Review Article
Selection of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes for Adoptive T-Cell Therapy Ralph Willemsen,1* Patrick Chames,2 Erik Schooten,1 Jan Willem Gratama,1 Reno Debets1
1
Department of Medical Oncology, Unit Clinical and Tumor Immunology, Tumor Immunology Group, Erasmus MC Daniel den Hoed Cancer Center, 3075 EA Rotterdam, The Netherlands
2
National Research Institute, CNRS LISM, 31 Chemin Joseph Aiguier, Marseille, France
Received 12 March 2008; Revision Received 9 June 2008; Accepted 11 August 2008 Additional Supporting Information may be found in the online version of this article. *Correspondence to: Ralph Willemsen, Department of Medical Oncology, Unit Clinical and Tumor Immunology, Tumor Immunology Group, Erasmus MC Daniel den Hoed Cancer Center, Groene Hilledijk 301, 3075 EA Rotterdam, The Netherlands Email:
[email protected] Published online 10 September 2008 in Wiley InterScience (www.interscience. wiley.com) DOI: 10.1002/cyto.a.20644 © 2008 International Society for Advancement of Cytometry
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Abstract Adoptive transfer of antigen-specific T-cells has shown therapeutic successes in the treatment of tumors in patients with metastatic melanoma. Tumor antigen-specific Tlymphocytes, however, occur only at low frequencies in a small proportion of patients. This low T-lymphocyte frequency together with the difficulties associated with in vitro generation of T-lymphocytes specific for cancers other than melanoma hampers adoptive T cell therapy. To make adoptive T-cell therapy more uniformly applicable, strategies were developed at transferring tumor-specificity to primary human T-lymphocytes via antibody (Ig) or T-cell receptor (TCR) molecules. We exploited the selection power of phage display that allows for the testing of tens of billions of individual clones with a high-throughput selection of Fabs with peptide/MHC complex binding capacity. Following in vitro selection, human ‘‘TCR-like’’ Fab fragments have been functionally expressed on human T-lymphocytes, resulting in MHC-restricted, tumor-specific lysis and cytokine production. Currently, we have extended our selections to a panel of class I and II MHC-restricted MAGE and other tumor-specific epitopes, and would like to propose that phage display represents a technology able to expand T-cell therapy to numerous tumor types. ' 2008 International Society for Advancement of Cytometry Key terms T-cell therapy; phage display; antibodies
GENOME -wide analyses and microarray studies result in the characterization of an ever-increasing number of tumor markers (1,2). Although some of these markers are membrane proteins, possibly displaying an immunogenic ectodomain, most of them are intracellular proteins, inaccessible to classical antibodies. These intracellular proteins are degraded by the proteasome or endo/lysosomes, with some of the resulting peptides getting associated with HLA class I or II complexes. These complexes are then displayed at the cell surface as peptide-MHC complexes (pMHC) where they provide targets for T-cell recognition via pMHC:T-cell receptor (TCR) interactions. Consequently, a large number of cancer immunotherapies are aimed at triggering or targeting T-cell responses toward a low number of cancer-specific pMHCs (3). Moreover, transcription of tumor marker(s), easily monitored by real time RT-PCR, does not guarantee that the corresponding pMHC is actually displayed at the cell surface and several escape mechanisms used by cancer cells have been described, such as downregulation of HLA molecules, 2-microglobulin, and TAP transporter protein (4). Antibodies able to bind pMHC in a peptide-specific way could be used to directly visualize the expression of a particular pMHC before or during a peptidebased immunotherapy. Such antibodies would represent valuable tools to detect the presence of T-cell epitopes. Generation of such antibodies by conventional hybridoma technology has proven to be very difficult, probably because T cells in contrast to B cells are educated to recognize pMHC class I or II through alternating negative and positive selection processes (5,6). The development of recombinant antibody
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Figure 1. Selection of TCR-like antibodies: A biotinylated peptide-MHC monomer is produced (1) immobilized on magnetic beads. These beads are used to select pMC binders from a large phage library of human Fab fragments (2). pMHC binders are subsequently screened by ELISA for their peptide specificity on various pMHCs. The DNA coding for the Fab fragment of TCR-like phage-antibodies is purified and reformated as chimeric T-cell receptors by fusion of variable domains (as scFv) with a transmembrane domain and a signaling domain. PBMCs or T cells are transfected by the construct and tested for cytotoxicity and specificity on tumor cells expressing or not expressing the relevant pMHC.
