It is widely accepted that the immune system has evolved cellular and humoral mechanisms that can evoke natural immune responses to tumours;1 however, in most instances, vaccines fail to induce rejection of established tumours.2 Adoptive T-cell transfer, a term coined by Billingham et al.,3 has the potential to overcome one of the significant limitations associated with vaccine-based strategies and specifically the requirement to activate de novo and expand a tumour antigen-specific T-cell response in patients who are often immunocompromised. Mitchison et al.4 first reported the targeting of cancer through the adoptive transfer of lymphocytes in rodent models over 50 years ago.
Application of the emerging discipline of synthetic biology to cancer, which combines elements of genetic engineering and molecular biology to create new biological structures with enhanced functionalities,5 is the focus of this article. In 1989, Gross et al.6 reported the first synthetic receptor expressed in lymphocytes. Shortly after, Irving and Weiss7 reported that a chimeric antigen receptor (CAR) comprising a CD8 and CD3ζ chain was sufficient to activate the T-cells. A coalescence of preclinical and clinical data support the premise that the principles of gene transfer combined with adoptive cellular therapy are poised to overcome the fundamental limitations associated with central and peripheral tolerance and enable the potent and efficient targeting of tumours at will.
There are many mechanisms that prevent the immune system of most patients from eliminating tumours.8 One major issue is the relatively low affinity of T-cell receptors (TCRs) for self antigens compared with foreign antigens. In humans, comparative analyses have revealed that the TCRs from T-cells that recognize self tumour antigens have a substantially lower affinity (approximately 1.5 logs) for cognate major histocompatibility complex (MHC)–peptide complexes than their virus-specific TCRs.9 Adoptive transfer using engineered TCRs and CARs is a promising approach to overcome this obstacle (Figure 1). The adoptive transfer of T-cells with endogenous TCRs is an effective therapy for virally induced tumours. As reviewed by Rooney et al.,10 the fraction of cancers known to be caused by tumour-associated viruses continues to increase. Because cytomegalovirus (CMV) appears to infect glioblastoma,11 clinical studies are using CMV-specific T-cells as a potential therapy.12,13
Adoptive transfer of tumour-infiltrating lymphocytes (TILs) following harvest from tumour and ex vivo expansion was pioneered by a group at the National Cancer Institute, under the premise that lymphocytic infiltrates at tumours are enriched for tumour antigen-specific T-cells. As reviewed by Hinrichs and Rosenberg,14 many factors influence the success of this approach, including culture technology and host conditioning with chemotherapy and ionizing radiation. TIL cultures for adoptive transfer are typically generated via short-term ex vivo expansion and screening for antitumour activity. TIL-based approaches have been primarily evaluated in the setting of melanoma, in part because melanoma biopsies are readily obtainable and in part because melanoma has long been considered an ‘immunogenic’ tumour. TIL therapy has been shown to result in durable tumour regression in a subset of patients with advanced metastatic melanoma.15 As reviewed by Linnemann,16 the response mechanisms of patients treated with TILs are the result of T-cells reacting to shared antigens as well as neoantigens created by tumour-specific mutations or by epitopes that are encoded by alternative open reading frames.17,18 Preliminary data suggest that some T-cell responses against neoantigens may be of a higher magnitude than T-cell responses against shared self antigens.19,20 The author believes that the major issue facing the field that prevents the widespread use of TIL therapy has been the infusions of high-dose interleukin 2 (IL-2) and the attendant off-target toxicities. A secondary obstacle is the challenging logistics of tumour harvest and TIL culture that have prevented investigators from conducting randomized clinical trials analysed with intention-to-treat endpoints.
Chimeric antigen receptors
Chimeric antigen receptors are modular polypeptides typically consisting of three distinct modules: an extracellular target-binding module, a transmembrane module anchoring the CAR into the cell membrane and an intracellular signalling module. The extracellular target-binding module is usually derived from single-chain variable fragment determinants isolated from antibodies and linked in a single chain through linker polypeptide sequences. Transmembrane modules are usually derived from molecules involved in T-cell function, such as the CD8 and CD4 coreceptor molecules.21 Recent contributions by Ruella and Kalos,22 Chmielewski et al.,23 Cheadle et al.,24 Dotti et al.25 and Jensen and Riddell26 focus on the status of CARs in clinical trials. The principal advantage of CAR-based strategies is that the target-binding moiety is derived from antibodies with affinities several orders of magnitude higher than TCRs. In addition, because CARs recognize intact cell surface proteins, targeting of target cells is neither MHC restricted nor dependent on processing and effective presentation of target epitopes and, therefore, CAR-based approaches are insensitive to tumour escape mechanisms related to MHC loss variants. Many groups have shown that CAR T-cells have potent antitumour effects against a variety of advanced haematological malignancies of the B-cell lineage. The central issue facing the field is whether or not the technology can be extended to non-B-cell-derived malignancies and, in particular, can this strategy work for carcinomas?
