Diphtheria-toxin based anti-human CCR4 immunotoxin for targeting human CCR4+ cells in vivo

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 4 5 8 e1 4 7 0

available at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/molonc

Diphtheria-toxin based anti-human CCR4 immunotoxin for targeting human CCR4D cells in vivo Zhaohui Wanga,1, Min Weia,1, Huiping Zhanga, Hongyuan Chena, Sharon Germanaa, Christene A. Huanga, Joren C. Madsena,c, David H. Sachsa,b, Zhirui Wanga,* a

Center for Transplantation Sciences, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA b TBRC Laboratories, Center for Transplantation Sciences, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA c Division of Cardiac Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

A R T I C L E

I N F O

A B S T R A C T

Article history:

CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic

Received 6 February 2015

drug target for CCR4þ tumor cells and Tregs. CCR4 is expressed on some tumor cells such

Received in revised form

as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL),

7 April 2015

adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is

Accepted 15 April 2015

also expressed on majority of Tregs, mainly effector Tregs. In this study we have success-

Available online 25 April 2015

fully developed three versions of diphtheria-toxin based anti-human CCR4 immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytom-

Keywords:

etry showed that all three versions of the anti-human CCR4 immunotoxins bound to the

CCR4

human CCR4þ tumor cell line as well as CCR4þ human PBMC. The bivalent isoform bound

Immunotoxin

stronger than its monovalent counterpart and the single-chain foldback diabody isoform

CCR4þ tumor

was the strongest among the three versions. In vitro efficacy analysis demonstrated that

Treg

the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein

Diphtheria toxin

synthesis in human CCR4þ tumor cells compared to the monovalent anti-human CCR4 im-

Pichia Pastoris expression

munotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4þ tumor-bearing mouse model. The immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor g /

(NSG) mice injected with human CCR4þ acute lymphoblastic leukemia cells compared

with the control group. This novel anti-human CCR4 immunotoxin is a promising drug candidate for targeting human CCR4þ tumor cells and Tregs in vivo. ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

* Corresponding author. Center for Transplantation Sciences, Massachusetts General Hospital and Harvard Medical School, MGH-East, Building 149-6113, 13th Street, Boston, MA 02129, USA. Tel.: þ1 617 643 1957; fax: þ1 617 726 4067. E-mail address: [email protected] (Z. Wang). 1 Co-first authors. http://dx.doi.org/10.1016/j.molonc.2015.04.004 1574-7891/ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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1.

Introduction

CCR4 receptor has attracted much attention for targeting CCR4þ tumors. CC chemokine receptor 4 (CCR4) is expressed on certain tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T cell leukemia-lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL) (Bayry et al., 2014). The first humanized antihuman CCR4 mAb, mogamulizumab was approved in Japan for treatment of human CCR4þ relapsed or refractory adult T cell leukemia/lymphoma (ATLL) in 2012 (Yoshi and Matsushima, 2015) and for relapsed or refractory CCR4þ peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) in 2014. These mAbs rely on competent host ADCC (antibody dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) pathways for their efficacy. However, for treatment of mAb-resistant CCR4þ cancer patients, alternative therapies are needed that are independent of the host ADCC or CDC. At the same time, clinicians and scientists are seeking efficient methods for the in vivo depletion of Tregs to facilitate cancer treatment as well as study the mechanisms of autoimmune diseases and transplantation tolerance (Pere et al., 2012). The Tregs sub-population of CD4þ T cells which suppress alloaggressive CD4þ and CD8þ T cell responses and is responsible for peripheral tolerance. CCR4 is expressed on the majority of Tregs and it is the unique receptor of CCL22 (macrophage derived chemokine) and CCL17 (thymus and activation-regulated chemokine). Tumor cells express chemokine CCL17 (TARC) and CCL22 (MDC) which attract CCR4þ effector Tregs to the tumor local environment. These recruited CCR4þ Tregs may, in turn, down-regulate the host’s anti-tumor response allowing the tumor cells to evade the host immune system. Thus, depletion of Tregs is a promising approach for combined cancer treatment (Nishikawa and Sakaguchi, 2014; Zou, 2006). CCR4 is specifically expressed by effector Treg, but not by na€ıve Treg cells or Th1 cells (Sugiyama et al., 2013). In healthy individuals, CCR4 was found to be highly expressed on Foxp3hi CD45RA effector-type Treg. In contrast, na€ıve Foxp3lo CD45RAþ Treg, CD8þ T cells, NK cells, CD14þ monocytes/macrophages, dendritic cells and B cells had barely detectable levels of CCR4 expression at the protein and mRNA level (Sugiyama et al., 2013). Given the need for an ADCC and CDC-independent therapy for CCR4þ cancer patients and the need to more effectively and specifically deplete Tregs in vivo, we have developed an anti-human CCR4 immunotoxin using the truncated diphtheria toxin DT390, which has been successfully used to build functional recombinant immunotoxins/fusion toxins for depleting specific cell populations in vivo (Woo et al., 2002; Kim et al., 2007; Wang et al., 2011; Peraino et al., 2013a, 2013b, 2014; Wei et al., 2014). The monovalent, bivalent and single-chain fold-back diabody anti-human CCR4 immunotoxins were expressed using yeast Pichia Pastoris expression system and their functions were assessed in vitro using protein synthesis inhibition and cellular proliferation inhibition assays. The binding affinity of these immunotoxins to human CCR4 was analyzed using flow cytometry. The binding specificity was determined using the anti-human CCR4

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immunotoxins as inhibitors to block the binding of the antihuman CCR4 antibody to human CCR4þ tumor cells by flow cytometry and utilizing the anti-human CCR4 antibody as an inhibitor to block the ability of the anti-human CCR4 immunotoxins to inhibit protein synthesis and cellular proliferation in human CCR4þ tumor cells in vitro. The in vivo efficacy was assessed using human CCR4þ tumor bearing NSG mouse model.

2.

Materials and methods

2.1.

Antibodies and cell line

Human CCR4þ acute lymphoblastic leukemia cell line CCRFCEM was purchased from ATCC (cat# CCL-119); non-CCR4 expressing tumor cell lines: MV3, M14 and MD-MBA-231 were generously provided by Dr. Soldano Ferrone (Massachusetts General Hospital). Human/rat CCR4 fluorescein mAb (clone 205410, cat# FAB1567F) and mouse IgG2B fluorescein isotype control (clone 133303, cat# IC0041F) were purchased from R&D Systems. PE-anti-human 194 (CCR4) mAb (Clone# L291H4), Alexa Fluor 647 anti-human Foxp3 mAb (clone# 150D, cat# 320014) and Alex Fluor 647 Mouse IgG1 k (clone# MOPC-21, cat# 400136) were purchased from Biolegend. BiscFv (1567)-Human Fc was produced using yeast Pichia Pastoris expression system in our lab.