techniques and in vitro selection methods such as phage display has opened new avenues in this field. For example, it has become feasible to select fully human antibodies from large naive libraries against a variety of antigens including toxic proteins or highly conserved proteins (7). In addition, several groups have established methods to produce large amounts of recombinant pMHC based on in vitro refolding of HLA molecules produced as inclusion bodies in E. coli (8). We and others have combined these two approaches to establish a protocol to reproducibly select a novel class of antibodies with TCR-like specificity, i.e., TCR-like antibodies (TCR-like Abs) (Ref. 9 and Fig. 1). We have used these antibodies to efficiently redirect primary human T-lymphocytes to tumors presenting 1094
the relevant pMHC complexes (10–12). Primary human Tlymphocytes expressing TCR-like Abs exert antigen-specific functions such as cytolysis and cytokine production. Phage display and selection of TCR-like Abs, and their use to genetically modify T cells is expected to extend T-cell therapy to tumor antigens and tumor types for which no TCRs are currently (or expected to become) available.
GENERATION OF TCR-LIKE Abs Production of Recombinant pMHC Complexes Immunization strategies as well as in vitro selections depend on the availability of hundreds of micrograms of
Selection of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes
REVIEW ARTICLE antigen. A major breakthrough was published in 1992, describing a method to produce large amounts of pMHC by in vitro refolding (8). This approach was based on the production of HLA chains (heavy chain and 2-microglobulin) as inclusion bodies in E. coli followed by their refolding in the presence of a synthetic peptide. They were first used in structural studies of the interaction between TCR and pMHC. Refolding was performed either by serial dilution of denaturing reagents such as urea or by rapid dilution of denatured proteins in a large volume of refolding buffer allowing thioldisulfide exchange for proper disulfide bond formation. A second breakthrough came a few years later when refolded pMHC were enzymatically tagged by site-specific biotinylation. E. coli-derived enzyme BirA biotinylates a single cytosolic protein biotin carboxyl carrier protein (BCCP) often used as a fusion partner of the protein of interest. Interestingly, a 15 amino-acid BCCP peptide was shown to be efficiently recognized and biotinylated by BirA in vitro. Altman et al. refolded and biotinylated pMHC monomers in this way, which they then mixed with a commercial reagent such as streptavidin-PE to obtain a fluorescently labeled tetrameric pMHC complex. These complexes were then used to monitor and sort a population of T cells expressing TCR capable of binding this particular pMHC (13). Over the next years, variations were added to the basic protocols, two of them being of particular interest. First, Reiter and coworkers linked the heavy chain and the 2microglobulin by a flexible linker and consequently only needed a single E. coli culture to produce and purify pMHCcontaining inclusion bodies (14). Importantly, the single chain nature of the construct circumvents any stoichiometry-related problems with respect to the heavy and 2-microglobulin chains during the refolding process. Second, our group successfully applied BirA biotinylation of pMHC monomers during the production process of inclusion bodies (11). In fact, overexpression of BirA during the formation of inclusion bodies was shown to lead to a very efficient in vivo biotinylation (15). Consequently, the refolded pMHC are already biotinylated, thereby decreasing the length and the cost of the synthesis procedure. Additional modifications of pMHC class I complexes are reported by David Price (16). These pMHC modifications allow discrimination between low- and high-affinity T-cells, and between CD8 dependent and independent pMHC-TCR interactions, and may have surplus value for selection of antibodies with such renewed properties. Methods to produce pMHC class II complexes at large scale are not yet widely available. Therefore, new developments as described by Giulia Casorati (17) may provide reagents that allow more extensive studies on the role of CD4 T-cells and may also be used to select for pMHC class II specific antibodies. Selection of TCR-Like Antibodies In vitro selection methods based on phage display allow selection of antibodies that are normally difficult to obtain in vivo (Fig. 1). These include antitoxin antibodies and antibodies directed against conserved proteins (7). An additional advantage is that libraries can be generated from human antibody fragments, resulting in retrieval of fully human antiboCytometry Part A 73A: 1093 1099, 2008
