BBAPAP-39361; No. of pages: 7; 4C: 2, 3, 4, 5, 6 Biochimica et Biophysica Acta xxx (2014) xxx–xxx
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Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions☆
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Jing Zhang, Terence H. Rabbitts
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Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
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Article history: Received 7 February 2014 Received in revised form 19 May 2014 Accepted 20 May 2014 Available online xxxx
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Keywords: Antibody Immunoglobulin Single domain VH IAC PPI Therapy Macrodrug Cancer
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Many proteins of interest in basic biology, translational research studies and for clinical targeting in diseases reside inside the cell and function by interacting with other macromolecules. Protein complexes control basic processes such as development and cell division but also abnormal cell growth when mutations occur such as found in cancer. Interfering with protein–protein interactions is an important aspiration in both basic and disease biology but small molecule inhibitors have been difficult and expensive to isolate. Recently, we have adapted molecular biology techniques to develop a simple set of protocols for isolation of high affinity antibody fragments (in the form of single VH domains) that function within the reducing environment of higher organism cells and can bind to their target molecules. The method called Intracellular Antibody Capture (IAC) has been used to develop inhibitory anti-RAS and anti-LMO2 single domains that have been used for target validation of these antigens in pre-clinical cancer models and illustrate the efficacy of the IAC approach to generation of drug surrogates. Future use of inhibitory VH antibody fragments as drugs in their own right (we term these macrodrugs to distinguish them from small molecule drugs) requires their delivery to target cells in vivo but they can also be templates for small molecule drug development that emulate the binding sites of the antibody fragments. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. © 2014 Published by Elsevier B.V.
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1. Introduction
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The specificity of antigen recognition by monoclonal antibodies (MAb) has been widely applied in bioscience research as well as medical applications including diagnosis and therapy [1]. Cancer therapies based on monoclonal anti-tumor antibodies have also been developed during the last 30 years. However, the tumor specific antigens, including EGFR, HER2, VEGF, and various clusters of differentiation (CD) antigens are limited to cell surface proteins of cancer cells [2,3]. Developments for the use of antibodies or antibody fragments inside cells [4,5] have seemed possible since it became clear that specific molecules could fold correctly within the reducing environment, to bind target antigens [6–8]. In particular, more recently the recognition that antibody fragments can effectively interfere with protein–protein interactions (PPI) [9,10] has highlighted their potential use in cell and disease biology. It is now an option in antibody-based cancer therapeutics to use these macromolecules (herein termed macrodrugs) as they are more readily isolated at high affinity than chemical compounds that bind to a protein and inhibit PPI.
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☆ This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
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2. Evolution of functional intracellular antibodies and antibody fragment isolation methods
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2.1. Antibody structure and derivation of antibody fragments
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The whole antibody is a large molecule with a molecular weight of about 150 kDa and is composed of two heavy chains and two light chains, which are held together by inter-chain disulfide bonds. In an IgG molecule, the two heavy chains are identical as well as the two light chains (Fig. 1A). Each chain, either heavy chain or light chain, is formed with two domains: variable or V regions (VH and VL respectively) and constant or C regions (CH and CL respectively). The V regions contribute to the antigen binding ability of a given antibody and each contains about 110 amino acids, whose sequence differs from those of other antibodies. Fragments of whole antibodies can be made by partial protease digestion into functionally distinct fragments known as Fab fragments (Fig. 1B), which are composed of VH and VL domains, a portion of CH domain (CH1), and CL domain. Fv fragments, however, have only VH and VL domains but still retain the antigen-binding ability (Fig. 1C). Novel antibody fragments have been made by protein engineering techniques, perhaps the most common linking the VL and VH domains with a short flexible linker to yield single chain Fv (scFv) [11] (Fig. 1D). The smallest fragment of antibody that retains specific antigen binding
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http://dx.doi.org/10.1016/j.bbapap.2014.05.009 1570-9639/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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Fig. 1. Antibody and antibody fragment structures. The whole antibody (IgG) comprises two heavy chains and two light chains (panel A). Each chain contains two distinct regions: a variable region (VL for light chain and VH for heavy chain) and a constant region (CL for light chain and CH1, CH2, CH3 for heavy chain). Fragments of antibody including Fab (B) and Fv (C) are respectively smaller in size compared with whole IgG while retaining full antigen binding ability. If the VL and VH are linked together with a short polypeptide, this creates a single chain Fv (scFv, panel D) or the V regions can be functional alone as VL or VH single domains (E). The structures are shown in ribbon form. The sizes of antibody/antibody fragments are given in approximate kilodalton (kDa).
