Protein Engineering, Design & Selection, 2016, vol. 29 no. 5, pp. 159–167
doi: 10.1093/protein/gzw002
Advance Access Publication Date: 10 March 2016
Original Article
Original Article
ASP2408 and ASP2409, novel CTLA4-Ig variants
with CD86-selective ligand binding activity and
improved immunosuppressive potency, created
by directed evolution
Shinsuke Oshima1,*, Erik E. Karrer2,*, Madan M. Paidhungat2,
Margaret Neighbors3, Steven J. Chapin3, Rong A. Fan3, Margaret A. Reed3,
Kuoting Wu3, Clifford Wong3, Yonghong Chen5, Marc Whitlow4,
Francisco A. Anderson2, Rujuta A. Bam2, Qian Zhang2, Brent R. Larsen5,
Sridhar Viswanathan6, Bruce H. Devens3, Steven H. Bass2,
and Yasuyuki Higashi1
1
Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma, Inc., Tsukuba, Ibaraki 3058585,
Japan, 2Department of Molecular Biology, Perseid Therapeutics, Redwood City, CA 94063, USA, 3Department of
Biology and Pharmacology, Perseid Therapeutics, Redwood City, CA 94063, USA, 4Colabrativ, Inc., Orinda, CA
94563, USA, 5Department of Protein Analytics and Pharmaceutics, Perseid Therapeutics, Redwood City, CA 94063,
USA, and 6Department of Process Development and Manufacturing, Perseid Therapeutics, Redwood City, CA
94063, USA
*To whom correspondence should be addressed. E-mail: [email protected] (S.O.), [email protected] (E.E.K.)
Edited by Frances Arnold
Received 15 September 2015; Revised 19 January 2016; Accepted 20 January 2016
Abstract
The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80,
yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that
confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold
enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity
to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potency in vitro and in vivo. Our results highlight the power of directed evolution methods to
obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective
CTLA4-Ig compounds that have entered clinical trials.
Key words: costimulation blockade, next-generation protein therapeutics, phage display, rheumatoid arthritis, transplantation
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected]
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Introduction
Adaptive immune responses are tightly regulated to balance immune activation with homeostasis (Bour-Jordan et al., 2011). During T cell activation, positive co-stimulatory signaling mediated by engagement of CD28
receptors on T cells with CD80/CD86 ligands on antigen-presenting cells
(APC) is counterbalanced by down-regulatory signaling via engagement
of CTLA-4 receptors on T cells with the same ligands (Salomon and
Bluestone, 2001; Walker and Sansom, 2011). This CD80/CD86–CD28/
CTLA-4 regulatory network represents substantial opportunities for
therapeutic intervention, either for immunosuppressive approaches blocking positive signaling through CD28 or immunostimulatory approaches
blocking down-regulatory functions of CTLA-4. To date, there are
three marketed biologics targeting this network: (a) abatacept (http://
www.pdr.net/drug-information/orencia?druglabelid=112), a wild-type
CTLA4-Ig fusion protein for rheumatoid arthritis (Keating, 2013); (b) belatacept (http://www.pdr.net/drug-information/nulojix?druglabelid=110),
a modified CTLA4-Ig with improved CD80/CD86 ligand binding affinity
for kidney transplantation (Wojciechowski and Vincenti, 2012); and
(c) ipilimumab, an anti-CTLA-4 monoclonal antibody for melanoma
(Trinh and Hwu, 2012).
The primary mechanism of action for CTLA4-Ig therapeutics is
binding to CD80/CD86 and thus blocking co-stimulatory signaling
through CD28 (Carman et al., 2009). Although both CD80 and
CD86 can engage CD28, structural (Stamper et al., 2001), biophysical
(Pentcheva-Hoang et al., 2004; Bhatia et al., 2005) and theoretical
modeling (Jansson et al., 2005) observations implicate CD86 as the
dominant co-stimulatory ligand for signaling through CD28. Cellbased assays in vitro confirm that immunosuppressive potency of
CTLA4-Ig variants correlates more with binding affinity to CD86 than
CD80 (Latek et al., 2009; Xu et al., 2012). As both abatacept and belatacept are selective for CD80 binding (Larsen et al., 2005; Bernett et al.,
2013), we employed DNA shuffling directed evolution methods to create
CTLA4-Ig variants with specific improvements in CD86 binding
affinity. Here, we provide the first description of CD86-selective
CTLA4-Igs with potential for improved clinical effectiveness.
Materials and methods
Library creation
Libraries of CTLA-4 ECD variants were created by synthetic DNA
shuffling (Ness et al., 2002) where overlapping oligonucleotides
(60-mers) encoding the desired diversity are assembled into full-length
genes by polymerase chain reaction (PCR) amplification using flanking
primers and cloned via SfiI/NotI into phage display vector pSB0124.
pSB0124 is a derivative of pBR322 (New England Biolabs, #N3033S)
encoding CTLA-4 ECD-phage M13 pIII fusion proteins targeted for
periplasmic secretion by the STII signal peptide (Picken et al., 1983)
and tightly regulated expression driven by the pBAD promoter
(Guzman et al., 1995) to facilitate monovalent phage display. Library
ligations in pSB0124 were transformed into TOP10 Escherichia coli
cells (Invitrogen, Inc., #C4040-50) by electroporation, and the cells
were cultured in Luria Broth (LB) media containing 50 µg/ml carbenicillin to make stocks of library DNA. Libraries of phage-displayed
CTLA-4-ECD proteins were created by transforming of E. coli TG-1
cells (Stratagene, #200123) with library DNA by electroporation.