dies. These antibodies are less immunogenic than the classical mouse or murinized antibodies and facilitate clinical translation of phage-display selected antibodies. The first human antibody directed against a class I peptide-MHC complex was isolated by Chames et al. (9) from a large (3.7 3 1010) phagedisplay library of human Fab fragments (18). This antibody, termed G8, specifically binds HLA-A1/MAGE-A1 complexes. The MAGE-A1 protein belongs to a family of cancer-germline gene products and is expressed in 40% of melanomas and other cancer. Antibody peptide fine-specificity was demonstrated by ELISA using various recombinant pMHCs complexes, and by flow cytometry using peptide-pulsed antigen-presenting cells (APCs) and importantly several melanoma cell lines expressing the relevant pMHC complex. The exact same approach was then extended to various other class I complexes to assess its reproducibility. The same library was used to select binders against several peptides displayed by HLA-A2, including peptides from tumor markers such as gp100, hTERT (telomerase catalytic subunit), MUC I, Melan-A, NY-ESO-1 or viral proteins such as HTLV-1 Tax, Influenza M1, EBV or HIV gag (19–25). Intriguingly, some HLA-A2 displayed peptides which predominantly led to the selection of phage clones that express high-affinity Fab’s despite the fact that the exposed surface of the peptide contributes only to a small part of the total pMHC surface. This phenomenon was not seen with peptide/HLA-A1 complexes and may be related to the haplotype of the donor cells used to build the library. Antibody Engineering Genes encoding for antibody fragments selected from phage-display libraries are readily available and allow subsequent molecular engineering of antibodies. This can be done rapidly and efficiently to modify ligand-binding affinity, specificity, size, and valency of a binder. For example, we have applied affinity maturation to increase the affinity of Fab-G8, an HLA-A1/MAGE-A1 binder of moderate affinity (250 nM) (11). To this end, we used a delicate process because it was estimated that the peptide only contributes 20% to the pMHC epitope’s immunogenicity. Newly selected binders to the nonpeptide epitope, namely the helixes of the HLA-A1 molecule, would result in a drastic decrease in peptide specificity. Two strategies were compared to generate the required diversity to obtain affinity-matured TCR-like Abs. First, the heavy chain of the G8-Fab was reshuffled with the available and light chain repertoire to optimize antibody chain pairing. Second, the G8 Fab heavy chain was targeted at CDR-H3 by spiking mutagenesis (1–4 residues of the CDR-H3 13 amino acid loop were randomly mutated). Phage display of both libraries, combined with various selection strategies such as limited antigen amounts, stringent washes, and competition in the presence of an excess of the original clone, resulted in several candidate antibodies with higher pMHC-binding affinity. Combining the heavy and light chains of the best mutations led to a binder termed HYB3 that displayed an 18-fold improved affinity. Further analyses demonstrated that the peptide fine-specificity of HYB3 was identical to that of the parental G8 clone. To 1095
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Figure 2. Various applications of TCR-like antibodies. Fab fragments (center: VL and CL domain of the lg light chain and VH and CH1 domain of the lg heavy chain) with MHC restricted specificity obtained by phage display can be used in different ways: (a) directly as diagnostic tools to check the expression of a specific pMHC complex; (b) fused to a toxin (immunotoxin); (c) fused to a radioisotope (radioimmunotherapy); (d) reformated as bispecific antibody binding simultaneously a pMHC and a receptor expressed by effector cells (CD3 on T cells, CD16 on NK cells); (e) fused to a signaling moiety to genetically retarget T cells toward cancer cells.
study the interaction between the Fab fragment HYB3 and its antigen in more detail, the three-dimensional structure of the complex was determined by X-ray crystallography (26). The HYB3-Fab binds the pMHC in a way similar to TCR. Strikingly, HYB3-Fab:pMHC binding conserves the diagonal binding mode adopted by TCRs. However, this Fab establishes surprisingly few direct interactions with the accessible peptide surface. Instead, it uses a large surface of interaction and achieves a very high surface complementarity. By this way, the antibody appears to sense modifications of exposed peptide residues and also subtle changes arising from mutations in buried peptide residues resulting in a slight modification of the HLA heavy chain surface. In total, the HYB3 clone represents a Fab with peptide specificity similar to that of a regular TCR, but with a ligand-binding affinity that is a 100–1,000fold higher than the average affinity of a TCR.