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2.2. Direct in-cell screening for antibody fragments
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Using monoclonal antibodies as the source of intracellular antibodies has proven laborious and unpredictable. Direct screening methods for intracellular antibodies using diverse libraries are theoretically more attractive since these screens would be performed in the reducing cellular environment and in the natural cellular milieu in which the target protein resides. The two-hybrid system developed by Fields and colleagues using yeast host cells [22], provides a suitable format for this approach and was adapted into Intracellular Antibody Capture (IAC) [6,7]. The system is based on the activation of downstream reporter genes (lacZ or β-galactosidase) by binding to the gene controlled by a
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ability is the single domain, either VH or VL (DAbs) [12] (Fig. 1D). Some animal species, such as camels, llamas and sharks, naturally have domain antibodies [13–15]. This minimal fragment binds antigens because the V region displays amino acid variability in short regions called complementarity determining regions (CDRs) that are displayed as external, antigen binding loops on the domain antibodies (Fig. 2A–C). Although the single domains do have conserved cysteine residues that give intra-chain disulfide bonds, these are not required for the correct folding of VH or VL since the crystal structures of single domains with or without these cysteine residues are almost identical [16]. This feature probably explains why single domains can fold and function intracellularly as minimal antibody fragments (iDabs) [17]. The use of antibodies inside cells that started life as whole antibodies injected into cells [18], evolved into a gene-based approach [4], to the use of scFv antibody fragments [19,20] and, with the finding that single domains are functionally active in cells, to the use of VH or VL polypeptide chains [21].
minimal promoter and a specific DNA binding site of a transcription factor complex formed from the bait protein (the target fused to a DNAbinding domain) and the prey protein (an antibody fragment fused to the VP16 transcriptional activation domain). The first generation of IAC [6,7,21] employed phage scFv libraries for in vitro phage display selection as a first step. All antigen-binding phage were collected to generate a sub-library of scFv in a yeast prey vector. The sub-library was screened in yeast using a target protein bait. Selected candidates from the scFv sub-library were characterized in yeast and mammalian cells for further identification [6]. In the second and third generation of IAC, the phage screen step was omitted and direct screening of yeast libraries was performed using intracellular scFv libraries in yeast [9]. The third generation of Intracellular Antibody Capture (IAC3) was thus developed using diverse libraries of single variable regions [9] directly screened in yeast (Fig. 3A, B). Typically, around 5 million yeast transfectants are screened in one experiment and we initially used VH libraries that were randomized only at CDR3. The yeast, in which the bait (antigen) and prey (VH) bind, grow on plates lacking histidine as the yeast strain used has a histidine synthesis reporter gene that can be activated by the bait–prey transcriptional complex (Fig. 3B, C). The interaction-specific his gene activation is confirmed by showing that a β-gal gene can be activated by the bait–prey transcriptional complex (Fig. 3D, E). The affinity of the selected VH can be improved by sequential randomization of CDR2 and re-screening, and then by similar treatment of CDR1 of selected clones (Fig. 2D). In this strategy, improvements in binding capacity is assessed by titration of yeast growth on increasing concentrations of 3-amino-1,2,4-triazole (3-AT, an inhibitor of the enzyme encoded by His3). The clones that
Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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grow at the highest 3-AT inhibitor concentration are selected and transferred to a mammalian two-hybrid (M2H) vector to test their effectiveness in binding to the target antigen (Fig. 4A, B). In the original incarnation of IAC3, we generated and screened a set of VH libraries with diversity at CDR3 only. The rationale was that CDR3
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Fig. 2. Single domain antibodies, libraries and affinity maturation. A. The β-sheets in the framework regions of antibody single variable domains (VH and VL) are shown as green ribbons and the complementarity determining regions (CDR1, CDR2 and CDR3) are shown in blue. B/C. The sequences of the CDRs for the VH (B) and VL (C) are shown with the non-variable framework regions represented as green lines. The amino acid sequences of CDRs are shown in the blue boxes. In IAC3, the libraries are initially only randomized in CDR3 for screening (the randomized amino acids are indicated by X). In the VH domains, naturally occurring antibodies can have CDR3 length variation from 2 to more than 16. D. The IAC yeast screening strategy (IAC is outlined in Fig. 3) allows for affinity maturation in iterative selections [9]. Initially, either a VH or a VL library is screened and clones obtained with yeast IAC may have low affinity for the target protein. One option for affinity maturation is to pool all positive clones from the first screen and randomize the CDR2 residues (not altering the selected CDR3) followed by rescreening. A similar further round of screening with randomized CDR1 is possible to potentially attain the maximum affinity single domain.
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Fig. 3. Intracellular antibody capture (IAC3). Isolation of intracellular antibody single domain fragments using IAC3 is illustrated. The initial screen in yeast involves either a VH library or a VL library (with randomized CDR3) cotransfected in yeast with a bait target protein (A). The yeast strain is deficient for growth in the absence of histidine and carries a his reporter gene that can be activated by the interaction of bait (target) and prey (single domain). Thus transfected yeast colonies can grow only on his-deficient medium if the his gene is activated (B, C). Clones growing on his-deficient medium are verified by plating on Xgal medium to generate β-galactosidase from a β-gal reporter gene and generate blue colonies (D, E).
Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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Fig. 4. Identifying intracellular single domain inhibitors of PPI. A mammalian (CHO) cell line has been developed carrying a Gal4-DBS-luciferase (Firefly) reported gene [9]. When this line is transfected with a vector expressing bait and prey protein (in this example, hypothetical proteins X &Y, panel A), Firefly luciferase is produced as a result of interaction of bait and prey and binding to the DBS (B). The dual bait and prey expressing vector also constitutively makes Renilla luciferase for normalization of Firefly luciferase in each transient transfection. Potential for a single domain to interfere with the interaction of proteins X and Y is determined by co-transfecting the dual bait–prey vector with a vector expressing the single domain (C) and comparison of luciferase levels with coexpressed single domain (iDab+) to those without expressing V region (iDab−) (D).
