Download The Dock and Lock Method: A Novel

The Dock and Lock Method: A Novel PlatformTechnology for
Building Multivalent, Multifunctional Structures of Defined
Composition with Retained Bioactivity
Chien-Hsing Chang,1,2 Edmund A. Rossi,2 and David M. Goldenberg1,2,3
Abstract
The idea, approach, and proof-of-concept of the dock and lock (DNL) method, which has
the potential for making a large number of bioactive molecules with multivalency and multifunctionality, are reviewed. The key to the DNL method seems to be the judicious application of a pair
of distinct protein domains that are involved in the natural association between protein kinase A
(PKA; cyclic AMP ^ dependent protein kinase) and A-kinase anchoring proteins. In essence, the
dimerization and docking domain found in the regulatory subunit of PKA and the anchoring
domain of an interactive A-kinase anchoring protein are each attached to a biological entity, and
the resulting derivatives, when combined, readily form a stably tethered complex of a defined
composition that fully retains the functions of individual constituents. Initial validation of the
DNL method was provided by the successful generation of several trivalent bispecific binding
proteins, each consisting of two identical Fab fragments linked site-specifically to a different
Fab. The integration of genetic engineering and conjugation chemistry achieved with the DNL
method may not only enable the creation of novel human therapeutics but could also provide
the promise and challenge for the construction of improved recombinant products over those
currently commercialized, including cytokines, vaccines, and monoclonal antibodies.
The impetus for developing the dock and lock (DNL) method
undoubtedly was due to the limitations of existing technologies
for the production of antibody-based agents having multiple
functions or binding specificities. For agents generated by
recombinant engineering, such limitations could include high
manufacturing cost, low expression yields, instability in serum,
formation of aggregates or dissociated subunits, undefined
batch composition due to the presence of multiple product
forms, contaminating side-products, reduced functional activities or binding affinity/avidity attributed to steric factors or
altered conformations, etc. For agents generated by various
methods of chemical cross-linking, high manufacturing cost and
heterogeneity of the purified product are two major concerns.
We, of course, recognize that innovative fusion proteins
created by recombinant technologies may be built into more
complex structures to gain additional attributes that are highly
desirable, yet not technically attainable, in the individual
engineered construct. Well-known examples include cytokines
modified with polyethylene glycol to increase serum half-lives
(1), biotinylated proteins to enable immobilization into
microarrays (2), and protein-DNA chimeras to quantify specific
Authors’ Affiliations: 1Immunomedics, Inc.; 2IBC Pharmaceuticals, Inc.; Morris
Plains, New Jersey ; and 3 Garden State Cancer Center, Center for Molecular
Medicine and Immunology, Belleville, NewJersey
Received 5/17/07; accepted 5/29/07.
Presented at the Eleventh Conference on Cancer Therapy with Antibodies and
Immunoconjugates, Parsippany, NewJersey, USA, October 12-14, 2006.
Requests for reprints: Chien-Hsing Chang, Immunomedics, Inc., 300 American
Road, Morris Plains, NJ 07950. Phone: 973-605-1330, ext. 108; Fax: 973-6051103; E-mail: kchang@ immunomedics.com.
F 2007 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-07-1217
molecules to which the protein binds (3). To date, these goals
are commonly achieved with varied success by judicious
application of conjugation chemistries. New strategies that are
based on the binding of enzyme to substrate (4) or inhibitor
(5), or the high-affinity interaction between two fragments of
human RNase I (6, 7), to tether two or more moieties of
distinct functions into covalent or quasi-covalent assemblies
have been reported; however, these methods are cumbersome
and therefore may limit their widespread use.
Protein Kinase A and A-Kinase Anchoring Proteins
The key to the DNL method is the exploitation of the specific
protein/protein interactions occurring in nature between the
regulatory (R) subunits of protein kinase A (PKA) and the
anchoring domain (AD) of A-kinase anchoring proteins (AKAP;
refs. 8, 9). PKA, which plays a central role in one of the best
studied signal transduction pathway triggered by the binding of
the second messenger cyclic AMP to the R subunits, was first
isolated from rabbit skeletal muscle in 1968 (10). The structure
of the holoenzyme consists of two catalytic subunits held in an
inactive form by the R subunits (11). Isozymes of PKA are
found with two types of R subunits (RI and RII), and each type
has a and h isoforms (12). The R subunits have been isolated
only as stable dimers and the dimerization domain has been
shown to consist of the first 44 NH2-terminal residues (13).