There are a number of limitations and challenges, both practical and theoretical, associated with CAR-based strategies. In terms of practical limitations, CAR-based approaches are restricted to the targeting of cell surface determinants to which antibodies can be generated in heterologous species. In addition, as CARs are chimeric molecules composed of distinct combinatorial modules that include unique junctional fragments, there is reasonable potential for CAR-modified T-cells to be targeted by patient humoral and cellular immune responses, which may be a clinically silent event or, in rare instances, can provoke anaphylaxis.27,28 In terms of theoretical limitations, because CARs are engineered to deliver TCR and costimulation-mediated signals independently from the physiological complex through which natural signalling occurs, it is possible that the signalling cascades initiated through CAR engagement are qualitatively and/or quantitatively distinct from those evoked by native TCR signalling. This could result in adverse effects such as uncontrolled lymphoproliferation, an event that has fortunately not occurred. However, the non-physiological signalling modules in CARs could also have beneficial effects. An example is that CAR T-cells may be less susceptible to regulation and, therefore, may have improved function in the tumour microenvironment.29 Chmielewski et al.23 describe a clever strategy of targeting the tumour stroma by recruiting innate immunity following the adoptive transfer of CAR T-cells engineered to secrete transgenic cytokines such as IL-12.
T-cell receptor engineering
The feasibility of transferring T-cell specificity into primary T-cells through transfer of TCR α and β chains was demonstrated almost 20 years ago.30,31 Tumour antigen-specific T-cells, expanded from both cancer patients and healthy volunteers, have been a primary source for isolating tumour-specific heterodimeric TCRs and, over the years, a large variety of approaches using both peptides and whole antigen have been implemented to expand such T-cells. Because of the low frequency of such T-cells in peripheral blood, the lack of effective culture and expansion methodologies, and the impact of central tolerance on the repertoire, T-cells have been isolated using these approaches, but with considerable difficulty; furthermore, such T-cells are in general of low affinity and demonstrate weak antitumour activity. A number of approaches to overcome these issues and generate more potent tumour antigen-specific T-cells have been developed. One recent and promising approach to overcome the issue of the intrinsically low affinity of TCR to self antigens has been to enhance the affinity of the TCR isolated from such T-cells by mutagenesis of the α and β receptor chains. Recent technological advances have facilitated elegant molecular and rational high-throughput genetic approaches to affinity enhance TCRs,32–34 and such efforts have resulted in the ability to reproducibly generate TCR with substantially higher affinities for target antigens.35 An alternative strategy to enhance TCR affinity follows from observations that enhanced functional avidity and improved recognition of tumour cells occurs following introduction of mutations that reduce N-glycosylation on TCR chains.36
As reviewed by Hinrichs and Rosenberg,14 Ruella and Kalos22 and Stromnes et al.,37 there are promising early results in a variety of tumours treated with T-cells expressing TCRs engineered by various approaches. However, there have also been on-target and off-target toxicities with engineered TCRs. In one trial, T-cells were engineered to express a TCR generated in HLA-A*0201 transgenic mice (i.e. not subjected to selection by the human immune system) and that recognized an epitope shared between MAGE-A3, -A9 and -A12.38 Of the nine patients treated, five demonstrated objective clinical responses, but three patients demonstrated serious adverse events associated with neural toxicity, including two deaths. Post-mortem analysis revealed a rare and previously unrecognized expression of MAGE-A12 in the brain tissue.38 Two trials that evaluated the use of affinity-enhanced HLA-A*01-restricted and MAGE-A3-specific TCR to target melanoma and myeloma were reported recently.39,40 The first patient treated in each of these trials experienced severe cardiac toxicity and each patient died within 7 days of T-cell infusion.39 Retrospective analysis demonstrated that the affinity enhancement of the TCR resulted in the off-target recognition of a related HLA-A*01-restricted epitope from the protein titin expressed in cardiac cells.40 These results highlight the potency of adoptively transferred T-cells with redirected specificity and the need to develop improved methods for preclinical screening of engineered TCRs.