(Zeng and THR, unpublished). This latter approach greatly simplifies and speeds up the screening of IAC3 single domain libraries.
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2.3. PPI target validation with intracellular antibodies
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Not all single domains selected by the IAC method will inhibit the PPI of the target molecule as this is dependent on the location of the antigenic epitope. We developed a simple secondary screening vector system to identify the PPI inhibitory single domains from the selected clones [9,24]. This comprises using a cell line carrying the Firefly luciferase reporter gene controlled by a minimal protein linked to a Gal4 DNA binding site. This line can be transfected with a vector expressing a Gal4DBD-target fusion (designated antigen X, bait) and a partner proteinVP16 activation domain fusion (designated protein Y, prey) (the Triplex vector, Fig. 4A). These cells are responsive to activation of Firefly luciferase signals due to interaction of bait and prey (Fig. 4B). The ability of individual VH single domains to inhibit the interaction of bait and prey can be assessed by co-expressing the bait–prey expressing vector with a vector encoding the specific VH (Fig. 4C). Competitive binding of the VH to the antigen bait will reduce or eliminate the production of Firefly luciferase (Fig. 4D). An additional approach to antibodies that interfere with PPI, called SPLINT, has also been described [10]. The molecular biology techniques outlined here facilitate the isolation of single domains that bind to target proteins and inhibit their PPI with natural partners. The purpose of this is to isolate inhibitory molecules that locate at specific sites on the target protein within the context of the intracellular environment of the cell. In this way, the objective is distinct from RNAi approaches that knock down all functions of a target protein by removing the mRNA. Both methods are important for target validation studies (i.e. is a target protein actually important for the implicated clinical indication) and encounter similar difficulties in their use as macrodrugs per se (see below).
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3. Exemplification of intracellular single domains as anti-cancer macrodrugs
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3.1. A VH single domain to interrogate the importance of mutant RAS-effector interactions in cancer
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The RAS family of oncogenes is among the most mutated in human cancers [25,26] and the mutant proteins are constitutively activated signaling molecules interacting with effector molecules such as RAF, PI3K, RALGDS [27–30]. We used the IAC approach to isolate an anti-RAS VH single domain and demonstrated that this VH could completely suppress RAS-dependent tumor formation in a mouse lung cancer model [31] and could halt the growth of RAS-mutant-carrying human tumor lines in a mouse xenograft colorectal tumor model [32]. The anti-RAS VH, which binds to activated forms of KRAS, HRAS and NRAS, has a nM affinity for binding to the RAS switch region where the natural effectors bind (these affinities range around μM) and competes for the binding site [31]. These data formally demonstrate for the first time that mutant RAS–effector interaction is necessary for cancer growth and that inhibiting RAS–effector interaction is a valid strategy for cancer therapy. Recently, it was confirmed that RAS-PI3K p110a interaction was required for lung tumor maintenance [33]. The VH single domain that we have used is therefore a drug surrogate for target validation and it is also a template for drug development (see below).
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3.2. The mechanism of PPI inhibition of an anti-LMO2 VH and target validation in T cell acute leukemia
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The inhibitory effect of the anti-RAS VH on RAS protein complexes and on RAS signaling occurs by direct competitive interactions of the VH with the RAS switch region [21]. We have carried out analogous studies with a single domain binding to the T cell oncogenic protein
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Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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4. Future prospectives: single VH domain macrodrug delivery options
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Sections 3.1 and 3.2 illustrate the potential for intracellular single domains to act as drug surrogates to interfere with PPI. Targeted macrodrug delivery has the potential to enhance the efficiency of inhibiting tumor cells while reducing the drug-induced toxicity and development of drug resistance. Can these antibody fragments be used as drugs (macrodrugs) per se rather than drug surrogates? Although macrodrugs, such as iDabs, are showing promising results in vitro, they are likely to be restricted as protein drugs in vivo because of fast elimination due to clearance, generation of immune responses and poor accuracy of uptake by target cells since these are macromolecules [38]. For these reasons, we have designated these protein ‘drugs’ as macrodrugs [39]. Rather than attempting to deliver these macrodrugs as proteins into cells, an alternative strategy that we are adopting is delivery of nucleic acid expression vectors to facilitate expression of the iDabs within target cells. The approach has produced promising results for delivery of RNAi molecules [40] and can potentially be exploited for protein macrodrugs. The use of viral delivery is another possible option but will not be discussed in this review of our IAC work.
