The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T cell ...

37 Pages·2014·2.31 MB·English

Author Manuscript Published OnlineFirst on October 14, 2014; DOI: 10.1158/1078-0432.CCR-14-0913
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and
generates T cell dependent tumor immunity

Sara M Mangsbo3, Sissela Broos2, Erika Fletcher3, Niina Veitonmäki1, Christina Furebring1,
Eva Dahlén1, Per Norlén1, Malin Lindstedt2, Thomas H Tötterman3, and Peter Ellmark1,2†
1Alligator Bioscience AB, Lund, Sweden
2Department of Immunotechnology, Lund University, Lund, Sweden
3Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
†Correspondence to: Peter Ellmark
Email: [email protected]
Alligator Bioscience AB, Medicon Village, 223 81 Lund, Sweden
Phone: [phone]
Running title: ADC-1013, a CD40 agonist for local immunotherapy of cancer
Keywords: Immunomodulatory antibody; CD40 agonist; Immunotherapy of cancer; Local
immunotherapy; Therapeutic antibody
Financial support: This study was sponsored by Alligator Bioscience AB and in part
supported by the Swedish research council (VR-IFA 621-2008-5903).
Potential conflicts of interest: Christina Furebring, Eva Dahlén, Per Norlén, Niina
Veitonmäki and Peter Ellmark are current employees of Alligator Bioscience AB. Thomas
Tötterman has received research funding from and is a consultant to Alligator Bioscience AB.
Word count: 5400 (Abstract, Introduction, Results, Discussion and Acknowledgments).
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Author Manuscript Published OnlineFirst on October 14, 2014; DOI: 10.1158/1078-0432.CCR-14-0913
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Translational relevance
Local administration of immune activating antibodies has the potential to increase the
efficacy and reduce the immune-related adverse events associated with systemic
immunotherapy of cancer. We have generated a human CD40 agonistic antibody (ADC-
1013) that represents the first immunomodulatory human antibody optimized for local
immunotherapy of cancer. Local treatment with ADC-1013 results in potent anti-tumor effects
and generation of a tumor specific immunity in a human CD40 transgenic mouse model. The
pre-clinical data strongly supports the initiation of the clinical investigation of ADC-1013 .
ADC-1013 is currently in late pre-clinical development.

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Abstract
Purpose: Local administration of immune-activating antibodies may increase the efficacy
and reduce the immune-related adverse events associated with systemic immunotherapy of
cancer. Here we report the development and affinity maturation of a fully human agonistic
CD40 antibody (IgG1), ADC-1013.
Experimental Design: We have used molecular engineering to generate an agonistic
antibody with high affinity for CD40. The functional activity of ADC-1013 has been
investigated in human and murine in vitro models. The in vivo effect has been investigated in
two separate bladder cancer models, both using human xenograft tumors in immune
deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40
transgenic mouse.
Results: Activation of dendritic cells (DCs) by ADC-1013 results in up-regulation of the co-
stimulatory molecules CD80 and CD86, and secretion of IL-12. ADC-1013 also activates
dendritic cells from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-
stimulated dendritic cells induce antigen-specific T cell proliferation in vitro. In vivo, treatment
with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant
anti-tumor effects and long-term tumor-specific immunity. Further, ADC-1013 demonstrates
significant anti-tumor effects in a human bladder cancer transplanted into immunodeficient
NSG mice.
Conclusions: Our data demonstrate that ADC-1013 induces long-lasting anti-tumor
responses and immunological memory mediated by CD40 stimulation. To the best of our
knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local
immunotherapy of cancer.