2.2.

Plasmid construction

As shown in Figure 1, anti-human CCR4 immunotoxins were constructed using the codon-optimized nucleotide sequences and contain two moieties; DT390 (Woo et al., 2002) and scFv (1567) (Chang et al., 2012 and Figure 2). A strategy previously employed to generate A-dm-DT390biscFv (2-6-15) (Wang et al., 2011) was applied to construct these anti-human CCR4 immunotoxins. DT390 and scFv (1567) or Bi-scFv (1567) domains are connected by a linker consisting of four glycines and a serine residue (G4S). The two scFv (1567) of the bivalent

Figure 1 e Schematic diagrams of the monovalent, bivalent and single-chain fold-back diabody anti-human CCR4 immunotoxin.

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Figure 2 e Codon-optimized anti-human CCR4 scFv (1567) DNA and amino acid sequence.

immunotoxin are joined by three tandem G4S linkers (G4S)3. Six histidines (6 His tag) were added to the C-terminus of each construct to facilitate protein purification. To construct DT390-scFv (1567), the codon-optimized scFv (1567) was synthesized by GenScript and cloned into pwPICZalpha-DT390 (Wang et al., 2011) between NcoI and EcoRI sites yielding the final construct DT390-scFv (1567) in pwPICZalpha. To construct DT390-BiscFv (1567), the first scFv (1567) was amplified using PCR primers CCR4-Nco carrying XhoI and NcoI sites þ CCR4-Bam1 carrying BamHI and EcoRI sites then cloned into pwPICZalpha between XhoI and EcoRI sites for sequencing confirmation. The insert was subsequently cut out with NcoI þ BamHI as insert I. The second scFv (1567) was PCR amplified using CCR4-Bam2 carrying XhoI and BamHI sites þ CCR4-Eco carrying an EcoRI site then cloned into pwPICZalpha between XhoI and EcoRI sites for sequencing confirmation. The insert was then cut out with BamHI þ EcoRI as insert II. The insert I

Table 1 e PCR primers used in this study.

carrying NcoI and BamHI sites þ insert II carrying BamHI and EcoRI sites [NcoI-scFv (1567)-BamHI/BamHI-scFv (1567)-EcoRI] were together cloned into pwPICZalpha-DT390 between NcoI and EcoRI yielding the final construct DT390-BiscFv (1567) in pwPICZalpha. To construct the single-chain fold-back diabody isoform, it is required to build a modified scFv (1567) with a short linker (one G4S) between VL and VH portions. The VL portion was amplified using PCR primers CCR4-Nco þ BamCCR4 carrying BamHI site and digested using BamHI. The VH portion was amplified using PCR primers BgL-CCR4 carrying BglII site þ CCR4-Bam1 and digested using Bgl II. The BamHIdigested VL portion and Bgl II-digested VH portion were ligated together for 4 h at room temperature as PCR template to amplify the scFv (1567) with short linker (one G4S) for constructing the single-chain fold-back diabody isoform following the construction procedure for DT390-BiscFv (1567). All PCR primers that were used are listed in Table 1.

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Protein expression in yeast Pichia Pastoris and subsequent purifications were performed as previously described (Wang et al., 2011; Peraino et al., 2013a). Western blot analysis, binding and blocking analysis by flow cytometry were all performed as previously described (Peraino et al., 2013a) using a

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human CCR4þ acute lymphoblastic leukemia cell line CCRFCEM (ATCC CCL-119). DT390 was used as negative controls for in vitro functional analysis. It was also expressed in the yeast Pichia Pastoris system in our lab. Protein synthesis inhibition and cell proliferation inhibition were performed as

Figure 3 e SDS PAGE, Western blot and HPLC analysis of the anti-human CCR4 immunotoxins. A) SDS PAGE analysis (4e12% NuPAGE, Invitrogen); B) Western blot analysis using a mouse anti-His mAb (clone#: 4A12E4, Invitrogen); C) Western blot analysis using a mouse antidiphtheria toxin mAb (clone# 3B6, Meridian). Lane 1: Protein marker; Lane 2e3: monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567), 70.26 kDa]; Lane 4e5: Bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567), 97.57 kDa]; Lane 5e6: single-chain foldback diabody anti-human CCR4 immunotoxin (96.31 kDa). DeF) HPLC analysis with Shimadzu HPLC system using Superdex 200 size-exclusion column, 10/300 GL (GE healthcare, Cat#: 17-5175-01): D) monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567)]; E) bivalent antihuman CCR4 immunotoxin [DT390-BiscFv (1567)]; F) single-chain fold-back diabbody anti-human CCR4 immunotoxin.

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described previously (Peraino et al., 2014). Using BiscFv (1567)human Fc anti-human CCR4 antibody as inhibitor to block the protein synthesis inhibition and cell proliferation inhibition of the anti-human CCR4 immunotoxins was also performed as described by Peraino et al. (2014). Isolation of human PBMC, in vitro binding and depletion analysis of the anti-human CCR4 immunotoxins to human CCR4 on PBMC using flow cytometry was performed as previously described (Peraino et al., 2013b, 2014).

2.3.

HPLC analysis

Anti-human CCR4 immunotoxins were analyzed with Shimadzu HPLC system using Superdex 200 size-exclusion column, 10/300 GL (GE healthcare, Cat#: 17-5175-01). The sample volume was 100 ml using 100 ml loop. The flow rate was 0.35 ml/min. The running time was 120 min and the running buffer was 90 mM Na2SO4, 10 mM Na3PO4, pH 8.0, 1 mM EDTA.

2.4.

