyzed for CD25 expression and CFSE dilution on day 6 of

culture. One culture was activated with anti-CD3 antibody while a second was not.

Figure 3 Tolerability of normal and mutant IL-2 based immunocytokines. Bl/6 or

SCID mice (3 per group) were injected i.v. with either PBS () or the indicated

immunocytokine, and survival (A and C) and body weight (B and D) were monitored. In





28

Bl/6 mice (A and B), doses of NHS-IL2 were given for 5 consecutive days at 50

μg/mouse () and for NHS-IL2LT, at 1000 μg/mouse (). Results shown were

representative of 3 experiments. In SCID mice (C and D), doses of 20 μg/mouse of

NHS-IL2 () or NHS-IL2LT () were given for 5 consecutive days, every week, until

overt toxicity occurred.

Figure 4 Pharmacological activity of NHS-IL2LT in cynomolgus monkey. Blood

samples of animals treated with 0, 1, 3 and 10 mg/kg/day NHS-IL2LT were analyzed for

total lymphocyte counts (A), CD3+ cells (B), CD8+ CD16+ cells (C), sIL2Rα (D), CD4+

CD25+ cells (E), and CD8+ CD25+ cells (F) at the indicated days. Data (except D) were

expressed as absolute counts (calculated based on total white blood cell count). Note that

animals were treated in a cyclic regimen on day (D) 0, 1, 2 and 21, 22, 23 and 42, 43, 44,

as indicated by arrows on the x-axis of (C) and (F). Female data as average are shown,

males showed a comparable response.

Figure 5 Efficacy in a syngeneic experimental metastasis model of non-small cell

lung cancer as assessed by both lung surface coverage and histolological examination.

Bl/6 mice were injected i.v. with LLC cells and then treated with the indicated dose of

NHS-IL2LT, the combination of the corresponding amounts of antibody and free IL-2, or

IL-2 alone for 5 consecutive days beginning 4 days later. Tumor outgrowth was assessed

on day 28 by histological staining of lung sections (A) or by measuring surface coverage

of lung metastases (B). The % surface coverage values of the lung sections shown in A





29

are indicated below a representative section stained with H&E. The results shown were

representative of three separate experiments.

Figure 6 NHS-IL2LT shows efficacy in experimental models of both

neuroblastoma and non-small cell lung cancer metastases. A. A/J mice were injected i.v.

with NXS2 neuroblastoma and treated with the indicated immunocytokine for 5

consecutive days beginning 4 days later. Tumor outgrowth in the liver was assessed by %

surface coverage as well as by weighing the diseased organ. The results shown were

representative of two separate experiments. B. Bl/6 mice were injected i.v. with LLC

cells and treatment began 4 days thereafter with NHS-IL2 (20 μg/mouse), NHS-IL2LT

(80 μg/mouse), or equimolar amounts of free IL-2 with or without the NHS antibody, for

5 consecutive days. Individual groups of mice were treated with depleting antibodies as

described in Materials and Methods. Tumor burden was assessed on day 28.





30

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Published OnlineFirst April 29, 2011.Clin Cancer Res Stephen D. Gillies, Yan Lan, Thore Hettmann, et al. activity despite its high degree of IL-2 receptor selectivityA low-toxicity IL-2 based immunocytokine retains anti-tumor

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