Binding of cyclic AMP to the R subunits leads to the release of
active catalytic subunits for a broad spectrum of serine/
threonine kinase activities, which are oriented toward selected
substrates through the compartmentalization of PKA via its
docking with AKAPs (14).
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Bioactive Structures by the DNL Method
Since the first AKAP, microtubule-associated protein 2, was
characterized in 1984 (15), more than 50 AKAPs that localize to
various subcellular sites, including plasma membrane, actin
cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum, have been identified with diverse structures in species
ranging from yeast to humans (9). The AD of AKAPs for PKA is
an amphipathic helix of 14 to 18 residues (16). The amino acid
sequences of the AD are quite varied among individual AKAPs,
with the binding affinities reported for RII dimers ranging from
2 to 90 nmol/L (17). Interestingly, AKAPs will only bind to
dimeric R subunits. For human RIIa, the AD binds to a
hydrophobic surface formed by the 23 NH2-terminal residues
(18). Thus, the dimerization domain and AKAP binding
domain of human RIIa are both located within the same
NH2-terminal 44-amino-acid sequence (13, 19), which is
termed the dimerization and docking domain (DDD) herein.
DDD of Human RIIA and AD of AKAPs as Linker
Modules
We envisioned a platform technology to exploit the DDD of
human RIIa and the AD of a certain amino acid sequence as an
excellent pair of linker modules for docking any two entities,
referred to hereafter as A and B, into a noncovalent complex,
which could be further locked into a stably tethered structure
through the introduction of cysteine residues into both the
DDD and AD at strategic positions to facilitate the formation of
disulfide bonds, as illustrated in Fig. 1. The general methods of
our DNL approach would be as follows. Entity A would be
constructed by linking a DDD sequence to a precursor of A,
resulting in a first component hereafter referred to as a. Because
the DDD sequence would effect the spontaneous formation of
a dimer, A would thus be composed of a2. Entity B would be
constructed by linking an AD sequence to a precursor of B,
resulting in a second component hereafter referred to as b. The
dimeric motif of DDD contained in a2 should create a docking
site for binding to the AD sequence contained in b, thus
facilitating a ready association of a2 and b to form a binary,
trimeric complex composed of a2b. This binding event could be
Fig. 1. Illustration of a stably tethered structure made by the DNL method. The a
helices involved in the natural binding interaction of PKA (blue) and AKAPs
(yellow) provide a preferred linker module for docking two types of entities, A and
B, which are further locked by disulfide linkages (shown as interlocking rings).
Such multivalent complexes always contain two copies of entity A.
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made irreversible with a subsequent reaction to covalently
secure the two entities via disulfide bridges, which might occur
very efficiently based on the principle of effective local
concentration because the initial binding interactions would
bring the reactive thiol groups placed onto both the DDD and
AD into proximity (20) to ligate site-specifically. By attaching
the DDD and AD away from the functional groups of the two
precursors, such site-specific ligations would also be expected to
preserve the original activities of the two precursors. This
approach would be modular in nature and potentially could be
applied to link, site-specifically and covalently, a wide range of
substances including peptides, proteins, and nucleic acids.
Bispecific Trivalent Structures Composed of
Three Stably Linked Fab Fragments
For proof-of concept, we used the DNL method to assemble
bispecific a2b complexes comprising three Fab fragments from
a panel of five fusion proteins, each containing either a DDD or
an AD of the sequence shown in Fig. 2A (21). The three DDDcontaining A entities were made recombinantly using as a
precursor the Fab fragment of the humanized monoclonal
antibody hMN-14 (22), which has binding specificity for
human carcinoembryonic antigen (CEACAM5). The first A,
C-DDD1-Fab-hMN-14, was generated by linking the DDD1
peptide sequence, which is composed of amino acids 1 to 44 of
human RIIa, to the COOH-terminal end of the Fd chain via a
14-residue flexible peptide linker (Fig. 2B). This construct was
modified by incorporation of a cysteine residue adjacent to the
NH2-terminal end of DDD1 to create C-DDD2-Fab-hMN-14
(Fig. 2C). The DDD2 sequence was moved to the NH2-terminal
end of the Fd to generate N-DDD2-Fab-hMN-14 (Fig. 2D).