A potential toxicity following the introduction of engineered TCRs is the production of mixed dimers comprising chains from the endogenous TCR with chains from the transgenic TCR.41 As reviewed by Cieri et al.42 and Sing et al.,43 a particularly elegant approach to prevent this complication involves TCR gene editing with zinc finger nucleases. Expression of the endogenous TCR α and β chains can be permanently abrogated using this approach, resulting in improved expression and function of the transgenic TCRs and CARs.44,45
In addition to receptor engineering, optimizing the effector function of engineered T-cells can also increase clinical efficacy. Previous disappointing results with adoptive transfer strategies were due to the use of cell culture approaches that resulted in a population of terminally differentiated effector cells. Recent results with CAR T-cells indicate that proliferative capacity of the infused T-cells is a predictive biomarker of clinical responses, as reviewed by Ruella and Kalos.22 It is now well recognized that stimulation of T-cells via their TCR without a second costimulatory signal induces tolerance and more recent CAR-based technologies have focused on overcoming this limitation. Thus, while first-generation CARs depended on intracellular transduction of the recognition signal via the CD3ζ chain alone, second- and third-generation CAR constructs have incorporated costimulatory signalling domains such as those derived from CD27, CD28, CD134 or CD137. In addition, culture systems that provide costimulation by immobilized ligands on beads have improved the function of adoptively transferred T-cells.46 Sophisticated artificial antigen-presenting cells that provide arrays of selected costimulatory molecules and cytokines have been developed,47,48 as reviewed by Butler and Hirano.49
A major controversy in the field is defining the optimal cell product for infusion. The issue is whether or not to purify selected subsets of cells for culture and subsequent genetic engineering or, more straightforwardly, to use bulk cell products that contain mixtures of CD4+ helper, CD8+ cytotoxic, naive, central memory, effector memory and other subsets. For example, cell culture conditions can be optimized to promote the expansion of T-central memory cells using anti-CD3- and anti-CD28-coated beads with IL-7 and IL-15.50 As summarized by Fowler,51 the blockade of the mechanistic target of rapamycin (mTOR) during culture has the potential to enhance adoptive therapy approaches. Manipulation of metabolic pathways with rapamycin and other mTOR kinase inhibitors can change the fate and function of adoptively transferred T-cells.52 Furthermore, CAR T-cells encoding a rapamycin-resistant mutant of mTOR have enhanced antitumour effects in preclinical models.53 The factors related to the desired composition of the adoptively transferred cells are reviewed by Jensen and Riddell.26 T-cells with stem cell-like properties have been described;54,55 however, it is not yet known if these cells are superior to central memory or naive T-cells. Ghosh et al.56 have focused on the development of T-cell-based immunotherapy for use in the context of allogeneic haematopoietic stem cell transplantation. They have reviewed a recent study57 on the development of ‘off the shelf’ cellular immunotherapies across MHC barriers, highlighting the key milestones in their development and use.57 In particular, they show that the adoptive transfer of precursor T-cells enhances T-cell reconstitution after allogeneic stem cell transplantation.57
A major issue with clinical adoptive cell transfer therapy is the avoidance of senescent and exhausted states in the infused cells. This issue was not predicted in mouse models because of substantial differences in telomere biology between the mouse and human immune systems.58 With TIL therapy, the telomere length of the transferred lymphocytes correlates with in vivo persistence and tumour regression in melanoma patients receiving cell transfer therapy.59 CD28 costimulation can augment telomerase activity and enhance telomere length during in vitro culture.60,61 One approach to circumvent this issue is the use of haematopoietic stem cells or induced pluripotent stem cells,62,63 as reviewed by Gschweng et al.64 Another approach to prevent terminal differentiation during culture is to uncouple cell proliferation from effector differentiation. Crompton et al.65 reviewed the cellular mechanisms that lead to progressive differentiation during the physiological immune response and they proposed the use of synthetic biology to uncouple proliferation from differentiation.