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4.1. Antibody-coupled vector delivery
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Conventional antibodies that bind to tumor cell surface antigens can carry cargos to local tumor sites [41,42]. By targeting a tumor surface antigen, antibody-mediated delivery [43] provides a potent method for macrodrug delivery (illustrated in Fig. 5A). When an antibody binds to an internalizing receptor on the cell surface, it triggers receptormediated uptake and is subsequently transferred into the cell interior, together with the cargo. In this case, the cargo can be a macrodrug expression vector, which will released from the carrier antibody due to reducing environment or pH change within the cell. One of the successful examples that have been reported using this approach is the delivery of small interfering RNAs (siRNAs). Antibodies targeting the HIV envelope, ErbB2 or CD7 were fused to either the nucleic acid-binding protein protamine or a 9-arginine peptide and loaded with siRNA to be successfully delivered into HIV infected cells, ErbB2-expressing cancer cells or CD7-expressing T cells respectively [42,44–46]. The success of this targeted siRNA delivery suggests the possibility of similarly delivering antibody-fragment macrodrug protein expression vectors (Fig. 5A). Antibody-mediated nucleic acid delivery systems, in principle, would avoid immune responses and enzymatic degradation while increasing cell-specific uptake efficiency.
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Fig. 5. Potential options for delivery of intracellular antibody expression vectors. Two potential methods for delivering single domain expression vectors into cells are illustrated. (A) Antibodies that recognize a surface antigen on the target cells (represented by the brown boxes) can be adapted to carry nucleic acids at their C terminus. After engagement with the specific receptor, internalization can occur and release of the vector internally to allow antigen-specific single domain expression within the cell. (B) An alternative is the use of nanoparticles to carry the intracellular antibody expression vectors to the cell of interest. Coating of the nanoparticle with specific antibody fragments binding to a cell surface antigen would enhance local concentration of the particle and the specificity of macrodrug delivery.
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LMO2, a LIM-domain protein acting as a protein interaction module in a multi-protein complex in T cell acute leukemia [34]. The anti-LMO2 single VH domain was isolated with the IAC3 method [35] and the VH prevented growth of LMO2-dependent T cell tumors in a mouse bone marrow transplantation assay [35,36]. Cell-based assays suggested that the VH was inhibiting LMO2-based PPI since there was evidence of interference with interactions with members of the LMO2 protein complex [37]. Clarification was obtained by solving the crystal structure of LMO2 in complex with the VH [36] as this showed that the shape of the bound LMO2 was different from its shape when bound to its natural protein partner LDB1. The effect of the VH binding was to bend and twist the LMO2 molecule around a short ‘hinge’ helix between the two LIM domains of LMO2 in such a way as to impede the natural interaction with LDB1. Thus the mechanism of VH inhibition of LMO2 PPI is by subverting the LMO2 molecule into an unnatural shape. This finding has implications for the way that natural LMO2 protein complexes form. The model is that initially LMO2 is produced as an unstructured protein and the formation of its multi-complex is facilitated by LMO2–LDB1 interaction that “fixes” LMO2 conformation to nucleate the formation of the complex [36]. The VH-induced shape of LMO2 hampers the formation of the natural complex.
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An alternative option for macrodrug delivery is the use of nanoparticles as carriers of cargos. In recent years, nanoparticles have attracted attention for clinical use due to their versatility and in vivo pharmacokinetic properties. By adjusting the properties of nanoparticles, drug loading can be achieved by encapsulating or attaching macromolecules thereby stabilizing them from degradation [47,48]. In addition, they are able to overcome limitations of conventional drugs such as low solubility and stability, as well as allowing targeted delivery and controlled release of the loaded drug [47]. Liposomes were one of the first nanoparticle formulations [49] applied in medicine and have been extensively developed for drug delivery in the past 15 years. Liposomal nanoparticles are closed spherical vesicles formed from lipid bilayers where a nucleic acid or drug cargo can be stored for systemic delivery and cell uptake via the plasma membrane [49,50]. Since the first clinical approved liposomal-drug formulation (Liposomal amphotericin B, AmBisome) in 1990, there are more than 17 liposomal anticancer drugs available in clinic [51,52]. Methods for coupling liposomes with antibodies or antibody fragments, which are specific to cell surface antigens, have also been developed extensively [53,54]. Encapsulation of single domains or vectors that are able to express single domains is a possible development to use nanoparticles to deliver these macrodrugs, as outlined in Fig. 5B. In addition to encapsulating vectors in liposomal nanoparticles, recent developments with gold particles [55] and nanoparticle polymers [56] coated with nucleic acids all promise to bring the drug surrogacy
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Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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Work underpinning this article has been supported by the Medical Research Council and Leukaemia and Lymphoma Research.
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The part of the single domain antibody fragment that binds to an antigen is the CDRs (Fig. 2A) and can be as few as 8–10 amino acids. The space occupied by the binding site of whole IgG is about 1500 Å2 [57] while drug-like small molecules range from 300 to 1000 Å2 [2]. The binding site of single domains (VH or VL) is less than 700 Å2, which is within the size range of small molecule drugs. Starting with a high affinity binding by single domain and a high-resolution structure of the single domain bound to its target molecule, this is a route to PPI inhibitor drug development (outlined in Fig. 6). The critical step is to delineate the anchor residues of the single domain CDRs to use as a template for small molecule design. The ease with which intracellular antibody fragments can be obtained using the IAC technology promises to provide a simple route from target validation through to drug development.
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of intracellular domain antibodies into mainstream drug use. This potentially opens a strategy for rapid development of antibody fragment inhibitors of PPI for drug use by implementing molecular biology methods such as described here.