Transformed cells were cultured in LB medium, infected with helper
phage M13KO7 (Invitrogen, #18311-019) at a multiplicity of infection
of 5–10 under dual selection for phagemid (carbenicillin at 50 µg/ml)
and helper phage (kanamycin at 70 µg/ml). Procedure in detail is
described in the Supplementary data.
Library screening
Phage libraries were panned for five rounds against soluble biotinylated ligands (Round 1) or immobilized ligands (Rounds 2–5). In the
first round, library phage were incubated in capture reactions containing biotinylated hCD80-Ig or hCD86-Ig. Ligand-bound phage were
captured by streptavidin-coated Dynal beads (Life Technologies,
#11205D). Bound phage were eluted by incubation with elution buffer
(0.2 M glycine pH 2.2 + 1% BSA) and neutralized by neutralization
buffer (1 M Tris pH 9.0). Escherichia coli TG-1 cells were infected
with eluted phage to generate enriched library phage for the next
round of selection. In Rounds 2–5, library phage were panned against
ligand immobilized on immunotubes (NUNC Maxi-Sorp, Thermo
Scientific, #470319) coated with CD80-Ig or CD86-Ig at 10 µg/ml.
Phage enriched from panning were characterized by phage ELISA.
Phage were captured by coated CD80-Ig or CD86-Ig on the plate and
detected by HRP-conjugated anti-M13 monoclonal antibody (GE
Healthcare, #27-9421-01). Phage displaying variant CTLA-4 ECDs
with improved ligand binding affinity were identified by further incubating with soluble competitor protein (0.3 µM abatacept for
CD80-Ig-coated plates or 1 µM abatacept for CD86-Ig-coated plates).
Differences in the ratio of ELISA signal in the presence or absence of
competitor protein were used to identify CTLA-4 variants with higher
affinity, as such variants would have the least reduction in ELISA signal after extended washing in the presence of competitor protein.
Procedure in detail is described in the Supplementary data.
Subcloning CTLA-4 variants into the IgG2-Fc fusion
expression vector
The IgG2-Fc fusion expression vector is a derivative of pCDNA3.1(+)
(Life Technologies, #V79020) engineered to express a fusion protein
comprising the human CTLA-4 protein from positions M1 to D161, including the signal sequence (Harper et al., 1991) and the human IgG2-Fc
domain (from positions E216 to K447 using the EU numbering system).
Unique AgeI and ClaI sites were introduced through silent mutations at
I31–P32–V33 and I115–D116 positions in the CTLA-4-ECD-encoding
region to facilitate cloning of PCR-amplified variants into the vector.
Production and purification of variant CTLA4-Ig fusion
proteins
COS-7 cells (ATCC, #CRL-165) were transiently transfected with plasmid DNA encoding variant CTLA4-Ig fusion proteins using FuGENE 6
transfection reagent (Roche, #11 815 075 001), as per the manufacturer’s recommended conditions. Following transfection, cells were cultured with DMEM medium (Invitrogen, #11964-142) containing 10%
fetal bovine serum (FBS) (Hyclone, #SV30014.03) and 1 × PSG (penicillin–streptomycin–glycine, Invitrogen, #10378-016) at 37°C and 5%
CO2 for 4 days. Variant CTLA4-Ig fusion proteins were purified by
Protein A affinity chromatography. SDS/PAGE analysis was used to assess identity and purity of variant CTLA4-Ig protein preparations. Size
exclusion chromatography (SEC) was used to measure the content of
aggregates in variant CTLA4-Ig protein preparations. Typical protein
preparations exhibited >90% monomeric peak area, where the monomeric species is a disulfide-linked homodimer comprised of two
CTLA4-Ig polypeptides.
Reagent and control proteins
Abatacept, the first-generation CTLA4-Ig comprising a fusion protein
of wild-type CTLA-4 with modified Fc domain of human IgG1
(Linsley et al., 1991), was purchased from Bristol-Myers Squibb
(Princeton, NJ). Belatacept, a next-generation CTLA4-Ig protein
Directed evolution of CD86-selective CTLA4-Ig
differing from abatacept by two mutations (A29Y and L104E) in the
CTLA-4 ECD (Larsen et al., 2005), was produced at Perseid
Therapeutics (Redwood, CA). As surrogate control proteins for binding assay, wild-type CTLA-4 or modified CTLA-4 with the two amino
acids mutations fused with Fc domain of human IgG2 was produced at
Perseid Therapeutics. CD80-mIg or CD86-mIg protein was produced
at Perseid Therapeutics.
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Astellas Pharma, Inc. (Tsukuba, Japan) that are awarded accreditation
status by the AAALAC International and performed in accordance
with the ethics criteria contained in the bylaws of the committee.
All the other experimental procedures performed on animals were
approved by the IACUC of Perseid Therapeutics and conducted in accordance with the current edition of the Guide for the Care and Use of
Laboratory Animals as standardly used by PHS/OLAW-assured and
AAALAC-accredited institutions.
Biacore measurements and analysis
Rabbit anti-mouse IgG antibody (GE Healthcare, #BR-1005-14) was
immobilized on CM-5 sensor chips (GE Healthcare, #BR-1000-14)
according to the manufacturer’s protocol. Human or murine
CD80-murine Ig (CD80-Ig) or CD86-murine Ig (CD86-Ig) proteins
were bound to antibody-coated sensor chips. CTLA4-Ig proteins
were diluted in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM
NaCl, 3 mM EDTA, 0.005% v/v surfactant P20, GE Healthcare,
#BR1001-88) and flowed over ligand-coated sensor chips for 2 min,
followed by 5- or 20-min incubation with HBS-EP buffer containing
no protein at the same flow rate, according to the purpose of analysis.