In vivo efficacy study

A breeding pair of NSG mice were purchased from Jackson laboratories and bred in our rodent barrier facilities for use in this study. All animal care procedures and experiments were approved by the Institutional Animal Care and Use

Committee (IACUC) of Massachusetts General Hospital (MGH). MGH is an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recognized research institution. The NSG mice were divided into one control group and three experimental groups: 1) C21 immunotoxin control group (a non-related diphtheria toxin-based immunotoxin as negative control) (n ¼ 7); 2) monovalent anti-human CCR4 immunotoxin group (n ¼ 7); 3) bivalent anti-human CCR4 immunotoxin group (n ¼ 8); 4) single-chain foldback diabody anti-human CCR4 immunotoxin group (n ¼ 7). All animals were IV injected with 10 million of human CCR4þ acute lymphoblastic leukemia cells (CCRF-CEM) via the tail vein. The immunotoxin was IP injected from day 0 on at 50 mg/kg, BID for 4 consecutive days as one course, two course total, and 3-day break between the two courses. The injected animals were observed daily for signs and symptoms of illness and scored weekly based on the parameters as previously reported by our lab (Peraino et al., 2013b): respiratory effort (0e3), weight loss/gain (0e2), fur integrity (0e3), provoked (0e3) and non-provoked activity (0e1), posture (0e3), abdominal distention (0e3), abdominal palpation (0e3) and body condition score (0e3). The highest score in each category represents the worst possible condition for that parameter. The highest possible score on the scoring system is a 24. The animals were humanely euthanized after a score of 12 or

Figure 4 e A) Binding analysis using flow cytometry of biotinylated monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567)] (left panel), bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567)] (middle panel), single-chain fold-back diabody anti-human CCR4 immunotoxin (right panel) to human CCR4D CCRF-CEM cells (acute lymphoblastic leukemia cell line). Cells incubated with only the secondary staining (PE-conjugated streptavidin) served as a negative control and human CCR4 fluorescein mAb (clone#205410, R&D systems, cat# FAB1567F) for the positive control, mouse IgG2B fluorescein for the isotype control. Biotin-labeled porcine CD3-εg (Peraino et al., 2012) was included as a negative control for background due to protein biotinylation. The data are representative of three individual experiments. B) KD Determination Using Flow Cytometry and Nonlinear Least Squares Fit. MFI was plotted over a wide range of concentrations of biotinylated 1) DT390-scFv (1567) (red curve); 2) DT390-BiscFv (1567) (blue curve) and 3) single-chain fold-back diabody anti-human CCR4 immunotoxin (green curve). The accompanying least-squares fits are shown based on the hyperbolic equation y [ m1 D m2 * m0/(m3 D m0) where y [ MFI at the given biotinylated anti-human CCR4 immunotoxin concentration, m0 [ biotinylated anti-human CCR4 immunotoxin concentration, m1 [ MFI of zero biotinylated anti-human CCR4 immunotoxin control, m2 [ MFI at saturation and m3 [ KD. A fitted KD of 5.66 nM was obtained for DT390-scFv (1567), 1.67 nM for DT390-BiscFv (1567) and 0.74 nM for single-chain fold-back diabody anti-human CCR4 immunotoxin.

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et al., 2011). A 6 His tag was added to the C-terminus of each immunotoxin construct to facilitate the downstream purification. The DT390 domain was genetically linked to the scFv (1567) domain by a linker containing four glycine residues and a serine residue (G4S). For the monovalent and bivalent version, the VL and VH were linked together by three tandom G4S linker (G4S)3 to generate the scFv (1567). For the singlechain fold-back diabody isoform, the VL and VH were linked by one G4S linker. The two scFv (1567) which make up the bivalent and the single-chain fold-back diabody isoform were also joined by three tandem G4S linkers (G4S)3. The anti-human CCR4 immunotoxins were expressed in a unique diphtheria-toxin resistant yeast Pichia Pastoris (Liu et al., 2003) expression system using 1 L Erlenmeyer flasks. The anti-human CCR4 immunotoxins were secreted into the extracellular supernatant then captured using a Nisepharose fast flow resin and further purified using strong anion exchange resin. The final purification yield was w25 mg per liter for the monovalent and w5 mg per liter for the bivalent and single-chain fold-back diabody anti-human CCR4 immunotoxins of the original harvested supernatant. The purified anti-human CCR4 immunotoxins were analyzed by SDS-PAGE (Figure 3A) and Western blot using a mouse anti-His monoclonal antibody (Figure 3B) and a mouse antidiphtheria toxin monoclonal antibody (Figure 3C) as well as HPLC analysis (Figure 3DeF). SDS PAGE, Western blot and HPLC analysis demonstrated that the three versions of the anti-human CCR4 immunotoxins were successfully expressed and purified with expected molecular weight of 70.26 kDa for the monovalent isoform, 97.57 kDa for the bivalent isoform and 96.31 kDa for the single-chain fold-back diabody isoform.

higher or when an animal lost more than 15% of its preinjection body weight. However this score system was actually almost not applied as the CCR4þ ALL is very aggressive to result in the animal death or euthanized within very short period of time. It was less than or about one day from completely normal to dead. To assess whether the immunotoxin alone is toxic to the experimental animals, mice (n ¼ 2) injected with the bivalent or single-chain fold-back diabody immunotoxin only (without tumor cells) were also included as controls.

2.5.

Statistical analysis

All P values were calculated using two-way ANOVA or Logrank (ManteleCox) Test of Prism. P < 0.05 was considered as significant. EC50 was determined using nonlinear regression (curve fit) of Prism.

3.

Results

3.1. Expression of the anti-human CCR4 immunotoxins using yeast Pichia Pastoris As shown in Figure 1, monovalent, bivalent and single-chain foldback diabody anti-human CCR4 immunotoxins were constructed so as to find a best isoform for in vivo depleting human CCR4þ cells. The codon-optimized scFv (1567) DNA (Figure 2) was cloned into the C-terminus of the DT390containing yeast Pichia Pastoris expression vector pwPICZalpha-DT390 between the NcoI and EcoRI sites (Wang

DT390-scFv (1567)

B

DT390-BiscFv (1567) Fold-back Diabody 9 1 0 7 1 0 5 1 1

0

b e r

1

0 8

0 9

0

Nu m

0 9 0 8 0 7

0 7

0 6

1

1 0

2

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1 0

4

1 0

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cont r ol - - >

2

1 0

3

1 0

2

1 0

3

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4

0

1 0

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Mouse IgG2B fluorescein isotype control, 20 nM

1 0 5 1

3

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1 0

1 0 1 0 0 7 0 5 0 3 0

1

si o t y p e c o n t r o l - - >

1 0

2

1 0

3

1

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1 0

4

si o t y p e c o n t r o l - - >

1

0

0

b e r

4

0

100

DT390-scFv(1567) DT390-BiscFv(1567) Fold-back Diabody

2 0 1

0

1

0

1

2

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2

3

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3

4

0

0

4

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0

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6

6 5

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7

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0

b e r

7

8

0

0

8

9

0

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9

1 0 9 0 8 0 7 0

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6 0 5

Nu m

0 4 0 3 0

1 0

Human CCR4 fluorescein mAb, 20 nM

0

1

0

0

4

0

0

0

0

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si o t y p e c o n t r o l - - >

