An artificial solution for adoptive immunotherapy

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An artificial solution for adoptive immunotherapy Harry Zemon Johns Hopkins University, Department of Pathology, 720 Rutland Avenue, Baltimore, MD 21205-2196, USA

Adoptive immunotherapy is a promising strategy in the battle against cancer and infectious diseases and the recently developed artificial antigen-presenting cell (aAPC) overcomes several obstacles for this therapy. The aAPC recapitulates the natural in vivo antigenpresenting cell (APC)–T cell interactions by coupling human leukocyte antigen –immunoglobulin fusion protein (HLA–Ig), a soluble multivalent HLA –Ig chimeric fusion protein, to beads. The natural ligand–receptor interaction promotes rapid sustained expansion and normal functioning of the antigen-specific T cells. Adoptive immunotherapy using CD8þ T cells is an exciting tool for the treatment of various diseases [1,2] and involves ex vivo stimulation and expansion of antigen-specific Tcells followed by infusion of these activated T cells back into patients. This T-cell specific (centric) therapy affords an individualized treatment plan with the opportunity of overcoming immunogenic tolerance and/or tumor evasion mechanisms by re-populating patients’ immune systems with antigen-specific, highly reactive T cells. The development of the artificial antigen-presenting cell (aAPC) by Oelke and colleagues [3] now allows barriers to the success of this therapy to be overcome. Antigen-specific adoptive immunotherapy was first shown to have clinical efficacy in humans using ex vivo-expanded cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) for treatment of CMV infection in immune compromised allogeneic bone marrow transplant (i.e. bone marrow donated by another person) recipients [4,5]. Neither CMV viremia nor CMV disease developed in any of the patients treated despite the usual high frequency of occurrence of life-threatening CMV diseases in the immune compromised patient population, indicating that protective T-cell immunity against CMV had been restored [4,5]. Following the success of CTL-based adoptive immunotherapy for CMV, adoptively transferred ex vivo-expanded Epstein–Barr virus (EBV) and HIV-specific CTLs were also used in treating EBV-related diseases and aspects of HIV infection [6– 9]. Rosenberg et al. [10] successfully expanded autologous tumor-specific T cells, referred to as tumor-infiltrating lymphocytes (TILs), ex vivo, and re-infused them into melanoma patients. They showed that re-infused TIL with the relevant antigen trafficked back to tumor sites and directly induced tumor shrinkage [10,11]. These encouraging initial experiments led investigators to explore adoptive immunotherapy for treatment of a variety of different cancers. Corresponding author: Harry Zemon ([email protected]). http://tibtec.trends.com

Current methodology A multitude of attempts to expand antigen-specific CTLs efficiently have been made. One frequently used approach involves autologous dendritic cells (DCs). This process can be separated into three steps: (1) generation of DCs; (2) induction of antigen-specific CTLs; and (3) expansion of antigen-specific CTLs. In the first step, CD14þ monocytes obtained by leukopheresis are cultured in a cytokine milieu and induced to differentiate into DCs. In the second (induction) step, DCs obtained are pulsed with the peptide of interest and co-cultured with naı¨ve CD8þ T cells. Subsequent stimulations of CD8þ T cells by DCs are performed weekly for 4 – 6 weeks using freshly generated DCs. Under these conditions, up to 2 – 5 £ 107 T cells that are ,70 – 80% antigen specific can be generated in 3– 4 weeks. Unfortunately, there are tremendous variations in the amount and quality of differentiated DCs, which are probably a function of the patient’s primary disease and pre-treatment with toxic agents that inhibit bone marrow production. As such, weekly stimulations are not always feasible. Because of these technical difficulties associated with obtaining enough DCs, the final expansion of the induced antigen-specific CTLs is often done by abandoning the antigen-specific stimulation and replacing it with nonspecific stimulation from anti-CD3 or anti-CD28 beads. Limitations to the current methodology Although limited success has been reported using DCs in combination with non-specific beads for CTL expansion, adoptive T-cell therapy has been impeded by the lack of a reproducible, economically viable method for generating therapeutic numbers of antigen-specific CTLs. The isolation of DCs is a time-intensive process that cannot reliably generate sufficient numbers of T cells for treating progressive disease under emergent conditions. Furthermore, the use of non-specific stimulation via anti-CD3based beads causes a significant decrease in antigenic specificity from 70 – 80% to 5 –20% during the CTL expansion step. It has yet to be determined why antiCD3 beads stimulate the non-antigen-specific component preferentially. In addition, anti-CD3 stimulation has the undesirable effect of causing T cells to cease expanding after 6 – 10 days [12]. Proper cell signaling during the expansion phase is also an essential component in producing effective, functioning CTLs following infusion. Excessive stimulation and improper or absent co-stimulation could result in deficient cytolytic activity, lack of persistence or apoptosis. Maus and colleagues addressed the issue of poor expansion after anti-CD3 or anti-CD28 beads by adding another