For more accurate comparison of mutant CTLA4-Ig fusion proteins
having very slow dissociation rates from ligands, kinetic assays were
conducted using 20 min dissociation times. No discernible dissociation of captured ligands was observed during the dissociation periods
(data not shown). Eight dilutions of CTLA4-Ig proteins ranging from
500 to 0.2 nM were analyzed against a blank reference (HBS-EP buffer alone). Ligand was removed from the chip surface between cycles
by incubation with 10 mM glycine, pH 1.7. Data were analyzed by
BIAevaluation software (v4.1, available from GE Healthcare) using
the ‘Fit kinetics, Simultaneous ka/kd’ function. Data were fit using
the 1:1 Langmuir model to determine the association rate constant
(ka) and the dissociation rate constant (kd) and to calculate the equilibrium dissociation constant, KD.
Sequence optimization
Protein sequences of CTLA-4 ECD variants were aligned using
VectorNTI (Invitrogen) and GeneDoc (http://www.nrbsc.org/gfx/
genedoc) software and ordered by relative CD86 binding affinity.
Individual mutations were weighted according to their frequency
among variants with high CD86 binding affinity relative to their frequency in the unselected library. Mutations were further weighted by
examining potential changes to the protein structure using available
NMR and X-ray crystal structures (Metzler et al., 1997; Schwartz
et al., 2001; Stamper et al., 2001) viewed using Cn3Dv4.1 (NCBI)
and Swiss-PdbViewer (ExPASy). Weighting information was used to
determine candidate amino acid positions for reversion to wild-type
sequence. Sequence-optimized clones were constructed by de novo assembly of overlapping oligonucleotides (60-mers with 30 nucleotide
overlap) encoding the full-length variant CTLA-4-ECD and cloned
into the IgG2-Fc fusion expression vector as described herein.
Homology modeling
The homology model was constructed from the Protein Data Bank coordinate set 1I8L of CTLA-4 bound to CD86 (Schwartz et al., 2001) using
Accelrys Discovery Studio Visualizer 2 to determine optimal fitting of
variant amino acid side chain rotamers within their local environments.
Animals
The experimental procedures on in vitro mice studies were approved
by the Institutional Animal Care and Use Committee (IACUC) of
T-cell proliferation assays
Human blood samples were obtained healthy volunteers (Palo Alto
Medical Foundation, CA) under IRB consent.
For human T-cell recall antigen-specific proliferation assay, peripheral blood mononuclear cells (PBMC) were freshly isolated from
the donors prescreened for reactivity to Mycobacterium tuberculosis
purified protein derivative (PPD) using a Ficoll (Sigma-Aldrich,
Histopaque, #10771) gradient separation as per the manufacturer’s
recommended protocol. PBMC were suspended in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin (Gibco,
#15140), and 1 × 105cells/well were added to cell culture plates with
PPD recall antigen (5 µg/ml, Mycos, #P-1000-001). Baseline control
wells contained PBMC only without PPD antigen.
For human mixed lymphocyte reactions (MLR) assay, human
PBMC were prepared as above. From one donor (responder),
1 × 105 PBMC were mixed with irradiated (26 Gy) 1 × 105 PBMC
from a second donor (stimulator) in 96-well round bottom culture
plates. Baseline control wells contained responder PBMC only.
For murine MLR assay, splenocytes of C57BL/6 (Charles River Labs,
Japan) and Balb/c (Charles River Labs, Japan) were isolated by standard
technique. Splenocytes of C57BL/6 were prepared in RPMI 1640 medium
supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml
streptomycin (Sigma-Aldrich, #P0781), 55 µM 2-mercaptoethanol
(Gibco, #21985-023) and 10% FBS, and 1.5 × 105 of splenocytes from
C57BL/6 (responder) were mixed with irradiated (20 Gy) 1.5 × 105 of
splenocytes obtained from BALB/c (stimulator) in 96-well round bottom
culture plates. Baseline control wells contained responder splenocytes
only.
For all T-cell response assays, test compounds were serially diluted
in the same medium and added to the wells. Culture plates were incubated for 2–5 days at 37°C and 5% CO2 with 1 µCi/well 3H thymidine
(GE Healthcare, Inc. or Moravek Biochemicals, Inc.) added to each
well for an additional 6–18 h. The cells were harvested with a cell harvester (Perkin Elmer, Waltham, MA) onto glass filter membranes
(Perkin Elmer). Cell proliferation was measured by incorporation of
radiolabel counted by a scintillation counter (Perkin Elmer) and reported as counts per minute (CPM). Assay data were analyzed by
Prism 5 (GraphPad Software, Inc., La Jolla, CA) with non-linear regression curve fitting using the sigmoidal dose–response (variable
slope) equation and least squares fit method with the top and bottom
parameters set to share the values from all the data sets. Geometric
mean IC50 for each test compound was generated from at least three
independent experiments.
In vivo mouse antibody response with KLH
immunization
Female BALB/c mice at the age of 8–12 weeks (Charles River Labs,
Hollister, CA) were immunized on day 0 by subcutaneous (SC) injection
with 200 µg of Keyhole Limpet Hemocyanin (KLH) (Sigma-Aldrich,
#H7017) at the base of the tail. To induce secondary antibody response,
mice were immunized with SC injection of 200 µg of KLH at the base of
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the tail after resting for at least 8 weeks from primary KLH immunization. Mice were treated with test compounds via SC at the doses indicated in parallel with the primary or secondary KLH immunization.
Mice were bled to detect anti-KLH antibody at 21 days after primary
immunization or at 14 days after secondary immunization, respectively.
Anti-KLH IgG in serum was measured by ELISA (Life Diagnostics,
#4010-1).