0

1

0

0

1

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7

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03

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03

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1

0

1 0

1 0

Cell only control

1

0

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b e r

1 0 0 9 0 8

Nu m

1

1

3

5

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7

0

0

1

1

7

9

0

1

9

A

1 0

2

1 0

3

1 0

3

1 0

a n t i h CCR 4 - CF S - - >

1 0

2

1 0

3

1 0

4 0

1

4

1 0

1

1 0

1

a n t i h CCR 4 - CF S - - >

1 0

2

1 0

3

1 0

4

1 0

2

1 0

3

1 0

4

07 6

0 4

0 0 4

0 3

03

0 2

0 2

1

1 0

2

1 0

3

1 0

4

0

1

0 0 9

1

a n t i h CCR4 - CF S + Z W 2 0 0 1 7 0 n M - - >

a n t i h CCR4 - CF S + Z W 2 1 0 1 7 0 n M - - >

1

4

9 0 7 1

1 0 8

1 0 9

09

Nu m

0

1

0

1

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1 1

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3

0

0

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9 0 8

b e r

0 7 0 6

7 5 0 1

1 0

1

1 0

2

1 0

3

1 0

1

a n t i h CCR4 - CF S + Z W 2 0 0 8 5 n M - - >

4

1 0

2

1 0

3

1 0

4

1 0

1 0

2

1 0

3

1 0

4

1 0 7

4 1

43 nM

0 0 1 0 1

7

1 0

2

1 0

3

1 0

4 6

1 0 0 0

40

0

20

1

1

1 0

2

1 0

3

1 0

4

1 0

1

a n t i h CCR4 - CF S + Z W 2 1 0 4 3 n M - - >

1 0

2

1 0

3

1 0

4

8

0

1

4

21 nM

0

6

2

1 1

0

0

4

1

0

0 1 1

0

0

1

1

0 1

60

0

1 0

a n t i h CCR4 - CF S + Z W 2 0 0 4 3 n M - - >

2 1 0 1 0 0

0

0 4 3 0

2

1 0

3

1 0

4 1

1 0

1

1 0

2

1 0

3

1 0

4

1 0

2

1 0

3

1 0

4

2

1

1

0

1

2

01

0

0

1

1

1

4

1 0

a n t i h CCR4 - CF S + Z W 2 1 0 2 1 n M - - >

85

1 0

0 9 0

Immuntoxin Conc. (nM)