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Fig. 6. Small molecule emulators of single domain binding sites. The CDR region of the single domain has a shape and the interaction surface, involved in binding, occupies less than 1000 Å2. When anchor residues within the CDRs are established, it reduces this binding surface to that suitable for a small molecule emulator. The development of small molecule CDR emulators ideally requires the structure of the V region bound to its target and some form of HTS (in silico or actual) to find hits for lead development. The purpose is to develop small molecules that can enter cells and find the target protein for binding and interference with the target PPI.
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Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions☆
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Jing Zhang, Terence H. Rabbitts
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Article history: Received 7 February 2014 Received in revised form 19 May 2014 Accepted 20 May 2014 Available online xxxx
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Keywords: Antibody Immunoglobulin Single domain VH IAC PPI Therapy Macrodrug Cancer
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Many proteins of interest in basic biology, translational research studies and for clinical targeting in diseases reside inside the cell and function by interacting with other macromolecules. Protein complexes control basic processes such as development and cell division but also abnormal cell growth when mutations occur such as found in cancer. Interfering with protein–protein interactions is an important aspiration in both basic and disease biology but small molecule inhibitors have been difficult and expensive to isolate. Recently, we have adapted molecular biology techniques to develop a simple set of protocols for isolation of high affinity antibody fragments (in the form of single VH domains) that function within the reducing environment of higher organism cells and can bind to their target molecules. The method called Intracellular Antibody Capture (IAC) has been used to develop inhibitory anti-RAS and anti-LMO2 single domains that have been used for target validation of these antigens in pre-clinical cancer models and illustrate the efficacy of the IAC approach to generation of drug surrogates. Future use of inhibitory VH antibody fragments as drugs in their own right (we term these macrodrugs to distinguish them from small molecule drugs) requires their delivery to target cells in vivo but they can also be templates for small molecule drug development that emulate the binding sites of the antibody fragments. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. © 2014 Published by Elsevier B.V.
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1. Introduction
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The specificity of antigen recognition by monoclonal antibodies (MAb) has been widely applied in bioscience research as well as medical applications including diagnosis and therapy [1]. Cancer therapies based on monoclonal anti-tumor antibodies have also been developed during the last 30 years. However, the tumor specific antigens, including EGFR, HER2, VEGF, and various clusters of differentiation (CD) antigens are limited to cell surface proteins of cancer cells [2,3]. Developments for the use of antibodies or antibody fragments inside cells [4,5] have seemed possible since it became clear that specific molecules could fold correctly within the reducing environment, to bind target antigens [6–8]. In particular, more recently the recognition that antibody fragments can effectively interfere with protein–protein interactions (PPI) [9,10] has highlighted their potential use in cell and disease biology. It is now an option in antibody-based cancer therapeutics to use these macromolecules (herein termed macrodrugs) as they are more readily isolated at high affinity than chemical compounds that bind to a protein and inhibit PPI.
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2. Evolution of functional intracellular antibodies and antibody fragment isolation methods
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2.1. Antibody structure and derivation of antibody fragments
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The whole antibody is a large molecule with a molecular weight of about 150 kDa and is composed of two heavy chains and two light chains, which are held together by inter-chain disulfide bonds. In an IgG molecule, the two heavy chains are identical as well as the two light chains (Fig. 1A). Each chain, either heavy chain or light chain, is formed with two domains: variable or V regions (VH and VL respectively) and constant or C regions (CH and CL respectively). The V regions contribute to the antigen binding ability of a given antibody and each contains about 110 amino acids, whose sequence differs from those of other antibodies. Fragments of whole antibodies can be made by partial protease digestion into functionally distinct fragments known as Fab fragments (Fig. 1B), which are composed of VH and VL domains, a portion of CH domain (CH1), and CL domain. Fv fragments, however, have only VH and VL domains but still retain the antigen-binding ability (Fig. 1C). Novel antibody fragments have been made by protein engineering techniques, perhaps the most common linking the VL and VH domains with a short flexible linker to yield single chain Fv (scFv) [11] (Fig. 1D). The smallest fragment of antibody that retains specific antigen binding
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http://dx.doi.org/10.1016/j.bbapap.2014.05.009 1570-9639/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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Fig. 1. Antibody and antibody fragment structures. The whole antibody (IgG) comprises two heavy chains and two light chains (panel A). Each chain contains two distinct regions: a variable region (VL for light chain and VH for heavy chain) and a constant region (CL for light chain and CH1, CH2, CH3 for heavy chain). Fragments of antibody including Fab (B) and Fv (C) are respectively smaller in size compared with whole IgG while retaining full antigen binding ability. If the VL and VH are linked together with a short polypeptide, this creates a single chain Fv (scFv, panel D) or the V regions can be functional alone as VL or VH single domains (E). The structures are shown in ribbon form. The sizes of antibody/antibody fragments are given in approximate kilodalton (kDa).