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Introduction
CD40 is a cell surface-expressed glycoprotein that belongs to the tumor necrosis factor
receptor (TNFR) superfamily and plays a central role in the immune system (1). It is
expressed on a variety of immune cells, such as B cells, dendritic cells (DC), monocytes and
macrophages, but also on several tumors, such as B cell lymphomas and carcinomas (1).
CD40 agonists trigger effective anti-tumor responses in pre-clinical models (2;3). These
responses occur via two distinct mechanisms: (i) tumor-specific immune responses, and (ii)
direct tumoricidal effects such as apoptosis and antibody-dependent cellular cytotoxicity
(ADCC) (2). The indirect anti-tumor immune effect, which is independent of the CD40 status
of the tumor, is mediated by tumor-specific cytotoxic T lymphocytes (4-6), and possibly
macrophages in certain tumor types (7). The direct tumoricidal effects, on the other hand,
depend highly on the CD40 expression of the tumor.
Several clinical trials have been reported for agonistic CD40 antibodies (2;8),
including CP-870,893 (IgG2) (9), ChiLob 7/4 (10) and SGN-40 (IgG1), the latter being a weak
CD40 agonist (11). Systemic administration of CD40 agonists may result in dose-limiting
immune-related adverse effects, such as cytokine release syndrome (12;13). Recently, it was
demonstrated that local administration of agonistic CD40 antibodies into tumor-bearing mice
resulted in improved anti-tumor effect, and caused less adverse immune-related events than
systemic treatment (4;14;15). The concept of intra-tumoral CD40 stimulation has also been
validated using local injection of adenoviral vectors expressing CD40L, demonstrating anti-
tumor effects in human and murine bladder cancer as well as in dog melanoma, with minimal
side effects (16;17).
To translate these findings into the clinic, we set out to engineer a CD40
agonistic antibody (18;19), ADC-1013, using Fragment INduced Diversity (FIND®) (20). The
affinity and potency of the antibody was increased since these properties are likely to be of
importance for local administration but also advantageous for systemic administration.
Initially, the anti-tumor effect of the optimized antibody was evaluated on a EJ human bladder

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cancer cell line in immunodeficient NSG mice, with or without transfer of human monocyte
derived DCs (moDCs) and T cells (21) (22). To further delineate the mode of action, we used
a human CD40 (hCD40) transgenic mouse (hCD40tg) on a C57BL/6 background to
demonstrate immune related anti-tumor effects of ADC-1013 in a fully immune-competent
host.

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Material and methods
Antibodies and cell lines
ADC-1013 was optimized as described in Supplementary methods (see also Supplementary
Table 1), S2C6 (23) (chimeric, human γ1/κ) and B44 (19) were produced by transient
expression in HEK cells and subsequent purification on protein A columns. An aglycosylated
variant of ADC-1013, referred to as ADC-1013-N297Q, was produced by introducing a single
N297Q mutation in the Fc-region. Endotoxin levels were analyzed using the Quantitative LAL
test using Endosafe – Portable Test System (PTS). Agonistic rat-anti-mouse CD40 (clone:
FGK4.5) and rat IgG2a (clone: 2A3) were purchased from Bio X Cell (West Lebanon, NH,
USA). The hCD40 positive Raji cell line was cultured in R10 medium (RPMI-1640
supplemented with L-glutamine and 10% FBS), at 37°C and 5% CO . The human EJ bladder
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cancer cell line (21) was cultured in R10 medium supplemented with 100 U/ml Penicillin–
Streptomycin (PEST) at 37°C and 5% CO . EJ cells were confirmed CD40 positive and HLA-
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A2 negative. The murine bladder transitional cell carcinoma cancer cell line Mouse Bladder-
49 (MB49; a kind gift from Dr. K. Esuvaranathan, National University of Singapore,
Singapore in 1996) and Lewis lung carcinoma-1 (LLC-1) (ATCC, CRL-1642) were cultured in
DMEM+ GlutaMax supplemented with 10% FBS, 0.1mM sodium pyruvate, 100U/ml
Penicillin–Streptomycin at 37°C and 5% CO . Human CD40 positive MB49 was transfected
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with human CD40 expressing vector under the control of CMV promotor. For cross-linking of
ADC-1013 in human moDC assays, goat anti human IgG (Jackson ImmunoResearch
Laboratories, Inc, PA, USA) was used. The structure models of B44 and ADC-1013 were
generated by Saromics Biostructures (Lund, Sweden) (Figure 1D).
Human cells obtained from peripheral blood mononuclear cells.
Buffy coats from peripheral blood were obtained from healthy blood donors at Uppsala
University Hospital Blood Center and Lund University Hospital in Sweden. Human PBMCs
were isolated by Ficoll-Paque density gradient centrifugation. CD14+ monocytes, CD19+ B