Results
Directed evolution strategy and library screening
The iterative directed evolution process employed consisted of multiple CTLA-4 variant libraries over three rounds of screening, in
which separate libraries were created to explore different types, frequency and location of amino acid diversity within the CTLA-4 protein structure, while keeping library sizes within the limits of phage
display screening throughput (∼1 × 109) (Supplementary Tables SI
and SII). Naturally occurring diversity was obtained from analysis
of orthologous CTLA-4 protein sequences from different mammalian
species (Supplementary Fig. S1), while random diversity was created
by incorporation of NNS degenerate codons (Supplementary
Table SI). Library 1 (Supplementary Table SI) interrogated orthologous diversity at CTLA-4 residues that were predicted to be at or
near the protein–protein interface based on structural studies
(Schwartz et al., 2001). As many of those residues mapped to regions
of CTLA-4 homologous to the complementarity-determining regions
(CDR1 and CDR3) of the variable-immunoglobulin domain, libraries
from Series 2 (Supplementary Table SI) interrogated randomized diversity in each of the variable-CDR-like loops of CTLA-4 (CRD1,
CDR2, CDR3 and C’‘D loop, Supplementary Table SI). Series 3 libraries were built to interrogate combinations of the most favored diversity
from previous libraries (1 and 2) with orthologous diversity in the
‘framework’-like regions of CTLA-4 variable-immunoglobulin-like
domain.
Libraries were screened by monovalent phage display panning
(Bass et al., 1990) against human CD86-Ig or CD80-Ig fusion proteins. Enriched phage was confirmed for ligand binding activity by
phage ELISA and for identity by DNA sequencing. Individual variants
were expressed as CTLA4-IgG2-Fc fusion proteins in transiently transfected COS cells, and the binding kinetics of variant CTLA4-Ig fusion
proteins to immobilized CD80-Ig or CD86-Ig were evaluated using a
surface plasmon resonance (Biacore) assay with a 5 min dissociation
phase. Amino acid changes and improvements in ligand binding affinity
relative to wild-type CTLA4-Ig (abatacept) for variants selected from
each round of library screening are shown in Fig. 1, Supplementary
Figures S2 and S3. Different profiles were observed for variants selected
from separate libraries, in which variants selected from Library 3 have a
wide range of different ligand binding selectivity (Fig. 1), with some variants preferentially improved for CD86 binding (such as 201685).
Sequence optimization
The CTLA4-Ig variants obtained from library screening have ∼20
amino acid changes per clone relative to the wild-type CTLA-4 sequence. In order to minimize potential immunogenicity risks associated
with sequence divergence from wild type, a sequence optimization process was adopted with the aim of reducing mutation load without compromising function. A large set of variants spanning a large binding
activity range (subset shown in Supplementary Fig. S2) was analyzed
using sequence-based (Fox et al., 2007) and structural analyses (data
not shown) to establish correlations between mutant residues and
Fig. 1 Ligand binding activity of CTLA4-Ig variant proteins selected from phage
screening of Library 3. Binding kinetics of CTLA-Ig variant proteins to
immobilized CD80-Ig and CD86-Ig proteins were measured using a surface
plasmon resonance (Biacore) assay with a 5 min dissociation phase. Binding
affinity is expressed as fold improvement in KD (equilibrium dissociation
constant) relative to wild-type CTLA4-Ig (abatacept) for binding to CD86
(black bars) or CD80 (gray bars). Data are representative of two independent
measurements.
binding activity. Potentially neutral mutations that correlated poorly
with binding activity were candidates for reversion during sequence optimization, while those that correlated strongly with binding activity
were preserved in the initial step. Clones 201685, 201731 and
201682 were selected as parent frameworks for this approach, as they
have high CD86 binding affinity and considerable sequence diversity represented among their 19–21 mutations (Supplementary Fig. S4).
Potentially neutral mutations in the parent clones were reverted to
wild type using site-directed mutagenesis. A total of four descendent
clones (D1–D4) were expressed as soluble CTLA4-IgG2 fusion proteins
and characterized for CD86 binding affinity by Biacore (Supplementary
Fig. S4 and Table SIII). Of the four descendants, Clone D3 retained the
highest level of CD86 binding affinity relative to its parent (0.86-fold
reduction), indicating that the seven reversions were likely neutral in
the context of the Clone 201731 framework. This sequence optimization process reduced the mutation load of Clone 201731 from 19 to
12 amino acid changes without significant loss of CD86 ligand binding
activity. In order to further optimize ligand binding activity relative to
mutation load, variants of Clone D3 (the D3 series) were generated with
different mutation loads by reversion and re-assortment of the 12 amino
acid changes (Supplementary Fig. S5). The D3 series variants were expressed as soluble CTLA4-IgG2 fusion proteins and characterized for
CD86/CD80 binding affinity by Biacore (Supplementary Fig. S6).
ASP2408 was identified as a variant with the highest CD86 binding selectivity with the lowest mutation load, while ASP2409 was identified as
a variant with the highest CD86 binding activity with reduced mutation
load relative to Clone D3 (Fig. 2a and b).
Directed evolution of CD86-selective CTLA4-Ig
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Fig. 2 Binding affinity on CD86, selectivity on ligands and mutation load of D3-related variants (the D3 series). (a) Relationship of fold improvement in the CD86
binding affinity of variants relative to abatacept vs mutation load. (b) Relationship of the CD86 binding selectivity of variants vs mutation load.