5 0 0 0

0

0

3

3

3

0

0

4

4

0

4

5

0

0

0

5

6

6

0

7

0

Nu m

0

7

0

Nu m

8

8

9

b e r

0

b e r

0

8 0 0 6 0

Nu m

7

b e r

0

1

1

0

9

0

0

0

0

11 nM

0

0

1

1

0

0

2

2

2 0 1 0 0 3

1 0

1 0

4

1

a n t i h CCR4 - CF S + Z W 2 0 0 1 0 n M - - >

1 0

2

1 0

3

1 0

4

a n t i h CCR4 - CF S + Z W 2 1 0 1 0 n M - - > 2 1

0 0

0

0

1

1

1

0

0

0

1

1 0

2

1 0

3

1 0

4

6

0

7

Nu m

0

0

1

1

0

0

2

2

0

0

3

3

0

0

4

4

0

0

5

5

0

0

6

0

7

Nu m

0

0

8

0

b e r

8

0

b e r

9

9

9 0 8

b e r

0 7 0 6

Nu m

0 5 0 4 0 3 0 2 0 1 0

1 0

a n t i h CCR4 - CF S + Z W 1 9 3 5 n M - - >

5 nM

0

0 1

1

1

1 0

2

2

0

0

0 1

a n t i h CCR4 - CF S + Z W 1 9 3 1 0 n M - - >

17 0

1

a n t i h CCR4 - CF S + Z W 2 0 0 2 1 n M - - >

42

3

1 0

4

21

1 0

1 0

11

2

3

5

1 0

1 0

0

0

1

2

0

1 0

1 0

4

1

0

1 0

a n t i h CCR4 - CF S + Z W 1 9 3 2 1 n M - - >

01

2

0

0

0

1

1

1

0

0

2

2

0

0

3

3

0

0

0

4

6

0

4

5

0

0

5

6

8

0

7

0

8

Nu m

0

Nu m

0

9

0 1

b e r

0

0

b e r

9 0 8

b e r

0 7 0 6 0

Nu m

80

0

1

1

1 0

a n t i h CCR4 - CF S + Z W 1 9 3 4 3 n M - - >

2

0 1

3

0

0

0

0

1

1

0

0

3

2

2

0

0

0

3

5

0

3

4

0

0

5

7

4

0

0

6

0

9

0

8

0

Nu m

9

Nu m

3

b e r

0 0 0 1

0 7 0 6 0 5

Nu m

b e r

8

b e r

0

1

1

5

2

9

0

0

1

0 1

0

01

9

1

1

0

6

0

1

a n t i h CCR4 - CF S + Z W 2 1 0 8 5 n M - - >

0

1 0

a n t i h CCR4 - CF S + Z W 1 9 3 8 5 n M - - >

0

0

0

1

0

1

0

3

0 2

2

0

0 3

3

0 4

0

0

0 5

4

0

0 6

0

5

07

0

Nu m

85 nM

1

0

3

5

1

0

0

0

1

0 1

5

1

0

1

1

0 1

7

0

0

0 3

1 0

Blocking Rate%

0 6 1 0 4 0 1

2

b e r

0 08

0

1

0

Nu m

0 9 0 8 0 7 0 6 0 5

0 4 0 3 0 2 0 1 0

1 0

a n t i h CCR4 - CF S + Z W 1 9 3 1 7 0 n M - - >

2

0 1

170 nM

1

0 1 1 0

Nu m

8 0 0 6 0 5

Nu m

7

b e r

0

b e r

9

0

1

1

3

0

0

01

1

1

1

5

8

0 1

0

0

2

1

2

0

2

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1

1 0

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1 0

a n t i h CCR 4 - CF S - - >

7

1

0

0

1 0 1 0

1 0

1

a n t i h CCR 4 - CF S + Z W 2 0 0 5 n M - - >

1 0

2

1 0

3

1 0

4

1 0

1

a n t i h CCR 4 - CF S + Z W 2 1 0 5 n M - - >

1 0

2

1 0

3

1 0

4

Figure 5 e Blocking analysis of the anti-human CCR4 immunotoxins for the human CCR4 receptor on human CCR4D CCRF-CEM leukemia cells. A) Flow cytometry histogram: unlabeled anti-human CCR4 immunotoxins were each incubated with CCRF-CEM cells at a range of concentrations for 15 min at 4  C in the dark. Subsequently, without washing the cells, anti-human CCR4 mAb 1567 was added to each tube containing cells in the presence of the unlabeled immunotoxins. Binding affinity of the anti-human CCR4 immunotoxins to the human CCR4 receptor on CCRF-CEM cells was measured by a decrease in anti-human CCR4 mAb staining in the presence of increasing concentrations of the unlabeled immunotoxins. Murine IgG2B fluorescein was included as an isotype control. B) Blocking rate (%) was plotted versus the concentration of the binding competitor (monovalent, bivalent or single-chain fold-back diabody anti-human CCR4 immunotoxin). The relative binding affinity for any two competitors can be estimated from the ratio of their concentration at equal inhibition rate values or parallel curve regions. X-axis: immunotoxin concentration; Y-axis: blocking rate [ (MFI value of the positive control e MFI value of the sample)/(MFI value of the positive control e MFI value of the isotype control) 3 100%. P < 0.0001 by two-way ANOVA (n [ 3). Error bars indicate ±SD.

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3.2. Binding affinity analysis of the anti-human CCR4 immunotoxins to human CCR4 by flow cytometry Anti-human CCR4 immunotoxins target the human CCR4þ cells via binding of the anti-human CCR4 scFv (1567) domain of the immunotoxins. Following the cellular internalization, the DT390 domain functions to inhibit protein synthesis resulting in the cell death (Murphy, 2011). Therefore, the first critical step in determining the functionality of the antihuman CCR4 immunotoxins was to analyze their binding affinity for human CCR4. The anti-human CCR4 immunotoxins were labeled with sulfo-EZ-link NHS biotin (Thermo Scientific) for binding analysis to human CCR4þ CCRF-CEM leukemia

cells using flow cytometry. As shown in Figure 4A, monovalent (left panel), bivalent (middle panel) and single-chain fold-back diabody (right panel) anti-human CCR4 immunotoxins bound to the human CCR4 in a dose-dependent manner. The binding affinity was quantified by calculating the dissociation constant (Kd ) for each anti-human CCR4 immunotoxin isoform from mean fluorescence intensity (MFI) (Peraino et al., 2013a). Consistent with previously developed recombinant immunotoxins (Woo et al., 2002; Kim et al., 2007; Wang et al., 2011) the bivalent isoform (Kd ¼ 1.67 nM, Figure 4B) bound stronger than the monovalent isoform (Kd ¼ 5.66 nM, Figure 4B) and the foldback diabody isoform was found to have the highest binding affinity (Kd ¼ 0.74 nM, Figure 4B).

B

A Inhibtion Rate (%)

100

DT390 DT390-scFv(1567) DT390-BiscFv(1567) Fold-back Diabody

80 60 40 20

-8

×

10

10

-9

0

06 1.

1.

06

×

10

-1

1.

06

×

10

-1

1

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× 06 1.

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06

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-1

× 06

1.

/o

Im

m

un

ot

10

io

xi

3

n

0

(-)

w

Protein Conc. (M)

D DT390-BiscFv(1567) DT390-BiscFv(1567) + BiscFv(1567)-Human Fc

20000

15000

15000

5000

5000

0

0

ot ox im m un

1.

m un

(-)

Immunotoxin Conc. (M)

w /o

im w /o (-)

10000

in 06 ×1 0 -1 3 1. 06 ×1 0 -1 2 1. 06 ×1 0 -1 1 1. 06 ×1 0 -1 0 1. 06 ×1 0 -9 1. 06 ×1 0 -8

CPM

20000

10000

Foldback Diabody Foldback Diabody + BiscFv(1567)-Human Fc

25000

ot ox in 06 ×1 0 -1 3 1. 06 ×1 0 -1 2 1. 06 ×1 0 -1 1 1. 06 ×1 0 -1 0 1. 06 ×1 0 -9 1. 06 ×1 0 -8

CPM

25000

1.

C

Immunotoxin Conc. (M)

Figure 6 e A) Anti-human CCR4 immunotoxin-mediated protein synthesis inhibition in human CCR4D CCRF-CEM cells in vitro: 1) monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567), red line]; 2) bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567), green line]; 3) single-chain fold-back diabody anti-human CCR4 immunotoxin (orange line); 4) DT390 alone (blue line). Y-axis: inhibition rate of the protein synthesis via the cpm value measuring incorporation of tritiated leucine. X-axis: plated anti-human CCR4 immunotoxin concentration. Cycloheximide (1.25 mg/mL) was used as a positive control. The negative control contained cells without immunotoxin. P < 0.0001 by two-way ANOVA (n [ 3). Error bars indicate ±SD. BeD) Binding specificity analysis of the anti-human CCR4 immunotoxin to the target human CCR4D CCRF-CEM cells in this in vitro protein synthesis inhibition assay using BiscFv (1567)-human Fc (1.06 3 10L7 M) as inhibitor: B) Monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567)] with (purple) and without (blue) inhibitor; C) bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567)] with (orange) and without (green) inhibitor. D) single-chain fold-back diabody anti-human CCR4 immunotoxin with (red) and without (green) inhibitor. Y-axis: cpm value measuring incorporation of tritiated leucine. X-axis: plated anti-human CCR4 immunotoxin concentration. Wells containing the inhibitor were incubated for 1 h at 37  C before addition of the immunotoxin. Cycloheximide (1.25 mg/mL) was used as a positive control. Cells without immunotoxin served as the negative control. p < 0.0001 by two-way ANOVA (n [ 3). Error bars indicate ±SD. Data are representative of multiple assays.