Both AD-containing B entities were generated recombinantly
using as a precursor the Fab fragment of the humanized monoclonal antibody h679 (23), which has binding specificity for
histamine-succinyl-glycine. The first B, C-AD1-Fab-h679, was
generated by linking the 17-residue amino acid sequence
derived from AKAP-IS, a synthetic peptide optimized for RIIselective binding with a reported dissociation constant (K d) of
0.4 nmol/L (17), to the COOH-terminal end of the Fd chain
via a 15-residue flexible peptide linker (Fig. 2E). The other B,
C-AD2-Fab-h679, was generated in the same fashion as C-AD1Fab-h679, except with the addition of cysteine residues to both
the NH2 and COOH-terminal ends of AD1 (Fig. 2F).
As expected, C-DDD1-Fab-hMN-14 and C-AD1-Fab-h679
were purified from culture media exclusively as a homodimer
(the a2 structure) and a monomer (the b structure) of Fab,
respectively. When C-DDD1-Fab-hMN-14 was combined with
C-AD1-Fab-h679, the formation of an a2b complex was readily
shown by size-exclusion high-performance liquid chromatography, with the K d determined by equilibrium gel filtration
analysis (24) to be f8 nmol/L, which is presumably too weak
of an affinity to keep the a2b complex intact at concentrations
typical (<1 Ag/mL) for in vivo applications.
To prevent the dissociation of the noncovalent complex
formed from C-DDD1-Fab-hMN-14 and C-AD1-Fab-h679 at
lower concentrations, thus allowing in vivo applications,
cysteine residues were added into the DDD and AD sequences
of the A (N-DDD2-Fab-hMN-14 and C-DDD2-Fab-hMN-14)
and B (C-AD2-Fab-h679) entities, respectively. We anticipated
that on mixing of the cysteine-modified entities, an a2b
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Fig. 2. A, DDD and AD amino acid sequences. B to F, schematic diagrams of C-DDD1-Fab-hMN-14 (B); C-DDD2-Fab-hMN-14 (C); N-DDD2-Fab-hMN-14 (D);
C-AD1-Fab-h679 (E); C-AD2-Fab-h679 (F). The heavy chain constant domain 1 (CH1) and the light-chain constant domain (CK) are shown in gray.Variable domains of the
heavy (VH) and light (VK) chains of hMN-14 and h679 are shown in blue and green, respectively. DDD and AD are shown in yellow and red, respectively. Peptide linker
sequences (L14, L15, and L12) consisting of GGGGS repeats indicate the number of amino acids in each linker. Disulfide bridges and free sulfhydryl groups are indicated
as SS and SH, respectively.
complex would promptly form, which could be further
stabilized by the formation of disulfide bridges. Two stably
tethered trivalent bispecific structures, referred to as TF1 for the
conjugate of N-DDD2-Fab-hMN-14 and C-AD2-Fab-h679 and
TF2 for the conjugate of C-DDD2-Fab-hMN-14 and C-AD2Fab-h679, were obtained in nearly quantitative yields following Tris (2-carboxy-ethyl)-phosphine-HCl reduction, DMSO
oxidation, and affinity purification. Similar results were
achieved by substituting Tris (2-carboxy-ethyl)-phosphineHCl and DMSO with reduced and oxidized glutathione,
respectively. TF1 and TF2 were each shown by size-exclusion
high-performance liquid chromatography to be a single peak
of the expected molecular size (f150 kDa), by BIAcore to be
bispecific for both histamine-succinyl-glycine and a rat antiidiotype monoclonal antibody to hMN-14 (25), and by
competition ELISA to be equivalent to hMN-14 immunoglobulin G (IgG) and h679 Fab, reflecting the full retention of
valency and binding affinity. Furthermore, TF1 and TF2 were
found to be stable for at least 7 days when incubated at 37jC
in human or mouse serum, and the superiority of TF2 as a
pretargeting agent for diagnostic imaging has been shown in
nude mice bearing carcinoembryonic antigen – expressing
human colonic cancer xenografts (21).