APPLICATIONS OF TCR -LIKE Abs Epitope Visualization An obvious and direct application of TCR-like Abs is visualization of their epitope, i.e. a pMHC (Fig. 2). TCR-like 1096
Abs can be used to follow intracellular generation, trafficking, and surface expression of pMHC complexes to estimate and quantify the density and stability of a particular pMHC on APCs. In addition, TCR-like Abs allows to check the availability of an antigen at the surface of tumor cells to predict and/or monitor an immunotherapy protocol. For example, Schuler and coworkers used the HLA-A1/MAGE-A1 HYB3-Fab to study and optimize exogenous peptide loading of monocytederived dendritic cells (DC) (27). Using flow cytometry, the antibody tools enabled the assessment of the best conditions to pulse DCs with peptide and enabled monitoring of the stability of the resulting complex in time. Similarly, Zehn et al. used TCR-like Abs to compare peptide display by mature DC versus other APCs (28,29), whereas Cohen et al. used an HLAA2/Tax specific antibody to study endogenous processing and presentation of HTLV-1 Tax in transfected JY B cells (22). The epitope density on APC or transfected cells is demonstrated to be several fold higher than the density of pMHC naturally expressed by tumor cells (see below). To increase the sensitivity of pMHC detection, one can apply BirA-based in vitro sitedirected biotinylation of monomeric TCR-like Abs, in analogy to that described for monomeric pMHC molecules, enabling the generation of fluorescent tetrameric antibodies upon addition of, for instance, streptavidin-PE (21). These multimeric antibodies have been validated by detection of tumor-related pMHC at the cell surface with high sensitivity. For example, the use of HLA-A2/MUC1 antibodies demonstrated that MUC1-expressing tumor cells only express several hundred MUC1 complexes per cell surface. In addition, HLA-A2/Tax binders were used to estimate the density of pMHC on HTLV1 infected cells, and studies demonstrated the expression of this complex on regulatory CD41CD251 T cells in patients with HTLV-1-associated neurological disease (30). As a last example, Held et al. used an HLA-A2/NY-ESO1 binder to study peptide presentation by tumor cells in more detail (24). In this study, the antibody allowed distinction between CTL responses against immunological meaningful or cryptic NYESO-1-derived peptides. Toxin and Radioisotope-Labeled Antibodies As for any antibody that targets a tumor marker, TCRlike Abs can be used to deliver a lethal compound such as a toxin or a radioisotope to tumor cells. This concept was exploited by fusing a murine HLA-A2/gp100 single chain Fv fragment to a truncated form of Pseudomonas exotoxin A (PE38), which killed human cancer cells in a pMHC-specific manner (31). Bispecific Antibodies to Retarget Immune Cells Phage display-selected TCR-like Abs can be molecularly engineered to create bispecific antibodies. The potential of this class of antibodies has been demonstrated (32), but their therapeutic application is most often limited by an inability to produce pure preparations of such proteins in large amounts. We and others have described several genetic approaches to create bispecific antibodies, and are currently using the HLAA1/MAGE-A1 antibody to create a bispecific molecule capable
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Figure 3. Overview of adoptive immunogene therapy.