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co-stimulatory 4-1BB ligand to their stimulation protocol, which prevented apoptosis [12]. However, the inability to maintain antigenic specificity remained an issue, as it is with all CD3 non-specific-based stimulations. In summary, the current methods, including use of DCs and anti-CD3 or anti-CD28 beads for induction and expansion suffer from variability in quantity, viability and functionality of the CTLs, as well as loss of antigen specificity of the expanded cytotoxic cells. These shortcomings ultimately affect therapeutic potential and impede standardization of a therapeutic protocol. aAPC versus dendritic cells A comprehensive solution to these issues is the ‘off-theshelf ’ aAPC developed by Oelke et al., which combines an immobilized dimeric form of human leukocyte antigen– immunoglobulin fusion protein (HLA –Ig) with co-stimulation from anti-CD28 antibody attached to a bead (Fig. 1) [3]. HLA – Ig is a fusion protein of a Class I HLA on an immunoglobulin scaffold. The multivalent dimer improves T-cell receptor avidity (i.e. how well and how long the ligand binds to the receptor) with HLA – peptide complexes [13]. CD28 is a co-stimulatory molecule expressed on resting and activated T cells, and is currently the most potent co-stimulatory molecule described [14]. T-cell receptor engagement, in conjunction with CD28 ligation, induces the production of interleukin-2 (IL-2), which is essential for ex vivo T-cell expansion [14,15]. Expansion of CTLs using this new aAPC technology has several important advantages over the methods currently used. Peptide-loaded HLA – Ig aAPCs induce rapid expansion of antigen-specific cells for both high and low affinity T cells, comparable to or better than the ‘gold standard’ DC-mediated expansion of antigen-specific CTLs. The antigen –HLA complex combined with an anti-CD28 antibody provides the appropriate stimulation and signaling necessary for promoting long-term expansion while also maintaining antigen specificity. CTL populations expanded with aAPC consist of up to 90% antigen-specific CTLs compared with 5 –20% obtained with current methods, and will enable expansion of precursor CTLs to numbers suitable for therapeutic use. The use of aAPC simplifies and shortens the ex vivo expansion process because leukopheresis is not needed and avoids the costly,

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(b) α2 Class 1 MHC α3

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time-intensive weekly process of DC differentiation. Because aAPCs are pre-formed acellular antigen-presenting beads, they have reproducible and reliable antigenpresenting activity and are always ready to use ‘off-theshelf ’ across a broad range of the patient population. Future generations of aAPCs The results of using this new aAPC are impressive and resolve the issues of rapid, reliable, and sustained expansion, while maintaining cytolytic function and antigenic specificity. However, successful expansion of the CTLs is just one step on the long road to successfully eradicating tumors and infectious diseases using adoptive immunotherapy. As we learn more about the in vivo response and the effects of differing amounts and types of co-stimulation on CTL function post-infusion, future generations of the aAPC might prove to be essential because the bead can easily adapt to the new stimulatory requirements by adding or removing different co-stimulatory complexes. In addition, new generations of aAPCs using a Class II HLA – Ig would facilitate expansion of tumor-specific CD4þ cells, providing crucial immunological ‘help’ for adoptive transfer. Several studies have indicated a crucial role for CD4þ cells in the in vivo induction of antigen-specific CTL [16,17]. Furthermore, these studies suggest that the reason for the inconsistent response seen in previous clinical trials of adoptive immunotherapy of infused CTLs was in part attributed to lack of functional in vivo CD4þ T-cell help. The combination of infusing both CD4þ and CD8þ T cells might improve CTL response and survival by optimizing secreted cytokines and co-stimulation [18– 20]. Further analysis of the data from Phase I trials involving the adoptive transfer of cells into melanoma patients showed that only a modest response was obtained from the adoptively transferred cells [10,21,22]. Significant work is still needed to understand the in vivo response to adoptive immunotherapy treatments. Meidenbauer and colleagues proved that transferred cells remained physiologically intact for several weeks in vivo and that CTLs were localized to the tumor site [21]. With multivalent HLA – Ig dimer or tetramer technology, we can now quantify the antigen-specific response and analyze the phenotype of the transferred effector cells [23].

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Fig. 1. (a) Class I HLA–Ig, (b) anti-CD28 antibody (c) aAPC with anti-CD28 antibody and Class I HLA–Ig covalently bonded to an epoxy bead. Abbreviations: HLA, human leukocyte antigen; Ig, immunoglobulin; aAPC, artificial antigen-presenting cell; HLA– Ig, human leukocyte antigen– immunoglobulin fusion protein. http://tibtec.trends.com

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Methods used to track transferred cells in vivo are an important aspect of future clinical trials. Advances in monitoring using dimer and tetramer technology can open a window into the cellular immune response with great precision. This window should facilitate development of new generations of aAPC through improved insight into the CTL interaction and mechanisms of tumor and infection evasion. Furthermore, in the context of minimal residual disease, accurate in vivo monitoring becomes all the more crucial because the obvious signs of tumor shrinkage will rarely be used as clinical end points to therapy. Overall, it appears that current and future generations of the HLA –Ig-based aAPC technology will probably play an important role in many future enhancements of the adoptive immunotherapy process. Acknowledgements H.Z. thanks Mathias Oelke and Jonathan Schneck for helpful discussions and insights into the world of adoptive immunotherapy and acknowledges grant support from NIH grant 5 T32 CA 67751.