A potential safety concern related to the infusion of engineered T-cells is virus integration-related insertional mutagenesis and cellular transformation, which has been demonstrated with the genetic engineering of haematopoietic stem cells (HSCs).66 This issue may also occur with non-viral-based integration using the Sleeping Beauty transposon system, as described by Sing et al.43 In patients with congenital and acquired immunodeficiency, genetically modified T-cells have been shown to persist after adoptive transfer in humans for more than a decade without adverse effects,67,68 indicating that the approach to genetically modify mature human T-cells is fundamentally safe, at least in part because lentiviral integration sites are not random and do not favour proto-oncogenes.69 Furthermore, unlike B-cells, T-cells are subject to clonal competition at the TCR level, which may explain the rarity of T-cell leukaemia and the relative resistance of T-cells to transformation.70
The development of mechanisms to control the lifespan of the transferred T-cells is yet another challenge for the field. Initial approaches attempted to introduce ‘suicide genes’ such as the herpes simplex virus thymidine kinase (TK) gene; however, these efforts revealed the strong potential for immunological rejection based on targeting of TK-derived sequences.71 More recently, an elegant and potentially powerful inducible system based on the use of a modified human caspase-9 fused to a human FK506 binding protein that permits conditional dimerization and delivery of apoptotic signals in response to small molecules that can permeate the T-cell plasma membrane is currently being evaluated in clinical trials.72 Approaches to regulate the persistence of engineered T-cells are discussed by Dotti et al.25 and by Jensen and Riddell.26
In this review I have highlighted two basic gene transfer approaches that are being pursued to bypass the effects of central and peripheral tolerance on the T-cell repertoire. Clinical data from my laboratory generated principally over the past 5 years suggest that we are at the threshold of a golden era for adoptive T-cell therapy, with a number of recent profound examples of the potency and promise of this approach to target cancer. Recent reports, using CAR T-cells with CD137 and CD3ζ signalling domains, which documented long-term functional persistence of T-cells engineered to target CD19, along with long-lasting clinical remissions and ongoing B-cell aplasia, have highlighted the potential for adoptive T-cell transfer to induce a profound long-term functional antitumour activity.73,74 Despite these early successes, a number of fundamental and important questions still remain to be resolved for the broad, reproducible and effective implementation of this approach to treat cancer beyond B-cell malignancies.
A few common themes emerge from these articles. First, identification of the optimal composition of the transferred cellular product requires clarification. Second, in ongoing clinical studies with CAR engineered cells that target CD19, patients remain disease free with persisting engineered T-cells for more than 3 years post treatment but also with ongoing B-cell aplasia owing to targeting of normal CD19-positive B cells, highlighting the practical necessity to eventually ablate engineered cells and enable normal B-cell reconstitution. Therefore, a central issue facing the field is the design and implementation of various approaches to control the fate of adoptively transferred cells. These findings are being translated into the clinic at a rapid pace and it is likely that engineered T-cell transfer will become established as an effective cancer therapy during the next decade. Finally, a challenge for adoptive T-cell therapy will be the necessity and rationale to combine the therapy with other antitumour therapies. In particular, we will require information to rationally combine with therapeutic vaccination, checkpoint inhibition, agonistic antibodies, small molecule inhibitors of tumours and the targeting of tumour stroma and neovasculature, as discussed by Yee.75
Conflict of interest
Carl H June is the inventor of technologies used to treat cancer with engineered T-cells. The patents are listed below under Inventions and detailed technology disclosure. This technology has been assigned to the University of Pennsylvania and the US Government, and some of the technology has been licensed by Novartis. The conflict is managed by the University of Pennsylvania according to policy and procedures.
Inventions and detailed technology disclosure
June CH, Thompson C, Kim S, inventors; US Government, ONR, assignee. Methods for modulating expression of exogenous DNA in T cells. Filed 7 June 1995, United States Patent and Trademark Office 08/435,095 and CIP issued as US 6692964 B1 17 February 2004.
June CH, inventor. Methods for modulating T cell responses by manipulating intracellular signal transduction. Filed 1 May 1995, Australia patent 706761, issued 1999.
June CH, Craighead N, inventors; US Government, ONR, assignee. Murine hybridoma and antibody binding to CD28 receptor secreted by the hybridoma and method of using the antibody. Filed 17 January 1996, US patent 5948893, issued 1999.
June CH, Thompson CB, Nabel GJ, Gray GS, Rennert PD, Freeman GJ, inventors. Methods for selectively stimulating proliferation of T cells. Filed 3 June 1994, US patent 5858358, issued 12 January 1999.
June CH, Thompson CB, inventors; US Government, ONR, assignee. Methods for modulating T cell survival by modulating Bcl-X L protein level. Filed 7 June 1995, US patent 6143291, issued 2000.