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2.2. Direct in-cell screening for antibody fragments
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Using monoclonal antibodies as the source of intracellular antibodies has proven laborious and unpredictable. Direct screening methods for intracellular antibodies using diverse libraries are theoretically more attractive since these screens would be performed in the reducing cellular environment and in the natural cellular milieu in which the target protein resides. The two-hybrid system developed by Fields and colleagues using yeast host cells [22], provides a suitable format for this approach and was adapted into Intracellular Antibody Capture (IAC) [6,7]. The system is based on the activation of downstream reporter genes (lacZ or β-galactosidase) by binding to the gene controlled by a
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ability is the single domain, either VH or VL (DAbs) [12] (Fig. 1D). Some animal species, such as camels, llamas and sharks, naturally have domain antibodies [13–15]. This minimal fragment binds antigens because the V region displays amino acid variability in short regions called complementarity determining regions (CDRs) that are displayed as external, antigen binding loops on the domain antibodies (Fig. 2A–C). Although the single domains do have conserved cysteine residues that give intra-chain disulfide bonds, these are not required for the correct folding of VH or VL since the crystal structures of single domains with or without these cysteine residues are almost identical [16]. This feature probably explains why single domains can fold and function intracellularly as minimal antibody fragments (iDabs) [17]. The use of antibodies inside cells that started life as whole antibodies injected into cells [18], evolved into a gene-based approach [4], to the use of scFv antibody fragments [19,20] and, with the finding that single domains are functionally active in cells, to the use of VH or VL polypeptide chains [21].
minimal promoter and a specific DNA binding site of a transcription factor complex formed from the bait protein (the target fused to a DNAbinding domain) and the prey protein (an antibody fragment fused to the VP16 transcriptional activation domain). The first generation of IAC [6,7,21] employed phage scFv libraries for in vitro phage display selection as a first step. All antigen-binding phage were collected to generate a sub-library of scFv in a yeast prey vector. The sub-library was screened in yeast using a target protein bait. Selected candidates from the scFv sub-library were characterized in yeast and mammalian cells for further identification [6]. In the second and third generation of IAC, the phage screen step was omitted and direct screening of yeast libraries was performed using intracellular scFv libraries in yeast [9]. The third generation of Intracellular Antibody Capture (IAC3) was thus developed using diverse libraries of single variable regions [9] directly screened in yeast (Fig. 3A, B). Typically, around 5 million yeast transfectants are screened in one experiment and we initially used VH libraries that were randomized only at CDR3. The yeast, in which the bait (antigen) and prey (VH) bind, grow on plates lacking histidine as the yeast strain used has a histidine synthesis reporter gene that can be activated by the bait–prey transcriptional complex (Fig. 3B, C). The interaction-specific his gene activation is confirmed by showing that a β-gal gene can be activated by the bait–prey transcriptional complex (Fig. 3D, E). The affinity of the selected VH can be improved by sequential randomization of CDR2 and re-screening, and then by similar treatment of CDR1 of selected clones (Fig. 2D). In this strategy, improvements in binding capacity is assessed by titration of yeast growth on increasing concentrations of 3-amino-1,2,4-triazole (3-AT, an inhibitor of the enzyme encoded by His3). The clones that
Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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grow at the highest 3-AT inhibitor concentration are selected and transferred to a mammalian two-hybrid (M2H) vector to test their effectiveness in binding to the target antigen (Fig. 4A, B). In the original incarnation of IAC3, we generated and screened a set of VH libraries with diversity at CDR3 only. The rationale was that CDR3
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Fig. 2. Single domain antibodies, libraries and affinity maturation. A. The β-sheets in the framework regions of antibody single variable domains (VH and VL) are shown as green ribbons and the complementarity determining regions (CDR1, CDR2 and CDR3) are shown in blue. B/C. The sequences of the CDRs for the VH (B) and VL (C) are shown with the non-variable framework regions represented as green lines. The amino acid sequences of CDRs are shown in the blue boxes. In IAC3, the libraries are initially only randomized in CDR3 for screening (the randomized amino acids are indicated by X). In the VH domains, naturally occurring antibodies can have CDR3 length variation from 2 to more than 16. D. The IAC yeast screening strategy (IAC is outlined in Fig. 3) allows for affinity maturation in iterative selections [9]. Initially, either a VH or a VL library is screened and clones obtained with yeast IAC may have low affinity for the target protein. One option for affinity maturation is to pool all positive clones from the first screen and randomize the CDR2 residues (not altering the selected CDR3) followed by rescreening. A similar further round of screening with randomized CDR1 is possible to potentially attain the maximum affinity single domain.
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Please cite this article as: J. Zhang, T.H. Rabbitts, Intracellular antibody capture: A molecular biology approach to inhibitors of protein–protein interactions, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.05.009
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Fig. 4. Identifying intracellular single domain inhibitors of PPI. A mammalian (CHO) cell line has been developed carrying a Gal4-DBS-luciferase (Firefly) reported gene [9]. When this line is transfected with a vector expressing bait and prey protein (in this example, hypothetical proteins X &Y, panel A), Firefly luciferase is produced as a result of interaction of bait and prey and binding to the DBS (B). The dual bait and prey expressing vector also constitutively makes Renilla luciferase for normalization of Firefly luciferase in each transient transfection. Potential for a single domain to interfere with the interaction of proteins X and Y is determined by co-transfecting the dual bait–prey vector with a vector expressing the single domain (C) and comparison of luciferase levels with coexpressed single domain (iDab+) to those without expressing V region (iDab−) (D).