of binding to pMHC, on the one hand, and FcRIII, an NK cell receptor responsible for antibody-dependent cell mediated cytotoxicity (ADCC), on the other hand (33). Advantages of using bispecific antibodies over Fc bearing antibodies to trigger ADCC include: (i) ease of engineering the affinity of the antibody/receptor interaction; (ii) ability to bind to activating Fc receptors but not inhibitory Fc receptors such as FcRIIB; and (iii) insensitivity to variation in FcR allele or antibody glycosylation pattern, parameters that have been shown to strongly affect the efficiency of many conventional antibodybased therapies. Genetic T-Cell Retargeting Adoptive T-cell therapy is based on the selection of tumor-specific T-cells and their expansion to clinically relevant numbers before infusion into the patient (Fig. 3). Recent successes obtained in melanoma patients demonstrate the potency of this therapy (34,35). Up to 50% of patients treated with autologous ex vivo expanded tumor-infiltrating lymphocytes (TIL) experienced objective clinical responses, including both complete and partial responses (34). However, in many cases, it is not possible to isolate TIL from cancer patients because either they are not present in sufficient numbers or cannot be expanded ex vivo. As an alternative, we and others developed genetic strategies to permanently graft primary human T-lymphocytes with tumor specificities (Figs. 2 and 3, Refs. 36 and 37). To this end, antibody-based receptors (36– 39) or TCRs (40–43) were transferred to human T-lymphocytes by means of retroviral infection and were analyzed for their ability to mediate antigen-specific functions such as tumor cell kill and cytokine release. Primary human T-lymphoCytometry Part A 73A: 1093 1099, 2008
cytes equipped with tumor-specific receptors, Ig and TCRbased, specifically kill tumor cells and more importantly were shown to have antitumor effects in vivo (39,44,45). More recently, adoptive transfer of genetically modified T-lymphocytes to cancer patients demonstrated safe and feasible translation of TCR and Ig gene therapy into the clinic (34,35,46–48). Many targeted tumor antigens expressed by tumors are autoantigens and, consequently, their tolerogenic nature prevents the generation and maintenance of high-avidity T-cell clones. To bypass the difficulty to select such T-cell clones, we propose the use of antibody phage display. Proof of principle for efficient T-cell retargeting with a phage display-derived TCR-like Ab was obtained by cloning the G8-Fab heavy and light chain fragments into a retroviral vector pBullet containing a leader sequence, transmembrane domain (CD4), and intracellular signaling domain (Fc(e)RIc chain). The resulting vectors VHCHCD4 and VLCLCD4 were retrovirally introduced into anti-CD3 mAb-activated primary human T-lymphocytes, and the membrane-expressed G8-Fab was shown to mediate HLAA1/MAGE-A1-specific immune functions such as tumor cell kill and cytokine release (10). As said earlier, one of the advantages of phage display-derived Fab fragments is the ability to modify the isolated Fab with respect to affinity. To evaluate whether or not TCR-like Abs with increased affinity would enhance antitumor activity of Fab-receptor transduced primary human T-lymphocytes, as would be expected based on the reported functional superiority of high- versus low-avidity T-cells (49), we compared G8 and (the affinity-matured) HYB3 Fab-transduced T-cells (11). Primary human T-lymphocytes equipped with the high-affinity Fab receptor HYB3CD4 were shown to exert higher levels of cytolysis and 1097
REVIEW ARTICLE cytokine production in response to HLA-A1/MAGE-A1-positive melanoma cells than T cells equipped with the low-affinity G8-CD4 receptor. In fact, as reported for high-avidity T cells, HYB3 receptor expressing T-lymphocytes also exerted lysis that showed enhanced sensitivity and kinetics. Next to affinity, receptors may be improved by providing costimulatory signaling capacity. We introduced a CD28 domain into the G8 and HYB3 receptors and evaluated primary human T-lymphocytes expressing these receptors for enhanced antitumor activity (12). As reported for T cells equipped with non-MHC restricted antibody-based receptors (44), the inclusion of a CD28 signaling domain into the G8 and HYB3-based receptors enhanced their antitumor activity, resulting in enhanced production of IL-2, TNF, and IFN. In contrast to T cells equipped with non-MHC restricted antibody-based receptors (36) or even TCR (50), G8-CD28- and HYB3-CD28-expressing T-lymphocytes also displayed higher levels of cytolysis (11). Importantly, it was observed that T-lymphocytes expressing HYB3-CD28 receptors, but not HYB3-CD4 or G8-CD28, killed HLA-A1/MAGE-A1-negative target cells, pointing to a downside of receptor modification and the necessity to check preservation of specificity of modified Fab receptors. These results indicate that high-affinity TCR(-like) interactions may result in cross-reactive binding to other pMHC complexes. Therefore, methods that aim to enhance the affinity of TCR or TCR-like antibodies may not always result in desired fine specificities. Apparently, the window that allows receptor modification while preserving specificity may be limited. In the end, the optimal affinity of TCR or TCR-like based chimeric receptors probably depends on various parameters including density and accessibility of the targeted pMHC. The possibility to create large spectrum (lM to pM) of affinity by protein engineering will have to be applied to chimeric receptors and tested in vivo in order to shed more light on this issue.
CONCLUDING REMARKS Phage display-derived TCR-like antibody fragments are considered valuable tools for genetic engineering of T-cell specificity. The TCR-like antibodies can be isolated with relative ease from large antibody libraries, provided that the repertoire is large enough, and can be used for multiple research, diagnostic, and (potentially) clinical applications.
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