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The binding specificity of the anti-human CCR4 immunotoxins were further analyzed by blocking the binding of the parent anti-human CCR4 mAb 1567 to its receptor on human CCR4þ CCRF-CEM leukemia cells. As shown in Figure 5A, monovalent (left panel), bivalent (middle panel) and singlechain fold-back diabody (right panel) anti-human CCR4 immunotoxins blocked the binding of anti-human CCR4 mAb 1567 to the cells in a dose-dependent fashion, which suggests that the anti-human CCR4 immunotoxins binding specifically to the human CCR4 receptor. The bivalent isoform blocked the binding about 10 times more strongly than the

A

monovalent version and the single-chain fold-back diabody is about 3 times stronger than the bivalent isoform (Figure 5B). To rule out the off-target effect of the immuntoxin, we have analyzed three irrelevant non-human CCR4-expressing tumor cell lines with biotin-labeled foldback diabody antihuman CCR4 immunotoxin: 1) MV3; 2) M14; 3) MD-MBA-231. The results demonstrated that there is no any binding activity with these non-human CCR4-expressing tumor cell lines (data not shown). We have also analyzed the viability of these three non-human CCR4-expressing tumor cell lines by flow cytometry using propidium iodide and Annexin V following DT390-scFv(1567) DT390-scFv(1567) + BiscFv(1567)-Human Fc

B 80 60

300000

CPM

40

200000 100000

20 0

(-)

-8

-9

1.

1.

06

06

×

×

10

10

-1 0

-1 1

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1.

06

06 1.

(-)

w

w /o

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× 06

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ot

06

im m un

m un Im

10

ox

in

ot io 1. xi 06 n × 10 13 1. 06 × 10 12 1. 06 × 10 11 1. 06 × 10 10 1. 06 × 10 9 1. 06 × 10 -

8

0

1.

Inhibtion Rate (%)

400000

DT390 DT390-scFv(1567) DT390-BiscFv(1567) Fold-back Diabdoy

100

Protein Conc.(M)

D

300000

300000

-8

-9

10 ×

06 1.

06

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10

-1 0

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1.

06

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10

-9

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10

-1 0

06 1.

×

×

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10

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10

-1 3

× 06 1.

06

×

10

xi ot o 1.

m un im /o

×

0

1.

0 -8

100000

-1 1

100000

-1 1

200000

06

CPM

400000

200000

Foldback Diabody Foldback Diabody + BiscFv(1567)-Human Fc

500000

400000

n

CPM

500000

1.

DT390-BiscFv(1567) DT390-BiscFv(1567) + BiscFv(1567)-Human Fc

1.

C

Immunotoxin Conc. (M)

Immunotoxin Conc. (M)

Immunotoxin Conc. (M)

Figure 7 e A) Anti-human CCR4 immunotoxin-mediated cellular proliferation inhibition in human CCR4D CCRF-CEM cells in vitro: 1) monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567), red line]; 2) bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567), green line]; 3) single-chain fold-back diabody anti-human CCR4 immunotoxin (orange line); 4) DT390 (blue line). Y-axis: inhibition rate of the cell proliferation via the cpm value measuring incorporation of tritiated thymidine. X-axis: plated immunotoxin concentration. Cycloheximide (1.25 mg/mL) was used as a positive control. The negative control contained cells without immunotoxin. P < 0.0001 by two-way ANOVA (n [ 3). Error bars indicate ±SD. BeD) Binding specificity analysis of the anti-human CCR4 immunotoxin to the target human CCR4D CCRFCEM cells during this in vitro cellular proliferation inhibition assay using BiscFv (1567)-human Fc (1.06 3 10L7 M) as inhibitor: B) Monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567)] with (red) and without (blue) inhibitor; C) bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567)] with (orange) and without (green) inhibitor. D) single-chain fold-back diabody anti-human CCR4 immunotoxin with (red) and without (green) inhibitor. Y-axis: cpm value measuring incorporation of tritiated thymidine. X-axis: plated anti-human CCR4 immunotoxin concentration. Wells containing the inhibitor were incubated for 1 h at 37  C before addition of the immunotoxin. Cycloheximide (1.25 mg/mL) was used as a positive control. Cells without immunotoxin served as the negative control. p < 0.0001 by two-way ANOVA (n [ 3). Error bars indicate ±SD. Data are representative of multiple assays.

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Figure 8 e In vitro binding and depletion analysis of the anti-human CCR4 immunotoxins to human CCR4D PBMC. A) Flow cytometry binding analysis of the anti-human CCR4 immunotoxins to the CCR4D cells within human PBMC. Human PBMC was stained with the biotinylated anti-human CCR4 immunotoxin as primary staining and PE-conjugated streptavidin as second staining. First panel: monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567)]; second panel: bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567)]; third panel: singlechain foldback diabody anti-human CCR4 immunotoxin; fourth panel: anti-human CCR4 antibody [BiscFv (1567)-human Fc]. Human PBMC with only the secondary staining (PE-conjugated streptavidin) served as the negative control and human CCR4 fluorescein mAb (clone#205410, R&D systems, cat# FAB1567F) for the positive control, mouse IgG2B fluorescein for the isotype control. Biotin-labeled porcine CD3-εg (Peraino et al., 2012) was included as a negative control for background due to protein biotinylation. The data are representative of three individual experiments. B) In vitro depletion of the CCR4D cells within human PBMC using the anti-human CCR4 immunotoxins. Human PBMC was incubated with the unlabeled anti-human CCR4 immunotoxin at 37  C for 48 h and analyzed by flow cytometry using biotinylated anti-human CCR4 antibody [BiscFv (1567)-human Fc] as primary staining and PE-conjugated streptavidin as second staining. Left panel: monovalent antihuman CCR4 immunotoxin [DT390-scFv (1567)]; middle panel: bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567)]; right panel: single-chain foldback diabody anti-human CCR4 immunotoxin. Human PBMC with only the secondary staining (PE-conjugated streptavidin)

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 4 5 8 e1 4 7 0

incubation with the foldback diabody anti-human CCR4 immunotoxin for 18 h. Their viabilities were not affected or minimally affected (data not shown).