(Zeng and THR, unpublished). This latter approach greatly simplifies and speeds up the screening of IAC3 single domain libraries.
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Not all single domains selected by the IAC method will inhibit the PPI of the target molecule as this is dependent on the location of the antigenic epitope. We developed a simple secondary screening vector system to identify the PPI inhibitory single domains from the selected clones [9,24]. This comprises using a cell line carrying the Firefly luciferase reporter gene controlled by a minimal protein linked to a Gal4 DNA binding site. This line can be transfected with a vector expressing a Gal4DBD-target fusion (designated antigen X, bait) and a partner proteinVP16 activation domain fusion (designated protein Y, prey) (the Triplex vector, Fig. 4A). These cells are responsive to activation of Firefly luciferase signals due to interaction of bait and prey (Fig. 4B). The ability of individual VH single domains to inhibit the interaction of bait and prey can be assessed by co-expressing the bait–prey expressing vector with a vector encoding the specific VH (Fig. 4C). Competitive binding of the VH to the antigen bait will reduce or eliminate the production of Firefly luciferase (Fig. 4D). An additional approach to antibodies that interfere with PPI, called SPLINT, has also been described [10]. The molecular biology techniques outlined here facilitate the isolation of single domains that bind to target proteins and inhibit their PPI with natural partners. The purpose of this is to isolate inhibitory molecules that locate at specific sites on the target protein within the context of the intracellular environment of the cell. In this way, the objective is distinct from RNAi approaches that knock down all functions of a target protein by removing the mRNA. Both methods are important for target validation studies (i.e. is a target protein actually important for the implicated clinical indication) and encounter similar difficulties in their use as macrodrugs per se (see below).
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3.1. A VH single domain to interrogate the importance of mutant RAS-effector interactions in cancer
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The RAS family of oncogenes is among the most mutated in human cancers [25,26] and the mutant proteins are constitutively activated signaling molecules interacting with effector molecules such as RAF, PI3K, RALGDS [27–30]. We used the IAC approach to isolate an anti-RAS VH single domain and demonstrated that this VH could completely suppress RAS-dependent tumor formation in a mouse lung cancer model [31] and could halt the growth of RAS-mutant-carrying human tumor lines in a mouse xenograft colorectal tumor model [32]. The anti-RAS VH, which binds to activated forms of KRAS, HRAS and NRAS, has a nM affinity for binding to the RAS switch region where the natural effectors bind (these affinities range around μM) and competes for the binding site [31]. These data formally demonstrate for the first time that mutant RAS–effector interaction is necessary for cancer growth and that inhibiting RAS–effector interaction is a valid strategy for cancer therapy. Recently, it was confirmed that RAS-PI3K p110a interaction was required for lung tumor maintenance [33]. The VH single domain that we have used is therefore a drug surrogate for target validation and it is also a template for drug development (see below).
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3.2. The mechanism of PPI inhibition of an anti-LMO2 VH and target validation in T cell acute leukemia
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The inhibitory effect of the anti-RAS VH on RAS protein complexes and on RAS signaling occurs by direct competitive interactions of the VH with the RAS switch region [21]. We have carried out analogous studies with a single domain binding to the T cell oncogenic protein
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4. Future prospectives: single VH domain macrodrug delivery options
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Sections 3.1 and 3.2 illustrate the potential for intracellular single domains to act as drug surrogates to interfere with PPI. Targeted macrodrug delivery has the potential to enhance the efficiency of inhibiting tumor cells while reducing the drug-induced toxicity and development of drug resistance. Can these antibody fragments be used as drugs (macrodrugs) per se rather than drug surrogates? Although macrodrugs, such as iDabs, are showing promising results in vitro, they are likely to be restricted as protein drugs in vivo because of fast elimination due to clearance, generation of immune responses and poor accuracy of uptake by target cells since these are macromolecules [38]. For these reasons, we have designated these protein ‘drugs’ as macrodrugs [39]. Rather than attempting to deliver these macrodrugs as proteins into cells, an alternative strategy that we are adopting is delivery of nucleic acid expression vectors to facilitate expression of the iDabs within target cells. The approach has produced promising results for delivery of RNAi molecules [40] and can potentially be exploited for protein macrodrugs. The use of viral delivery is another possible option but will not be discussed in this review of our IAC work.
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Conventional antibodies that bind to tumor cell surface antigens can carry cargos to local tumor sites [41,42]. By targeting a tumor surface antigen, antibody-mediated delivery [43] provides a potent method for macrodrug delivery (illustrated in Fig. 5A). When an antibody binds to an internalizing receptor on the cell surface, it triggers receptormediated uptake and is subsequently transferred into the cell interior, together with the cargo. In this case, the cargo can be a macrodrug expression vector, which will released from the carrier antibody due to reducing environment or pH change within the cell. One of the successful examples that have been reported using this approach is the delivery of small interfering RNAs (siRNAs). Antibodies targeting the HIV envelope, ErbB2 or CD7 were fused to either the nucleic acid-binding protein protamine or a 9-arginine peptide and loaded with siRNA to be successfully delivered into HIV infected cells, ErbB2-expressing cancer cells or CD7-expressing T cells respectively [42,44–46]. The success of this targeted siRNA delivery suggests the possibility of similarly delivering antibody-fragment macrodrug protein expression vectors (Fig. 5A). Antibody-mediated nucleic acid delivery systems, in principle, would avoid immune responses and enzymatic degradation while increasing cell-specific uptake efficiency.