Table I. Ligand binding activity of ASP2408, ASP2409 and control proteins to immobilized human CD80-Ig and CD86-Ig
CTLA4-Ig
CD86_human
CD80_human
ka (1/Ms)
Abatacept
Belatacept
ASP2408
ASP2409
kd (1/s)
KD (M)
−2
6
1.08 × 10
9.52 × 10−4
2.17 × 10−4
8.23 × 10−5
1.62 × 10
2.25 × 106
2.69 × 106
2.74 × 106
Fold
−9
6.66 × 10
4.23 × 10−10
8.05 × 10−11
3.00 × 10−11
1
16
83
220
Selectivity
ka (1/Ms)
kd (1/s)
0.054
0.083
3.0
2.1
KD (M)
−4
6
1.32 × 10
1.79 × 106
1.33 × 106
1.27 × 106
4.75 × 10
6.26 × 10−5
3.20 × 10−4
8.12 × 10−5
−10
3.61 × 10
3.50 × 10−11
2.41 × 10−10
6.42 × 10−11
Fold
Selectivity
1
10
1.5
5.6
18
12
0.33
0.47
Binding kinetics were measured using Biacore assay with a 5 or a 20 min dissociation phase. ka represents the association rate constant, kd represents the dissociation
rate constant and KD represents the equilibrium dissociation constant. Values are expressed as the mean of 2 or more independent measurements. Fold indicates the
fold difference in KD relative to abatacept. Selectivity is expressed as the ratio of KD for each ligand. Binding kinetics for belatacept, ASP2408 and ASP2409 were
obtained using a 20 min dissociation time, while those for abatacept were obtained using a 5 min dissociation time.
Table II. Ligand binding activity of ASP2408, ASP2409 and control proteins to immobilized murine CD80-Ig and CD86-Ig
CTLA4-Ig
CD86_murine
ka (1/Ms)
Abatacept-IgG2
Belatacept-IgG2
ASP2408
ASP2409
5
8.23 × 10
6.16 × 105
1.41 × 106
1.13 × 106
CD80_murine
kd (1/s)
KD (M)
−4
5.63 × 10
2.19 × 10−3
6.65 × 10−5
1.46 × 10−4
Fold
−10
6.84 × 10
3.55 × 10−9
4.70 × 10−11
1.29 × 10−10
1
0.19
15
5.3
Selectivity
0.17
N.D.
1.1
2.5
ka (1/Ms)
5
9.81 × 10
N.D.
1.44 × 106
9.50 × 105
kd (1/s)
KD (M)
−4
1.16 × 10
7.92 × 10−4
7.77 × 10−5
3.02 × 10−4
−10
1.19 × 10
N.D.
5.40 × 10−11
3.18 × 10−10
Fold
Selectivity
1
N.D.
2.2
0.37
5.7
N.D.
0.87
0.41
Abatacept-IgG2 and belatacept-IgG2, which consist of the same CTLA-4 sequence as abatacept and belatacept, respectively, fused with Fc domain of human IgG2,
were assessed as a surrogate protein. Binding kinetics were measured using Biacore assay with a 5 or a 20 min dissociation phase, and the values are indicated as in
Table I. N.D., not determined.
Ligand binding activity of ASP2408 and ASP2409
Binding activity for human ligands of ASP2408 and ASP2409 is
shown in Table I. ASP2408 and ASP2409 have binding affinity to
human CD86 with KD of 80.5 and 30.0 pM, respectively, which represent approximately 83-fold and 220-fold improvements in binding
affinity relative to abatacept, respectively. The CD80 binding affinity
of ASP2408 is similar to that of abatacept (KD of 241 pM for
ASP2408 vs 361 pM for abatacept), whereas ASP2409 has 5.6-fold
higher affinity (KD of 64.2 pM for ASP2409 vs 361 pM for abatacept).
ASP2408 and ASP2409 are selective for CD86 binding relative to abatacept, as the affinity of ASP2408 for CD86 is 3.0-fold higher than that
for CD80 (80.5 vs 241 pM) and the affinity of ASP2409 for CD86 is
2.1-fold higher than that for CD80 (30.0 vs 64.2 pM). This selectivity
profile is opposite to that observed for abatacept and belatacept, which
are selective for CD80 binding (18-fold and 12-fold, respectively).
We also examined the binding affinity of ASP2408 and ASP2409
with murine CD80 and CD86 (Table II). ASP2408 and ASP2409
also showed improved binding affinity for murine CD86 relative to
a surrogate protein of abatacept. In contrast to the binding activity
for human CD86, ASP2408 showed more potent affinity to murine
CD86 relative to ASP2409 (47.0 vs 129 pM, respectively). The potential for off-target binding of ASP2408 and ASP2409 was assessed by
immunohistochemical staining of a wide range of normal tissues from
human, cynomolgus monkey, rat and mouse, with no unexpected
staining observed (data not shown).
Modeling of ASP2408 and ASP2409 amino acid changes
to the CTLA-4 protein structure
There are 6 and 10 amino acid changes present in the ECD of ASP2408
and ASP2409, respectively, relative to wild type (Fig. 3a). The location
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S.Oshima et al.
Fig. 3 Homology model of ASP2408 and ASP2409 in complex with CD86. (a) Sequence alignment of ASP2408, ASP2409 and wild-type CTLA4-IgG2. Numbering is
relative to the start of the mature CTLA-4 ECD (Peach et al., 1994), identity to the wild-type CTLA4-IgG2 is represented by dots and differences are indicated by single
letter amino acid code. (b and c) Views of the homology model of ASP2408 and ASP2409 in complex with CD86 to illustrate the location of mutations relative to the
ligand binding interface. The solvent accessible surface of CD86 has been color according to its electrostatic potential; blue and red areas having positive and
negative potentials at their surface, respectively. The alpha carbon (Ca) trace of ASP2408 and ASP2409 is shown in light blue. The Ca trace of the highly
conserved ligand binding motif (M97YPPPY102) is highlighted in green. The carbon atoms of mutations are color coded gold for hydrophobic residue (V32I,
A50M, S64P, S70F), blue for positively charged residues (M54K), red for negatively charged residues (A24E, G55E, N56D, L104E) and purple for hydrophilic
non-charged residues (T30N, I65S). (d) An expanded view of CTLA4/CD86 binding interface region, with the CTLA-4 residues known to directly participate in
CD86 binding (E31 R33, T51, M97YPPPYY103) are shown in green (Schwartz et al., 2001). The carbon atoms of hydrophobic V32I, A50M, S70F and negative-charged
L104E side chains are shown in gold, purple, yellow and red, respectively, and proximal CTLA-4 hydrophobic side chains of residues Y52, L58, F60, V75, V94 and
L96 are shown in light blue.