Multivalent Binding Complexes Composed of Six
Fab Fragments
Since the generation of TF1 and TF2, several additional
trivalent bispecific Fab-based complexes (the TF series) have
been produced. We have also applied the DNL method to
successfully generate hexavalent, antibody-based complexes
(the DNL series) that are either monospecific (Fig. 3A) or
bispecific (Fig. 3B and C). The complexes in the DNL series, all
of which comprise an IgG-(AD2)2 module coupled to two FabDDD2 modules, were made using the C-DDD2-Fab derived
from hLL2 (epratuzumab, anti-CD22 IgG; ref. 26), hA20
(humanized anti-CD20 IgG; ref. 27), or hMN-14, and the
IgG-(AD2)2 modules derived from hLL2 or hA20. To evaluate
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Bioactive Structures by the DNL Method
whether the four extra Fab moieties would affect the functions
of the Fc, we produced two types of IgG-(AD2)2 modules, with
AD2 fused to each COOH terminus of the heavy chain (C-AD2IgG) or to each NH2 terminus of the light chain (n-AD2-IgG).
Preliminary results indicate that a hexavalent complex with
AD2 placed at the NH2 termini seems to have a functional Fc
but may selectively lose the binding activity of the IgG-AD2
module, depending on the epitope. In contrast, a hexavalent
complex with AD2 placed at the COOH termini may not have a
functional Fc but preserves the binding activity of the IgG-AD2
module. In both types of hexameric complexes, each of the four
Fab moieties contributed from the Fab-DDD2 module remains
active, however. Additional findings are that the monospecific
hexameric complex, designated Hex-hA20 (composed of four
hA20 Fab tethered to the COOH termini of hA20 IgG), and the
two bispecific hexameric complexes, designated DNL1 and
DNL2 (composed of four hA20 Fab tethered to hLL2 IgG and
four hLL2 Fab tethered to hA20 IgG, respectively), show much
greater potency in inhibiting growth of human Burkitt
lymphoma cell lines in vitro than the parental monoclonal
antibodies alone or combined (28). The bispecific DNL2 and
Hex-hA20 showed >100-fold and >10,000-fold more potent
antiproliferative activity, respectively, than hA20 IgG on Daudi
cells (Fig. 4A). For Raji cells, Hex-hA20 displayed potent
antiproliferative activity, whereas DNL2 showed only minimal
activity (Fig. 4B). For Ramos cells, both DNL2 and Hex-hA20
showed potent antiproliferative activity (Fig. 4C). In contrast,
Hex-hLL2 (composed of four hLL2 Fab tethered to the COOH
termini of hLL2 IgG) and the two bispecific controls, DNL1-c
(substituting hA20 Fab in DNL1 for hMN-14-Fab) and DNL2-c
(substituting hLL2 Fab in DNL2 for hMN-14 Fab), had little
antiproliferative activity under the conditions used (data not
shown).
The Advantages of DNL
Based on our experience with the tri-Fab and hexa-Fab
constructs, the advantages of the DNL method that distinguish
it from other site-specific conjugation methods (1 – 7) are
briefly summarized as follows.
DNL is modular. Each DDD- or AD-containing entity is a
module and any DDD module can be paired with any AD
module. Such modules can be produced independently, stored
separately ‘‘on shelf’’, and combined ‘‘on demand,’’ without
Fig. 3. Schematics of IgG-based
hexavalent DNL structures. A, monospecific
derived from C-AD2-IgG and C-FabDDD2; B, bispecific derived from C-AD2IgG and C-DDD2-Fab; C, alternative
bispecific derived from n-AD2-IgG and
C-DDD2-Fab.
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expression system, certain pairs of DDD and AD modules
may be coexpressed in the same host cell without affecting the
formation of the DNL conjugates. Furthermore, DDD or AD
can be coupled to the NH2-terminal or COOH-terminal end or
positioned internally within the fusion protein, preferably with
a spacer containing an appropriate length and composition of
amino acid residues, provided that the binding activity of the
DDD or AD and the desired activity of the polypeptide fusion
partners are not compromised.