3.3. In vitro protein synthesis inhibition analysis of the anti-human CCR4 immunotoxins The immunotoxin efficacy was assessed in vitro using protein synthesis inhibition assay through incorporating the tritiated leucine. As shown in Figure 6A, all three versions (monovalent, bivalent and single-chain fold-back diabody) of antihuman CCR4 immunotoxins are potent to inhibit the protein synthesis of human CCR4þ CCRF-CEM leukemia cells. The bivalent version (EC50 ¼ 1.53  1011 M) is about 40 folds stronger than its monovalent counterpart (EC50 ¼ 6.67  1010 M). The single-chain fold-back diabody format (EC50 ¼ 9.42  1013 M) is about 16 folds stronger than its bivalent counterpart and about 700 folds stronger than its monovalent isoform. The bivalent and fold-back diabody strategies significantly improved the efficacy as other bivalent and fold-back diabody recombinant immunotoxins (Woo et al., 2002; Kim et al., 2007; Wang et al., 2011). In order to confirm that the human CCR4þ CCR-FEM leukemia cells are being targeted specifically through the interaction of the anti-human CCR4 scFv (1567) domain on the immunotoxins and the human CCR4 receptor on the cell surface, we assessed the immunotoxins’ ability to halt protein synthesis in the presence of the anti-human CCR4 antibody [BiscFv (1567)-human Fc]. Target cells that were incubated with immunotoxin in the presence of the anti-human CCR4 antibody [BiscFv (1567)-human Fc] showed a marked increase in protein synthesis compared to cells which were cultured with the corresponding concentration of immunotoxin only. The anti-human CCR4 antibody [BiscFv (1567)-human Fc] acted as an inhibitor of immunotoxin as it prevented the monovalent (Figure 6B), bivalent (Figure 6C) and the foldback diabody (Figure 6D) anti-human CCR4 immunotoxins from targeting the human CCR4þ cells.

3.4. In vitro cell proliferation inhibition analysis of the anti-human CCR4 immunotoxins The potency of the anti-human CCR4 immunotoxins was further in vitro assessed using cell proliferation inhibition assay in DNA level through incorporating the tritiated thymidine. As shown in Figure 7A, all three versions of the antihuman CCR4 immunotoxins are potent in inhibiting the

1467

proliferation of the target cells and the bivalent isoform is about 20 folds more effective than the monovalent isoform and the fold-back diabody isoform is about 10 folds more effective than the bivalent isoform and about 200 folds more effective than the monovalent isoform, which is consistent with the previous protein synthesis inhibition analysis. Again, to double confirm the anti-human CCR4 immunotoxins bound to the target cells via interaction of the cell surface human CCR4 receptor with the anti-human CCR4 scFv (1567) domain of the immunotoxins in this cell proliferation inhibition assay, we observed the ability of the anti-human CCR4 immunotoxins to inhibit the cellular proliferation in the presence of human CCR4 inhibitor, BiscFv (1567)-human Fc. Consistently, BiscFv (1567)-human Fc drastically affected the ability of the anti-human CCR4 immunotoxins to obstruct cellular proliferation in target cells (Figure 7BeD).

3.5. In vitro binding and depletion analysis of the antihuman CCR4 immunotoxins to human PBMC To further characterize the anti-human CCR4 immunotoxins, we performed the in vitro binding and depletion analysis of the immunotoxins to human PBMC. As shown in Figure 8A, three versions of the biotinylated anti-human CCR4 immunotoxins bound to CCR4þ human PBMC in a dose-dependent fashion. The bivalent isoform bound stronger than the monovalent isoform and the fold-back diabody isoform is the best, which are consistent with the previous binding analysis using the human CCR4þ CCRF-CEM leukemia cell line. Based on this positive binding data, we further performed the in vitro depletion assay to the CCR4þ human PBMC using the immunotoxins. As shown in Figure 8B, CCR4þ human PBMC was in vitro depleted in a dose dependent manner. The bivalent and fold-back diabody isoforms are better than the monovalent version and the fold-back diabody version is the best for depleting CCR4þ human PBMC. The depletion profile was double confirmed using another anti-human CCR4 mAb, PE-antihuman 194 (CCR4) mAb (Clone# L291H4, Biolegend) by flow cytometry (data not shown). One of the main expected applications of this immunotoxin is to specifically deplete CCR4þ Tregs in vivo. Therefore we further analyzed the binding of the immunotoxins to the CCR4þ Foxp3þ human PBMC. As shown in Figure 8C, the immunotoxins bound to the CCR4þFoxp3þ Tregs within human PBMC also in a dose dependent manner. The bivalent isoform bound stronger than the monovalent isoform and the foldback diabody isoform is the best.

served as the negative control and anti-human CCR4 antibody [BiscFv (1567)-human Fc] for the positive control. Biotin-labeled porcine CD3-εg (Peraino et al., 2012) was included as a negative control for background due to protein biotinylation. The data are representative of three individual experiments. C) Flow cytometry binding analysis of the anti-human CCR4 immunotoxins to the Foxp3DCCR4D human PBMC. Human PBMC was stained with Alexa Fluor 647 anti-human Foxp3 mAb (clone# 150D, Biolegend, cat# 320014) and biotinylated anti-human CCR4 immunotoxin. First panel: monovalent anti-human CCR4 immunotoxin [DT390-scFv (1567)]; second panel: bivalent anti-human CCR4 immunotoxin [DT390-BiscFv (1567)]; third panel: single-chain foldback diabody anti-human CCR4 immunotoxin; fourth panel: anti-human CCR4 antibody [BiscFv (1567)-human Fc]. Human PBMC with only the secondary staining (PE-conjugated streptavidin) served as the negative control and anti-human CCR4 antibody [BiscFv (1567)-human Fc] for the CCR4 positive control, Alexa Fluor 647 anti-human Foxp3 mAb for the Foxp3 positive control, Alex Fluor 647 Mouse IgG1 k (clone# MOPC-21, Biolegend, cat# 400136) for the isotype control of Alexa Fluor 647 anti-human Foxp3 mAb. Biotin-labeled porcine CD3-εg (Peraino et al., 2012) was included as a negative control for background due to protein biotinylation. The data are representative of three individual experiments.

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3.6. In vivo efficacy assessment of the anti-human CCR4 immunotoxins using a CCR4þ tumor-bearing NSG mouse model Human CCR4þ CCRF-CEM tumor-bearing NSG mouse model was used to assess the in vivo efficacy of the anti-human CCR4 immunotoxins. NSG mice were IV injected with 1  107 human CCR4þ CCRF-CEM tumor cells and treated (IP) with the anti-human CCR4 immunotoxin at 50 mg/kg BID for 4 consecutive days as one course, two course total, three day break between the two courses. This dosing schedule was based on our previous experience (Peraino et al., 2013a, 2013b) and given this CCR4þ ALL is extremely aggressive. As shown in Figure 9, C21 immunotoxin (a non-related DT390 based immunotoxin) was injected as negative control (n ¼ 7). Both bivalent and single-chain fold-back diabody anti-human CCR4 immunotoxins significantly prolonged the animal survival from median 20 days of the negative control to 30 days using the bivalent version (n ¼ 8) and 32 days using the fold-back diabody version (n ¼ 7). The bivalent version was significantly more effective (p < 0.0001) than the negative control. The fold-back diabody version is even more effective than the bivalent version (p ¼ 0.0057). The monovalent version did not prolong the animal survival with median survival time of 19 days using this dosing schedule. Mice received the antihuman CCR4 immunotoxin alone did not show any evidence of toxicity (data not shown). All animals that were injected with the human CCR4þ CCRF-CEM tumor cells succumbed to tumors, demonstrated by growth pathology and histopathology (data not shown). Human CCR4þ CCRF-CEM ALL was very aggressive to result in the animal death with only median 20 days, which speeded up our entire in vivo efficacy assessment.