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cells and CD56+ NK cells were isolated using magnetic bead separation (MACS) according
to the manufacturer’s protocol (Miltenyi Biotec, Bergish Gladbach, Germany). Monocytes
were cultured for 6-7 days in R10 with recombinant human IL-4 and GM-CSF with medium
changed every second day and moDCs were confirmed to be CD1ahigh and CD14- by flow
cytometry. T cells for the in vivo experiments were isolated using Dynabeads Untouched
Human T cells-kit according to the manufacturer’s protocol (Invitrogen Life Technologies,
Carlsbad, CA, USA).

Flow cytometry
The flow cytometry used to analyze the human cells in vitro has been described previously
(24). Briefly, cells were blocked with ChromPure Mouse IgG (Jackson ImmunoResearch
Laboratories) and incubated with anti-human Abs for 15 min at 4°C: anti-CD1a, anti-CD14,
anti-CD19, anti-CD86 (Dako, Stockholm, Sweden), anti-CD80 (BD Biosciences, San Jose,
CA, USA) and anti-CD19 (ImmunoTools, Friesoythe, Germany) antibodies. Cells in live gate
were acquired on a FACS Canto II (BD Biosciences).
For analysis of murine cells by flow cytometry, the spleens were cut into small
pieces and incubated in serum free media with Liberase TL (Roche Applied Science,
Penzberg, Germany). Subsequently, cells were stained with antibodies binding to CD11b,
F4/80, CD11c, B220, CD3, NK1.1, CD8a and to hCD40 or to murine CD40 antibodies. Cells
from a CD40-/- mouse strain (25) were used to define the expression level. Conventional
DCs were gated as CD11chigh, B220- and subsequently as CD8a+ CD11blow or CD8a-
CD11b+. Macrophages were gated as CD11b+, F4/80+, CD11c- and B220-. B cells were
gated as B220+, CD11b-, CD11c-. T cells were gated as CD3+, NK1.1-. NK cells were gated
as CD3-, NK1.1+. NKT cells were gated as CD3+, NK1.1+. Antibodies were obtained from
Biolegend (San Diego, CA, USA).
In vitro cell assays in human cells

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Dendritic cell assay. DC were derived from human peripheral blood monocytes (24). The
moDCs and dilution series of stimulating antibodies or isotype control antibody (IgG1) were
cultured for 48 h in R10 medium with IL-4 and GM-CSF. Up-regulation of the activation
markers CD86 and CD80 were analyzed by flow cytometry. Secretion of IL-12 (p40) was
measured by ELISA (Biolegends, San Diego, CA, USA). IL-12p70 was measured using
ELISA (eBioscience, San Diego, CA, USA, and R&D Systems Minneapolis, MN, USA). For
cross-linking experiments, the antibodies were cross-linked using anti-human goat antibody
(Jackson ImmunoResearch Laboratories, Inc, PA, USA).
B cell proliferation assay. The CD19+ cells (5 x 104/well, >95% purity) were cultured in R10
medium with 10 ng/ml of IL-4 and dilution series of antibodies. After 48 to 72 h, the metabolic
activity was measured with CellTiter-Glo (Promega, WI, USA).
ADCC assay. CD56+ NK cells were mixed with Raji cells in a 5:1 ratio in ADCC-medium
(RPMI-1640 + 10% Ultra low IgG FCS + 10 mM Hepes + 1mM Na-pyruvate). The cytotoxicity
was measured after 2 h of incubation using aCella-Tox Kit (Cell Technology, Inc., CA, USA)
according to the manufacturer’s instructions.
In vivo experiments
Female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ) mice were obtained from the Jackson
Laboratory (Maine, USA, 005557). Cryopreserved human T cells were thawed and grown
with human recombinant IL-2 one day prior to the start. EJ cells (3x106), alone or mixed with
1x105 moDCs and 5x105 T cells were injected subcutaneously. Antibodies were administered
together with the cell solution on day 0 and thereafter peritumorally on day 7 and 14. Tumor
size was measured with caliper and weighed at the end-point day (day 23). The treatment
groups with moDC and T cells (DC/T) consisted of 8 mice per group, for which T cells and
DCs from two donors were used (4 mice/group/human donor). The experiment using EJ cells
alone consisted of 6 mice per group.