of each mutation within the protein structure was visualized by creating
homology models of ASP2408 and ASP2409 in complex with CD86
(Fig. 3b and c). The amino acid changes in ASP2408 and ASP2409 are
positioned throughout the CTLA-4 domain, with some changes such as
S64P well removed from the CD86 ligand binding surface. The remaining
changes are positioned in proximity to the ligand binding interface,
where they may participate more directly in enhancing ligand binding.
Several of the proximal changes are hydrophobic in nature (A50M,
S70F in both, V32I in ASP2409). An expanded view of these hydrophobic
changes reveals that they are clustered in a hydrophobic region adjacent
to the ligand binding loop M97YPPPY102, with their side chains orientated away from the ligand binding interface. In addition, L104E, which
is a key mutation in belatacept (Larsen et al., 2005), is also present in
ASP2409 and likely contributes directly to ligand binding.
In vitro immunosuppressive activity of ASP2408
and ASP2409
The immunosuppressive activity of ASP2408 and ASP2409 was
measured in vitro using several primary human and murine T-cell
proliferation assays (Table III). Co-stimulatory signal is provided by endogenous APC within the PBMC population. In the M. tuberculosis
PPD stimulation assay, modulation of memory T cells was assessed
using PBMC harvested from donors prescreened for reactivity to PPD
antigen and cultured in the presence of PPD to recall antigen-experienced
T cells. CTLA4-Ig variants inhibited memory T-cell proliferation in a
dose-dependent manner (Supplementary Fig. S7a). In human and murine MLR assays, modulation of allo-reactive T-cell was evaluated, where
co-stimulatory signal was provided by endogenous APC within the allogeneic PBMC or splenocyte population. Allogeneic T-cell proliferation
was also inhibited in a dose-dependent manner by the CTLA4-Ig variants (Supplementary Fig. S7b and c). Suppressive potency for each
CTLA4-Ig variant was calculated as the geometric mean IC50 and is
summarized in Table III. In all assay formats tested, the improved immunosuppressive potency of ASP2408, ASP2409 and belatacept relative
to abatacept correlates with their improved CD86 binding affinity. Of
note, ASP2408 was more potent than ASP2409 in inhibiting murine
T-cell proliferation, consistent with the higher binding affinity of
ASP2408 vs ASP2409 to the murine CD86 ligand.
Directed evolution of CD86-selective CTLA4-Ig
165
Table III. IC50 values of CTLA4-Ig variants in each in vitro T-cell stimulation assay
CTLA4-Ig
PPD_human
IC50, M (95% CI)
Abatacept
Belatacept
ASP2408
ASP2409
−9
2.2 × 10
(7.2 × 10−10–6.6 × 10−9)
1.8 × 10−10
(9.3 × 10−11–3.3 × 10−10)
3.6 × 10−11
(2.1 × 10−11–6.0 × 10−11)
1.7 × 10−11
(6.3 × 10−12–4.6 × 10−11)
MLR_human
Fold
1
12
60
130
IC50, M (95% CI)
−9
7.3 × 10
(3.3 × 10−9–1.6 × 10−8)
4.2 × 10−10
(1.3 × 10−10–1.3 × 10−9)
4.2 × 10−11
(1.4 × 10−11–1.3 × 10−10)
2.2 × 10−11
(6.9 × 10−12–7.1 × 10−11)
MLR_murine
Fold
1
18
170
330
IC50, M (95% CI)
−9
Fold
8.6 × 10
(1.7 × 10−9–4.3 × 10−8)
>1.3 × 10−7
1
4.5 × 10−10
(1.2 × 10−10–1.7 × 10−9)
2.5 × 10−9
(4.6 × 10−10–1.3 × 10−8)
19
N.D.
3.5
IC50 values were determined from concentration–response curves and are represented as the geometric mean with the 95% confidence interval (CI) from at least
three experiments. N.D., not determined.
Fig. 4 Immunosuppressive effects of ASP2408 and ASP2409 on primary and secondary anti-KLH antibody responses in mice. Female BALB/c mice were immunized
with 200 µg KLH, and anti-KLH IgG antibody responses were measured as described in the Materials and Methods section. Test compounds were administered
subcutaneously at the doses indicated on the same day of primary or secondary KLH immunization (n = 3–8/group). Anti-KLH antibody responses were
measured at 21 days after primary immunization with KLH and treatment with (a) ASP2408 or (b) ASP2409. Anti-KLH antibody responses were measured at 14
days after secondary KLH immunization and treatment with (c) ASP2408 and (d) ASP2409. The hashed line indicates the arithmetic mean value of the group.
The error bars show the standard error of the mean. Statistical significance was analyzed by using Dunnett’s multiple comparison test. *P < 0.05 when
compared with primary or secondary KLH immunized animals untreated with test compounds.