June C, Thompson C, Nabel GJ, Gray G, Rennert P, inventors. US Government, ONR, assignee. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells. Filed 10 March 1995, US patent 6352694 B1, issued 2002.
June CH, inventor. Methods for modulating T cell responses by manipulating intracellular signal transduction. Filed 29 April 1994, US patent 6632789, issued 2003.
June CH, Thompson CB, Nabel GJ, Gray GS, Rennert PD, inventors; US Government, ONR, assignee. Methods for selectively stimulating proliferation of T cells. Filed 4 May 1995, US patent 6534055, issued 2003.
June CH, Kim S, Thompson CB, inventors; US Government, ONR, assignee. Methods for transfecting T cells. Filed 7 June 1995, US patent 6692964 B1 issued 2004.
Thompson CB, June CH, inventors; US Government, ONR, assignee. Methods of treating autoimmune disease via CTLA-4Ig. Filed 7 February 1995, US patent 6685941, issued 2004.
June CH, Thompson C, Nabel GJ, Gray GS, Rennert P, inventors; US Government, ONR, assignee. Methods for selectively stimulating proliferation of T cells. Filed 26 January 1996, US patent 6905680, issued 2005.
Carroll RG, Shan X, Danet-Desnoyers G, June CH, inventors. Modulation of regulatory T-cells by human IL-18. Filed 14 September 2007, PCT Application PCT/US2007/01995, application pending.
June CH, Thompson CB, Kim S, inventors. Methods for transfecting T cells. Filed 20 April 2004, US patent 7172869 B2, issued 2007.
June CH, Thompson CB, Nabel GJ, Gray GS, Rennert PD, inventors. Methods for selectively stimulating proliferation of T cells. Filed 2 March 2006, US patent 7175843 B2, issued 2007.
June CH, Thompson C, Nabel GJ, Gray GS, Rennert P, inventors; US Government, ONR, assignee. Methods for treating HIV infected subjects. Filed 22 October 2008, United States Patent and Trademark Office 12/255,861, US patent application pending.
Blazar B, June CH, Godfrey W, Carroll R, Levine B, Riley J, Taylor P, inventors; Trustees of the University of Pennsylvania, assignee. Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses. Filed 29 December 2009, United States Patent and Trademark Office 12/649,101, US patent, issued 6 March 2012.
June CH, Riley J, Maus M, Thomas AK, inventors; The Trustees of the University of Pennsylvania, assignee. Activation and expansion of T-cells using an engineered multivalent signaling platform. Filed 3 January 2003, US patent 7638326, issued 2009.
June CH, Thompson C, Nabel G, Gray G, Rennert P, inventors; Genetics Institute, Regents of the University of Michigan, The United States of America as represented by the Secretary of the Navy, assignee. Methods for selectively enriching TH1 and TH2 cells. Filed 5 January 2006, US patent 7479269, issued 2009.
Riley J, June CH, Blazar B, Hippen KL, inventors. Inducible regulatory T-cell generation for hematopoietic transplants. Filed 19 June 2009, PCT/US2009/047887, application pending.
Blazar B, June CH, Godfrey W, Carroll R, Levine B, Riley J, Taylor P, inventors; The Trustees of the University of Pennsylvania, assignee. Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses. Filed 19 April 2004; US patent 7651855, issued 2010 .
Fowler DH, Jung U, Gress RE, Levine B, June CH, inventors. Rapamycin-resistant T cells and therapeutic uses thereof. Filed 9 December 2005, US patent 7718196 B2, issued 2010.
June CH, Levine B, Porter D, Kalos M, inventors; University of Pennsylvania, assignee. Use of chimeric antigen receptor-modified T cells to treat cancer. Filed 9 December 2010, US Provisional Patent Appl. No. 61/421,470, application pending.
June CH, Riley J, Maus M, Thomas A, Vonderheide R, inventors; The Trustees of the University of Pennsylvania, assignee. Activation and expansion of T-cells using an engineered multivalent signaling platform as a research tool. Filed 13 June 2003; US patent 7745140, issued 2010.
Riley J, June CH, Maus M, inventors; The Trustees of the University of Pennsylvania assignee. System and methods for promoting expansion of polyclonal and antigen-specific cytotoxic T lymphocytes in response to artificial antigen presenting cells. Filed 3 January 2003, US patent 7670781, issued 2010.
Riley J, June CH, Vonderheide R, Aqui N, Suhoski M, inventors; The Trustees of the University of Pennsylvania assignee. Artificial antigen presenting cells and uses therefore. Filed 3 January 2003; US patent 7754482 B2, issued 2010.