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The human CD40 transgene mice contains the hCD40 sequence downstream
of the murine CD40 promotor and regulatory elements (26) and do not express murine CD40
(27) (Supplementary Methods). The mice were analyzed for human CD40 expression by
immunohistochemistry using tissue cryo sections from spleen, lymph node, pancreas, CNS,
liver, lung and kidney from one female and one male hCD40tg mouse. Human and mouse
spleen was used as positive and negative controls. Briefly, the sections were blocked in 5%
normal mouse serum (Jackson ImmunoResearch Laboratories) and stained with mouse anti-
hCD40-FITC (BD Biosciences). The section was subsequently stained with rabbit anti-FITC
(Abcam, Cambridge, UK) and anti-rabbit-HRP (Brightvision poly-HRP rabbit, ImmunoLogic
Immunologic BV, Duiven, Netherlands). The slides were analyzed in a Leica DMRX-e
microscope and representative photos were taken from all tissues. CD40-/- C57BL/6 mice
were obtained from the Jackson Laboratory (002928).
For in vivo pharmacokinetic experiments, female SCID mice (Taconic, Ry,
Denmark) were used. 107 Raji cells were injected s.c. in the left flank. Tumors at approx. 500
mm2 were injected with antibodies (30 μg, in 60 μl) in the tumor mass and blood was
collected upon termination 4 h post injection. For intratumoral retention studies, hCD40+
MB49 cells were inoculated subcutaneously in male hCD40tg mice and antibodies were
injected intratumorally in 20µl when the tumor reached approximately 150 mm2. The tumors
were homogenized in T-PER (Thermo Scientific, Rockford, IL USA). The antibody levels
were measured by sandwich ELISA.

In vitro immune activation profiling in cells from hCD40tg mice

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Bone marrow derived DCs (BMDCs) were generated by extraction of cells from the bone
marrow and subsequent culturing with mGM-CSF for 8 days. Immature BMDCs were
stimulated with a concentration range of ADC-1013 or isotype control antibody (IgG1), or
LPS (Sigma Aldrich, Stockholm, Sweden). Activation was measured by IL12p40 production
or CD86 expression after 48 hours of stimulation using an in house ELISA assay or flow
cytometry (see Supplementary Methods).
For T cell activation, transgenic pmel T cells (TCR specific for human gp100 in
H-2Db, obtained from the Jackson Laboratory 005023), were isolated from splenocytes by
CD8 negative selection (Dynal® invitrogen). BMDCs were generated as described above
and loaded with suboptimal concentration (1 pg/ml) of human gp100 (KVPRNQDWL) and
stimulated with ADC-1013 or an isotype control antibody. After 24 hours BMDCs and CFSE
labeled (Invitrogen) CD8+ pmel cells were mixed and T cell proliferation was assessed by
flow cytometry after 3 days.
BMDCs were additionally stimulated with or without LPS (1µg/ml) in vitro and
pulsed with hgp100 (1µg/ml) before injected i.v into naïve wt C57BL/6 mice (1x106
cells/mouse), which were injected i.v the day before with CFSE labeled splenocytes (11 x106
cells/mouse) isolated from pmel mice. The in vivo proliferation of pmel CD8+ cells was
analyzed using flow cytometry four days post BMDC injection.

In vivo experiments in syngeneic MB49 bladder cancer model

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