In vivo immunosuppressive activity of ASP2408
and ASP2409
T-cell-dependent B cell differentiation requires co-stimulatory signal, as
demonstrated by inhibition of antigen-specific antibodies when
co-stimulation is blocked using CTLA4-Ig (Linsley et al., 1992). To
test the in vivo immunosuppressive effects of ASP2408 and ASP2409,
we investigated murine primary and secondary antibody responses induced by KLH immunization. As shown in Fig. 4, ASP2408 and
ASP2409 with a single SC dose at the time of the last KLH
immunization inhibited the level of anti-KLH antibody generation in
primary and secondary antibody responses in a dose-dependent fashion. In primary anti-KLH antibody response, significant suppression
was observed by ASP2408 at the 0.4 mg/kg dose (Fig. 4a), while a
2 mg/kg dose of ASP2409 (Fig. 4b) was required to achieve a significant
effect. Complete suppression of primary anti-KLH antibody response
was observed by ASP2408 treatment at 1 mg/kg (Fig. 4a), and
ASP2409 was not able to achieve complete suppression at dose levels
up to 2 mg/kg (Fig. 4b). In secondary anti-KLH antibody response,
166
higher dose levels of ASP2408 or ASP2409 were required to observe suppressive effects relative to the primary response. For ASP2408, although a
significant suppressive effect was observed at the 0.5 mg/kg dose, complete suppression was not observed at dose levels up to 10 mg/kg
(Fig. 4c). For ASP2409, a significant suppressive effect was observed at
the 5 mg/kg dose and complete suppression was not observed at dose levels up to 20 mg/kg (Fig. 4d). These findings indicate that in vivo immunosuppressive effect of ASP2408 was more potent relative to
ASP2409 in mice, which was consistent with in vitro immunosuppressive
effect using murine splenocyte and with affinity to the murine CD80 and
CD86 ligands (shown in Tables II and III and Supplementary Fig. S7).
Discussion
We employed a directed evolution approach known as
MolecularBreeding™ or DNA shuffling (Stemmer, 1994; Minshull
and Stemmer, 1999) to improve the ligand binding properties of a
class of protein therapeutics approved for rheumatoid arthritis and kidney transplantation indications. DNA shuffling methods harness the
power of iterative genetic recombination and have proved versatility
in dissecting protein structure–function relationships (Schürmann
et al., 2013) and improving diverse protein properties such as cytokine
activity (Chang et al., 1999), enzymatic activity (Fox et al., 2007), thermal stability (Ruller et al., 2008), ligand binding affinity (Luginbühl
et al., 2006), expression level and solubility (Keenan et al., 2005).
Here we used synthetic DNA shuffling (Ness et al., 2002) to improve
CD86 binding affinity and generate the first examples of CD86-selective
CTLA4-Ig proteins that have entered into clinical trials.
Multiple CTLA-4 variant libraries containing over 3 billion unique
clones were screened iteratively over three rounds in order to effectively sample the large sequence space (Crameri et al., 1998) represented
within natural CTLA-4 orthologs (∼6 × 1021 unique clones,
Supplementary Fig. S1). In the initial rounds of screening (Libraries
1 and 2, Supplementary Table SI), natural or random diversity was explored in the region of the well-characterized CTLA-4 ligand binding
interface (Peach et al., 1994; Metzler et al., 1997; Schwartz et al.,
2001; Stamper et al., 2001). Comparing results from these different
approaches (Supplementary Fig. S3a and b), libraries created from natural orthologous diversity yielded CTLA4-Ig variants with higher
CD86 binding affinity and selectivity. This result is consistent with
the observation that re-assortment of naturally occurring diversity
can provide accelerated directed evolution relative to random mutagenesis (Crameri, et al., 1998). Although neither library generated variants with substantially improved activity relative to belatacept,
beneficial mutations were selected that could be recombined in further
library iterations.
In later rounds of screening (Library 3, Supplementary Table SI),
beneficial mutations from previous rounds were combined with natural
orthologous diversity located throughout the CTLA-4 protein structure.
Results from Library 3 screening underscore the power of the family
shuffling approach, as dramatic improvements in CD86 binding affinity
and selectivity were observed (Fig. 1). The majority of variants selected
from Library 3 have CD86 affinity improved substantially beyond that
of belatacept, with several clones displaying >5-fold higher affinity relative to belatacept and up to 50-fold higher affinity relative to abatacept.
Improvement in CD86 binding affinity generally correlated with improved CD86 binding selectivity, with several clones showing strong
(5- to 6-fold) CD86 binding selectivity profiles.
A sequence optimization process was employed to reduce the mutation load of top variants selected from Library 3. Of the four reversion constructs created, only Clone D3 retained sufficient ligand
S.Oshima et al.
binding activity (Supplementary Table SIII), emphasizing the inherent
uncertainty in predicting relative contributions of multiple amino acid
changes within a 3D protein structure. Based on the amino acid
changes in Clone D3, further sequence optimization was performed.
Among these variants of Clone D3 (the D3 series), ASP2408 was identified as a variant with the highest CD86 binding selectivity with the
lowest mutation load, while ASP2409 was identified as a variant with
the highest CD86 binding activity with reduced mutation load relative
to Clone D3 (Fig. 2a and b).
Several amino acid mutations are common for ASP2408 and
ASP2409, such as hydrophobic changes (S64P, S70F), positive charge
changes (M54K) and neutral changes (T30N, A50M). Unique mutations
are also present in ASP2408 (G55E: negative charge change) and in
ASP2409 (A24E, N56D and L104E: negative charge changes; V32I and
I65S: neutral changes), respectively (Fig. 3a). Homology modeling reveals
several mutations V32I, A50M, S70F form a core hydrophobic patch
below the ligand binding loop M97YPPPY102 (Fig. 3d). These mutations
appear to interact with adjacent hydrophobic residues to increase the
overall hydrophobicity of the region, which could act to stabilize a
more productive binding conformation for the CD86 ligand relative to
CD80. Further experiments would be required to test this hypothesis.