References 1 Melief, C.J. and Kast, W.M. (1995) T cell immunotherapy of tumors by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol. Rev. 145, 167– 177 2 Riddell, S.R. and Greenberg, P.D. (1995) Principles for adoptive T cell therapy of human viral diseases. Annu. Rev. Immunol. 13, 545 – 586 3 Oelke, M. et al. (2003) Ex vivo induction and expansion of antigenspecific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat. Med. 9, 619 – 625 4 Riddell, S.R. et al. (1994) Selective reconstitution of CD81 cytotoxic T lymphocyte responses in immunodeficient bone marrow transplant recipients by the adoptive transfer of T cell clones. Bone Marrow Transplant. 14 (Suppl 4), S78 – S84 5 Walter, E.A. et al. (1995) Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T cell clones from the donor. New Engl. J. Med. 333, 1038– 1044 6 Heslop, H.E. et al. (1996) Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat. Med. 2, 551 – 555 7 Heslop, H.E. et al. (1994) Donor T cells to treat EBV-associated lymphoma. New Engl. J. Med. 331, 679 – 680 8 Brodie, S.J. et al. (1999) In vivo migration and function of transferred HIV-1-specific cytotoxic T cells. Nat. Med. 5, 34 – 41

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9 Brodie, S.J. et al. (2000) HIV-specific cytotoxic T lymphocytes traffic to lymph nodes and localize at sites of HIV replication and cell death. J. Clin. Invest. 105, 1407 – 1417 10 Rosenberg, S.A. et al. (1988) Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. New Engl. J. Med. 319, 1676– 1680 11 Dudley, M.E. et al. (2002) Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298, 850 – 854 12 Maus, M.V. et al. (2002) Ex vivo expansion of polyclonal and antigenspecific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T cell receptor, CD28 and 4-1BB. Nat. Biotechnol. 20, 143– 148 13 Greten, T.F. et al. (2002) Peptide-beta2-microglobulin-MHC fusion molecules bind antigen-specific T cells and can be used for multivalent MHC-Ig complexes. J. Immunol. Methods 271, 125– 135 14 June, C.H. et al. (1994) The B7 and CD28 receptor families. Immunol. Today 15, 321 – 331 15 Jenkins, M.K. and Johnson, J.G. (1993) Molecules involved in T cell costimulation. Curr. Opin. Immunol. 5, 361 – 367 16 Baxevanis, C.N. et al. (2000) Tumor-specific CD41 T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor. J. Immunol. 164, 3902– 3912 17 Marzo, A.L. et al. (2000) Tumor-specific CD41 T cells have a major ‘posT licensing’ role in CTL mediated anti-tumor immunity. J. Immunol. 165, 6047– 6055 18 Pardoll, D.M. and Topalian, S.L. (1998) The role of CD41 T cell responses in antitumor immunity. Curr. Opin. Immunol. 10, 588 – 594 19 Topalian, S.L. (1994) MHC class II restricted tumor antigens and the role of CD41 T cells in cancer immunotherapy. Curr. Opin. Immunol. 6, 741– 745 20 Lin, K.Y. et al. (1996) Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 56, 21 – 26 21 Meidenbauer, N. et al. (2003) Survival and tumor localization of adoptively transferred Melan-A-specific T cells in melanoma patients. J. Immunol. 170, 2161– 2169 22 Yee, C. et al. (2002) Adoptive T cell therapy using antigen-specific CD8þ T-cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc. Natl. Acad. Sci. U. S. A. 99, 16168 – 16173 23 Yee, C. (2003) Adoptive T cell therapy – immune monitoring and MHC multimers. Clin. Immunol. 106, 5 – 9 0167-7799/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2003.08.005

Silent, but deadly – eliminating reservoirs of latent HIV Michael S. Kay Department of Biochemistry, University of Utah School of Medicine, MREB 211, 50 N. Medical Drive, Salt Lake City, UT 84132, USA

HIV treatment with highly active anti-retroviral therapy cannot eliminate latent reservoirs of HIV. In a recent paper, Root and Hamer describe a novel targeting mechanism for killing HIV-infected cells. They show that 5-helix (a gp41-binding protein) fused to a Pseudomonas exotoxin fragment can selectively kill cells Corresponding author: Michael S. Kay ([email protected]). http://tibtec.trends.com

displaying envelope proteins on their surface from a broad range of HIV strains. HIV/AIDS has been transformed from a fatal infection into a chronic infection for those who can afford and tolerate the current cocktail of reverse transcriptase and protease inhibitors. Although this treatment often reduces viral loads to undetectable levels, HIV lurks in poorly understood