Modules may also be made synthetically to contain peptides,
peptide mimetics, oligonucleotides or polynucleotides, small
interfering RNA, polyethylene glycol, chelators with or without
radioactive or nonradioactive metals, drugs, dyes, oligosaccharides, natural or synthetic polymeric substances, nanoparticles,
fluorescent molecules, or quantum dots, depending on the
intended applications.
DNL manufacture is easy. The DNL method is basically a
one-pot preparation and requires three simple steps to recover
the product from the starting materials: (a) combine DDD and
AD modules in stoichiometric amounts; (b) add redox agents
to facilitate the self-assembly of the DNL conjugate; and (c)
purify by an appropriate affinity chromatography.
DNL results in quantitative yields of a homogeneous product
with a defined composition and in vivo stability. The facile
binding between the DDD and AD modules effects nearly
100% conversion of each into the desired DNL product. The
site-specific conjugation also ensures that the full activity of
each module is preserved, the molecular size is homogeneous,
the composition is defined, and in vivo stability is sustained.
Potential Limitations
Fig. 4. Comparison of in vitro antiproliferative activity of the hexavalent
(Hex-hA20) and bispecific (DNL2) constructs with parental antibody, hA20, in
Daudi (A), Raji (B), and Ramos (C) lymphoma cell lines. Cell viability was
assessed by the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cells were plated at 5,000 per well
in 96-well plates and incubated for 4 d at 37jC with the test samples at the
indicated concentrations before adding MTS.
requiring purification before mixing. There is essentially no
limit on the types of precursors that can be derived into a DDD
or AD module, so long as the resulting modules do not interfere
with the dimerization of DDD or the binding of DDD to AD. In
addition to the DDD sequence of human RIIa, other DDD
sequences may be selected from human RIa, human RIh, or
human RIIh. The DDD sequence of choice will be matched
with a highly interactive AD sequence, which can be deduced
from the literature (29) or determined experimentally.
DNL is versatile. Modules may be made recombinantly or
chemically. Recombinant modules, which may be produced in
mammalian or microbial systems, may include derivatives of
antibodies or antibody fragments, cytokines, enzymes, natural
carrier proteins (such as human serum albumin and human
transferrin), or a variety of natural or artificial non-antibody
binding or scaffold proteins (30 – 33). Although each recombinant module would usually be produced in a separate
The yield of the precursor protein may predetermine that of
its recombinant DDD or AD module. We have found that
modules based on IgG or Fab fragments are expressed at high
levels similar to the precursor monoclonal antibody, whereas
recombinant proteins expressed at low levels result in DNL
modules also expressed at comparably low levels. Thus, the
optimal expression system for high-level production of a DDD
or AD module should be based on what would be best suited
for production of the precursor.
Although all of the DNL complexes that have been generated
to date are highly stable, it is possible that some other DNL
structures may be susceptible to proteolytic degradation in vivo,
and therefore, the serum stability of each new DNL complex
will need to be evaluated.
In an effort to minimize immunogenicity, the DDD and AD
used for the DNL method consist of the smallest functional
peptides derived from human protein sequences. However,
the DDD2 and AD2 peptides possess additional cysteine
residues, which are not part of the natural domains, and both
are fused to precursor proteins via flexible Gly-Ser linker
peptides, which are foreign but generally considered to be
largely nonimmunogenic. Whether the DNL constructs would
be immunogenic in humans can only be assessed in future
clinical trials.
Conclusions
We believe that the superior preclinical results obtained, to
date, with the trivalent bispecific Fab-based complexes as a
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Bioactive Structures by the DNL Method
pretargeted imaging agent will likely be translated successfully
into clinical trials. Present challenges lie in showing that
the DNL method is also useful as a tool for creating a new
class of cytokines with improved pharmacokinetic properties
and enhanced in vivo potency, as well as applicable to
developing a new paradigm for more effective vaccines
composed of multiple subunits, each being well defined.
When such challenges are met, the prospects of the DNL
method may indeed be endless and limited only by our
imagination.
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The Dock and Lock Method: A Novel Platform Technology
for Building Multivalent, Multifunctional Structures of
Defined Composition with Retained Bioactivity
Chien-Hsing Chang, Edmund A. Rossi and David M. Goldenberg
Clin Cancer Res 2007;13:5586s-5591s.
Updated version
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