Further characterization of ASP2408 and ASP2409 binding profile
using Biacore conditions optimized for measuring slow dissociation
rates reveals dramatic improvements in human and murine CD86
binding relative to abatacept (Table I and II). Importantly, the ligand
binding selectivity profile for ASP2408 and ASP2409 (3.0-fold and
2.1-fold selective for CD86, respectively) has been completely reversed
relative to abatacept and belatacept, which are 18-fold and 12-fold
selective for CD80, respectively. Selectively improved CD86 binding
affinity correlates with improved immunosuppressive potency as measured by in vitro T cell proliferation assays (Table III). Further, we
found that the immune modulatory effect of CTLA4-Igs in mouse
KLH immunization models was clearly consistent with improved
binding affinity for CD86 (Fig. 4). These findings support and extend
previous observations where immunosuppressive potency of CTLA4Ig variants correlates more with binding affinity to CD86 than CD80
(Vincenti, 2008; Latek et al., 2009; Xu et al., 2012).
Structure-based rational design and high-throughput mutagenesis
methods have been previously used to engineer CTLA4-Igs for improved CD80/CD86 ligand binding (Larsen et al., 2005; Xu et al.,
2012; Bernett et al., 2013). Although these studies have provided fundamental advances in the understanding of the molecular interactions
between CTLA-4 and its ligands, none have generated CTLA4-Igs
with such pronounced and selective improvements in CD86 ligand
binding affinity as represented by ASP2408 and ASP2409. The magnitude of improvement in CD86 binding from previous studies is in the
range of 16-fold (Larsen et al., 2005), 17-fold (Xu et al., 2012) and
23-fold (Bernett et al., 2013) relative to abatacept, while ASP2408
and ASP2409 have substantial further improvements of 83-fold and
220-fold, respectively. Of note, ASP2408 and ASP2409 represent the
first examples of CTLA4-Igs with binding selectivity for CD86, having
3.0-fold and 2.1-fold higher affinity for CD86 vs CD80, respectively.
CTLA4-Igs from previous engineering efforts with the highest improvements in CD86 binding are still selective for CD80, having 12-fold
(Larsen et al., 2005), 6-fold (Xu et al., 2012) and 2-fold (Bernett
et al., 2013) higher CD80 binding affinity relative to CD86. It is anticipated that such attributes will enable ASP2408 and ASP2409 to have
improved dosing convenience and potentially improved efficacy and
safety profiles relative to previous generations of modified CTLA4-Igs.
Improvements in CD86 binding affinity and selectivity are hypothesized to confer clinically meaningful advantages relative to
Directed evolution of CD86-selective CTLA4-Ig
current CTLA4-Ig therapies by several interrelated mechanisms.
Because improved CD86 affinity correlates with improved immunosuppressive potency, CTLA4-Igs with improved CD86 binding affinity
would be able to provide therapeutic levels of CD86 occupancy at substantially lower serum drug concentrations. Thus, increased convenience
of drug administration could be realized by allowing lower dose levels
and less frequent dosing intervals. This concept is reinforced by the observation that ASP2408 or ASP2409 requires between 60- and 360-fold
lower protein concentration relative to abatacept to achieve similar inhibition of primary human T cell proliferation in vitro (Table III). In addition, increased affinity to CD86 could enable increased therapeutic
efficacy by virtue of providing more complete saturation of the dominant
co-stimulatory ligand CD86 throughout the dosing cycle.
In a related set of hypotheses, CD86-selective CTLA4-Igs are anticipated to occupy substantially less CD80 ligand at therapeutic dose levels. Increased CD80 availability could provide increased efficacy by
enhancing regulatory functions involved in the maintenance of immune
tolerance. Because Tregs require CD28 signaling for development and
survival (Tang et al., 2003), and CD80 can be a productive ligand for
CD28 (Jansson et al., 2005), it follows that increased CD80 availability
could enhance Treg cell generation via increased CD80–CD28 interactions. Treg function could also be improved by increased CD80 availability, as CTLA-4 is critical for Treg function (Walker and Sansom,
2011) and CD80 is the preferred ligand for CTLA-4 (Jansson et al.,
2005). Additionally, increased CD80 availability could allow increased
CD80-mediated immune functions known to be associated with antiviral responses (Lumsden et al., 2000), anti-B cell lymphoma (Suvas
et al., 2002), antitumor responses in CNS (Ando et al., 2002) and maintenance of bone marrow plasma cells (Rozanski et al., 2011). Increased
preservation of such immune responses may reduce immunosuppressive
complications of standard CTLA4-Ig therapy such as infection and
malignancy (Wojciechowski and Vincenti, 2012; Keating, 2013).
In conclusion, we employed DNA shuffling directed evolution
methods and created CTLA4-Ig variants, ASP2408 and ASP2409,
with specific improvements in CD86 binding affinity. We provide
the first description of CD86-selective CTLA4-Igs with potential for
improved clinical effectiveness.
Author contribution
S.O. and E.K. equally contributed to make this manuscript. E.K. was project lead. S.O. led in vitro (murine cells) studies. M.P. led library design,
screening and sequence optimization. M.N. led murine KLH studies. S.C.
led Biacore analysis, R.F. led in vitro human PPD and MLR assays, M.R.
performed Biacore measurements, and K.W. performed human PPD and
MLR assays. C.W. performed murine KLH studies. Y.C. led protein production and characterization. M.W. created homology models of
CTLA-4/CD86 interactions. F.A., R.B. and Q.Z. performed construct design, protein production and library design/screening. M.N., B.L., S.V.,
B.D., S.B. and Y.H. provided scientific leadership.
Supplementary data
Supplementary data are available at PEDS online.
Acknowledgements
We thank Anithakumari Dulapalli, Tasnim Kothambawala, Judy Gao, Edward
Pascua and William Ngo for protein production and characterization. This
paper and all the work associated with this manuscript are dedicated to the
memory of our wonderful colleague, B.R.L., who recently passed away.
167
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