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Fig. 5. Potential options for delivery of intracellular antibody expression vectors. Two potential methods for delivering single domain expression vectors into cells are illustrated. (A) Antibodies that recognize a surface antigen on the target cells (represented by the brown boxes) can be adapted to carry nucleic acids at their C terminus. After engagement with the specific receptor, internalization can occur and release of the vector internally to allow antigen-specific single domain expression within the cell. (B) An alternative is the use of nanoparticles to carry the intracellular antibody expression vectors to the cell of interest. Coating of the nanoparticle with specific antibody fragments binding to a cell surface antigen would enhance local concentration of the particle and the specificity of macrodrug delivery.
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LMO2, a LIM-domain protein acting as a protein interaction module in a multi-protein complex in T cell acute leukemia [34]. The anti-LMO2 single VH domain was isolated with the IAC3 method [35] and the VH prevented growth of LMO2-dependent T cell tumors in a mouse bone marrow transplantation assay [35,36]. Cell-based assays suggested that the VH was inhibiting LMO2-based PPI since there was evidence of interference with interactions with members of the LMO2 protein complex [37]. Clarification was obtained by solving the crystal structure of LMO2 in complex with the VH [36] as this showed that the shape of the bound LMO2 was different from its shape when bound to its natural protein partner LDB1. The effect of the VH binding was to bend and twist the LMO2 molecule around a short ‘hinge’ helix between the two LIM domains of LMO2 in such a way as to impede the natural interaction with LDB1. Thus the mechanism of VH inhibition of LMO2 PPI is by subverting the LMO2 molecule into an unnatural shape. This finding has implications for the way that natural LMO2 protein complexes form. The model is that initially LMO2 is produced as an unstructured protein and the formation of its multi-complex is facilitated by LMO2–LDB1 interaction that “fixes” LMO2 conformation to nucleate the formation of the complex [36]. The VH-induced shape of LMO2 hampers the formation of the natural complex.
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An alternative option for macrodrug delivery is the use of nanoparticles as carriers of cargos. In recent years, nanoparticles have attracted attention for clinical use due to their versatility and in vivo pharmacokinetic properties. By adjusting the properties of nanoparticles, drug loading can be achieved by encapsulating or attaching macromolecules thereby stabilizing them from degradation [47,48]. In addition, they are able to overcome limitations of conventional drugs such as low solubility and stability, as well as allowing targeted delivery and controlled release of the loaded drug [47]. Liposomes were one of the first nanoparticle formulations [49] applied in medicine and have been extensively developed for drug delivery in the past 15 years. Liposomal nanoparticles are closed spherical vesicles formed from lipid bilayers where a nucleic acid or drug cargo can be stored for systemic delivery and cell uptake via the plasma membrane [49,50]. Since the first clinical approved liposomal-drug formulation (Liposomal amphotericin B, AmBisome) in 1990, there are more than 17 liposomal anticancer drugs available in clinic [51,52]. Methods for coupling liposomes with antibodies or antibody fragments, which are specific to cell surface antigens, have also been developed extensively [53,54]. Encapsulation of single domains or vectors that are able to express single domains is a possible development to use nanoparticles to deliver these macrodrugs, as outlined in Fig. 5B. In addition to encapsulating vectors in liposomal nanoparticles, recent developments with gold particles [55] and nanoparticle polymers [56] coated with nucleic acids all promise to bring the drug surrogacy
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Work underpinning this article has been supported by the Medical Research Council and Leukaemia and Lymphoma Research.
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The part of the single domain antibody fragment that binds to an antigen is the CDRs (Fig. 2A) and can be as few as 8–10 amino acids. The space occupied by the binding site of whole IgG is about 1500 Å2 [57] while drug-like small molecules range from 300 to 1000 Å2 [2]. The binding site of single domains (VH or VL) is less than 700 Å2, which is within the size range of small molecule drugs. Starting with a high affinity binding by single domain and a high-resolution structure of the single domain bound to its target molecule, this is a route to PPI inhibitor drug development (outlined in Fig. 6). The critical step is to delineate the anchor residues of the single domain CDRs to use as a template for small molecule design. The ease with which intracellular antibody fragments can be obtained using the IAC technology promises to provide a simple route from target validation through to drug development.
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Fig. 6. Small molecule emulators of single domain binding sites. The CDR region of the single domain has a shape and the interaction surface, involved in binding, occupies less than 1000 Å2. When anchor residues within the CDRs are established, it reduces this binding surface to that suitable for a small molecule emulator. The development of small molecule CDR emulators ideally requires the structure of the V region bound to its target and some form of HTS (in silico or actual) to find hits for lead development. The purpose is to develop small molecules that can enter cells and find the target protein for binding and interference with the target PPI.
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