Riley J, June CH, Vonderheide R, Aqui N, Suhoski M, inventors; Trustees of the University of Pennsylvania, assignee. Novel artificial antigen presenting cells and uses therefore. Filed 8 June 2010, United States Patent and Trademark Office 12/796445, US patent.
Fowler DH, Jung U, Gress RE, Levine B, June CH, inventors; DHHS and Trustees of University of Pennsylvania, assignee. Rapamycin-resistant T cells and therapeutic uses thereof. Filed 30 March 2010; US patent 8075921, issued 2011.
June CH, Levine B, Porter D, Kalos M, inventors. Use of chimeric antigen receptor-modified T cells to treat cancer. Filed December 9 2011, PCT/US11/64191, application pending.
June CH, Levine B, Porter D, Kalos M, inventors. Compositions and methods for treatment of chronic lymphocytic leukemia. Filed 29 June 2011, US Prov’l Patent Appl. No. 61/502649, application pending.
June CH, Carroll RG, Riley JL, St. Louis DC, Levine BL, inventors; US Government, ONR, assignee. Methods for downregulating CCR5 in T cells with anti-CD3 antibodies and anti-CD28 antibodies. Filed 20 February 1998, US patent 7927595, issued 2011.
Riley J, June CH, Blazar B, Hippen KL, inventors. Methods to expand a T regulatory cell master cell bank. Filed 28 March 2011, WO2011126806 A1, application pending.
Riley J, Paulos CM, June CH, Levine B, inventors. ICOS critically regulates the expansion and function of inflammatory human Th17 cells. Filed 2 April 2011, PCT/US11/23744, application pending.
Zhao Y, June CH, inventors. Switch costimulatory receptors. Filed 29 July 2011; WO2013019615 A2, application pending.
Zhao Y, June CH, inventors; University of Pennsylvania, assignee. RNA engineered T cells for the treatment of cancer. Filed 16 September 2011; WO2013040557 A2, application pending.
Blazar B, June CH, Godfrey W, Carroll R, Levine B, Riley J, Taylor P, inventors; Trustees of the University of Pennsylvania, assignee. Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses. Filed 29 December 2009, US patent 8129185, issued 2012.
Frigault MJ, Scholler J, Zhao Y, June CH, inventors. Compositions and methods for generating a persisting population of T cells useful for the treatment of cancer. Filed 22 February 2012; PCT/US2013/027337, application pending.
Guedan S, Zhao Y, Scholler J, June CH, inventors. Use of ICOS based CARs to enhance antitumor activity and CAR persistence. Filed 21 February 2012, WO2013126733 A1, PCT/2013/027366, application pending.
Levine B, June CH, inventors. Methods for treating progressive multifocal leukoencephalopathy. Filed 24 February 2010, US patent 8415150, issued 9 April 2013.
June CH, Zhao Y, Liu X, inventors. Enhancing activity of CAR T cells by co-introducing a bispecific antibody. Filed 12 July 2013. Patent No: WO2014011988 A2, application pending.
June CH, Levine BL, Kalos MD, Zhao Y, inventors. Epitope spreading associated with CAR T cells. Filed 12 July 2013. Patent No: WO2014011993 A2, application pending.
Aqui N, June CH, Riley JL, Suhoski M, Vonderheide RH, inventors. Novel artificial antigen presenting cells and uses therefore. Filed 25 May 2005. WO2005118788 A3, application pending.
Carroll RG, June CH, Riley JL, inventors. Methods, compositions and kits relating to antigen presenting tumor cells. Publication date Filed 30 August 2007, WO2008027456 A3, application pending.
June CH, Carroll R, Riley J, Louis DS, Levine B, inventors. Methods for modulating expression of an HIV-1 fusion cofactor. Filed 10 March 2010. Published by United States Patent and Trademark Office, 14 September 2006, US20060204500 A1 US Patent.
June CH, Riley JL, Maus M, Thomas A, Vonderheide R, inventors. Activation and expansion of T-cells using an engineered multivalent signaling platform as a research tool. Filed 10 May 2010. US patent US8637307 B2, issued 2014.
Riley J, June CH, Maus M, inventors. Activation and expansion of T-cells using an agent that provides a primary activation signal and another agent that provides a co-stimulatory signal. Filed 3 January 2003. US patent number US7670781 B2, issued 2010.