4.

Discussion

We have successfully developed three versions (monovalent, bivalent and single-chain fold-back diabody) of a diphtheria toxin-based, anti-human CCR4 immunotoxins for targeting human CCR4þ cells in vivo. In vitro and in vivo efficacy characterization using a human CCR4þ ALL cell line demonstrated that the bivalent and fold-back diabody versions are promising drug candidates. The potential applications include: 1) directly depleting human CCR4þ tumor cells such as T-cell ALL, ATL, PTCL and CTCL; 2) depleting CCR4þ Tregs for combined cancer treatment; 3) treatment of allergic diseases such as asthma through depleting CCR4þ Th2 cells. In vivo efficacy analysis in this study demonstrated that the bivalent and single-chain fold-back diabody anti-human CCR4

100

Percent Survival

To further rule out the off-target effect of the immunotoxin, we have also analyzed other human PBMC populations following incubation with the foldback diabody anti-human CCR4 immunotoxin for 48 h: CD8 T cell (CD3þCD8þ), B cell (CD19þ), NK cells (CD16þCD8þ) and monocyte (CD14þCD16þ). The data demonstrated that there was no effect on other human PBMC populations (data not shown). In contrast, as expected, CD4 T cell (CD3þCD4þ) was depleted in a dosedependent manner as most of CCR4þ cells belong to this sub-population (data not shown).

C21 Immunotoxin Control Monovalent Bivalent Foldback Diabody

80 60 40 20 0

0

5

10

15

20

25

30

35

Survival Days Immunotoxin (IP, 50 μg/kg) Tumor Cells (CCR4+ ALL) (IV, 1 x107 )

Figure 9 e In vivo efficacy analysis of the anti-human CCR4 immunotoxins. NSG mice were IV injected with 1.0 3 107 human CCR4D CCRF-CEM leukemia cells and treated from day 0 on with the anti-human CCR4 immunotoxin at 50 mg/kg BID for 4 consecutive days as one course, two course total and 3 day break between the two courses. 1) C21 immunotoxin control group (a nonrelated diphtheria toxin-based immunotoxin as negative control) (n [ 7, red curve) with a median survival time of 20 days; 2) monovalent anti-human CCR4 immunotoxin group (n [ 7, green curve) with a median survival time of 19 days; 3) bivalent anti-human CCR4 immunotoxin group (n [ 8, black curve) with a median survival time of 30 days; 4) single-chain foldback diabody anti-human CCR4 immunotoxin group (n [ 7, purple curve) with a median survival time of 32 days. The schedule of drug and tumor cell injection is pictured in the schematic below the survival curve. The vertical arrows indicate the days on which the tumor cells or antihuman CCR4 immunotoxins were injected.

immunotoxins are effective for depleting human CCR4þ ALL. However this dosing schedule did not completely eradicate the tumor cells in vivo. In order to improve the in vivo efficacy, it will probably be necessary to optimize the dosing schedule as well as use a combined approach with other chemotherapy agents or specific signal pathway inhibitors. It is also needed to test this immunotoxin under therapeutic set-up such as in mouse model with established tumor. Recently some groups have been exploring alternative approach to target CCR4 receptor particularly small molecule CCR4 antagonists as vaccine adjuvants to block the interaction between CCR4þ Tregs and DCs (dendritic cells) to improve the vaccines’ cellular and humoral immune response. Mature and activated DCs secrete CCL17 and CCL22 in lymphoid and non-lymphoid tissues to recruit CCR4þ Tregs towards DCs to suppress DC-mediated immune response via inhibiting DC maturation and expression of co-stimulatory molecules, which are required to activate effective T cell, as well as inhibiting stable contact between DCs and effector cells. The CCR4 antagonists blocked these negative regulations and enhanced the DC-mediated immune response. Their data demonstrated that CCR4 receptor is a promising vaccine adjuvant target (Davies et al., 2009; Bayry et al., 2008). Combined with vaccines CCR4 antagonists inhibited the Tregs, induced antigenspecific anti-self CD8þ T cells and inhibited the tumor growth (Pere et al., 2011). CCR4 antagonists also blocked the interaction between DC-derived CCL22 and steady state migratory DC-induced iTregs in cutaneous lymph nodes (Vitali et al., 2012).

M O L E C U L A R O N C O L O G Y 9 ( 2 0 1 5 ) 1 4 5 8 e1 4 7 0

Targeting Treg through the high affinity IL-2Ra chain, CD25 using IL-2 fusion toxins such as Denileukin diftitox or our recently developed bivalent porcine IL-2 fusion toxin (Peraino et al., 2013b) results in depletion of CD25þ activated effector T cells too. NK cells were also depleted by IL-2 fusion toxin since IL-2Rb (CD122) and the common g-chain (CD132) are expressed on NK cells (Yamada et al., 2012 and our unpublished data). This specificity problem contributed to our desire to develop an anti-human CCR4 immunotoxin that depleted Tregs in vivo more specifically. Of note, CCR4 is also expressed on Th2 cells (T-helper type 2) (Honjo et al., 2013). Therefore, future studies will determine the in vivo effects of the CCR4 immunotoxin. Further specificity may be achieved base on the report that CCL17 and CCL22, which are conformationselective ligands of the CCR4 receptor, interact with their unique CCR4 receptor using different mechanism (Viney et al., 2014). The diphtheria toxin based CCL17 and CCL22 fusion toxins are under construction. We expect that the CCL17 fusion toxin will mainly target Th2 cells and CCL22 fusion toxin, mainly target Tregs.

Acknowledgments We would like to thank Angimmune LLC for kindly providing the diphtheria toxin resistant yeast Pichia Pastoris strain and the codon-optimized DT390 DNA. We would like to thank Ruan Zhang for his excellent technical help on in vivo tumor-bearing mouse model. R E F E R E N C E S

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