Adoptive Cellular Therapy With Synthetic T Cells as an “Instant Vaccine” for Cancer and Immunity

Chapter 30

Adoptive Cellular Therapy With Synthetic T Cells as an “Instant Vaccine” for Cancer and Immunity Carl H. June, MD Center for Cellular Immunotherapies, Abramson Cancer Center; University of Pennsylvania, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, Philadelphia, PA, United States

Chapter Outline 1 Introduction 1.1 History of Adoptive Cell Transfer 1.2 Lymphocyte Cell Transfer 1.3 Emergence of ImmunoOncology 2 Discovery of T Cells with Stem-Cell-Like Qualities 3 Approaches to Engineer Lymphocytes Using Synthetic Biology

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4 Design of CAR T Cells 5 Lessons from Clinical trials on Patients with Chronic Infection 6 Clinical trials with CAR T Cells Directed Against B-Cell Malignancies 7 Toxicities and Management 8 Conclusions and Future Challenges References

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1 INTRODUCTION The fields of vaccinology and adoptive cell transfer (ACT) converge in the shaping of the acquired cellular and humoral responses of the B- and T cells of the immune system toward desired antigens on pathogens or self-targets. However, the approaches differ markedly, with vaccines often requiring immunizations and boosting over a period of months, whereas ex vivo culture of T cells and gene transfer can generate sufficient T cells in less than a week (Fig. 30.1). The process of ACT is more complex but the power of the resultant T cells may justify the expense and effort in some areas such as cancer and chronic infection, where vaccines often fail. The Vaccine Book. http://dx.doi.org/10.1016/B978-0-12-802174-3.00030-8 Copyright © 2016 Elsevier Inc. All rights reserved.

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FIGURE 30.1  General approach for trials with ACT. The patient donates lymphocytes by phlebotomy, and the cells are genetically modified and expanded in numbers in the manufacturing facility. In an optional step, the patient may receive chemotherapy before intravenous infusion of the CAR T cells.

1.1  History of Adoptive Cell Transfer A scientific and technological revolution in the war against cancer has taken place in the past century. Paul Ehrlich was probably the first to envision targeted therapies with “magic bullets,”1 an idea that was actualized by the invention of monoclonal antibodies and has inspired generations of scientists to devise powerful molecular cancer therapeutics. In this review the potential uses of chimeric T cells, modified to express antibody fragments is discussed. The idea that passive transfer of primed immune cells can generate immunity in the recipient of this transfer is a relatively old idea in the history of immunology. As first proposed by Billingham, Brent, and Medawar in 1954, who coined the term “adoptive immunity,”2 numerous animal studies have demonstrated the effectiveness of adoptive transfer of immunity toward cancer and infectious disease. Immunity has remarkable specificity toward its targets, and specificity can be controlled through strategies such as in vivo and ex vivo priming and genetic engineering to install receptors of defined specificities. Moreover, it has the potential to induce longstanding effects via the establishment of immunologic memory.

1.2  Lymphocyte Cell Transfer In early studies, the transfer of pig and nonhuman primate lymphocytes were tested and found inadequate for ACT due to transient engraftment.3,4 However,

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with the advent of efficient tools for genetic editing, it is not inconceivable that the use of xenogeneic cells for ACT might be reconsidered. Currently, human T cells with alpha beta T-cell receptors (TCRs) are currently the cell of choice for most clinical trials, reviewed in Ref. 5. Gene transfer of MHC class I-restricted TCRs can “convert” a population of polyclonal CD8+ T cells to cytotoxic Tlymphocytes (CTLs) of monoclonal TCR specificity.6 The adoptive transfer of engineered CD4+ T cells has promise for adoptive therapy of cancer and HIV, reviewed in Refs. 7,8. Recent advances in the understanding of the biology of γδ T cells suggest that these cells have promise for ACT. For example, human γδ T cells can prime αβ T cells with an efficiency similar to that of dendritic cells.9 Human γδ T cells can be engineered to express chimeric antigen receptors (CARs) and αβ TCRs.10,11 The use of γδ T cells may provide a significant safety feature over the use of TCR-engineered αβ T cells, taking advantage of the finding that α and β TCR chains cannot pair with γ and δ TCR chains. Natural killer T (NKT) cells are functionally related to γδ T cells since they also bridge innate and adaptive immune responses and can enhance or suppress immunity.12 The best characterized human NKT cell subpopulation, referred to as invariant NKT (iNKT) cells, expresses CD161 and an invariant Vα24Jα18 TCR that recognizes α-galactosylceramide presented by the MHC class I-like molecule CD1d. After activation, iNKT cells have MHC-independent cytotoxic activity against various tumors and secrete high levels of interferon-γ, although this function becomes impaired in patients with cancer.13 Compared with mouse NKT cells, human NKT cells are rare and comprise <1% of total lymphocytes. However, human NKT cells, unlike mouse NKT cells, can undergo substantial expansion in vitro.14 The first ACT studies of iNKT cells have been conducted and show safety and some evidence of antitumor activity.15

1.3  Emergence of Immuno-Oncology Targeting of disease through the adoptive transfer of lymphocytes was first reported more than 50 years ago in rodent models.16 However, it is only recently that the widespread use of immune-oncology has been accepted into the daily practice of medicine. This is best illustrated by the striking success of immune checkpoint blocking antibodies, which have achieved the complete and sustained remission of advanced solid tumors,17 and the use of genetically engineered T cells for melanoma and leukemia.18 The field of ACT has been made possible by improved understanding of T-cell biology, including the mechanisms for T-cell activation and recognition of targets, the role of accessory surface molecules and signal transduction pathways involved in the regulation of T-cell function and survival, as well as the identification and cloning of soluble T-cell growth factors, has facilitated the ability to expand ex vivo large numbers of highly potent T cells for adoptive immunotherapy. There are several excellent reviews of the experimental basis for ACT therapy of tumors and chronic infections.19–21

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2  DISCOVERY OF T CELLS WITH STEM-CELL-LIKE QUALITIES Mouse syngeneic tumor models have been essential for the identification and preclinical optimization of many tumor therapies. However, mouse tumor models in general have not been a good predictor of responses to ACT for cancer although they have been quite useful for chronic infections. In part this is likely due to the fact that most tumor models in mice don’t mirror the mutational loads and chronic inflammation found in humans, and due to the fact that the onset human T-cell senescence is quite different from mice, reviewed in Ref. 20. The seminal discovery of telomerase22 and the developing field of telomere biology identified fundamental differences in mechanisms of immune senescence between mice and humans23–25 that have important implications for ACT. In human T cells, a decrease in mean telomere restriction fragment length was shown for increasing age.26,27 As one consequence of the appreciation of the differences in biology between mouse and human T cells, the duration of culture for human T cells used in adoptive transfer has decreased from a month or longer to days to a few weeks, resulting in preservation of telomere length in infused T cells. More recent studies in the mouse indicate that a memory stem cell subset of CD8+ T cells exists.28 Similar T cells with stem cell-like qualities have been identified in humans.29,30 In elegant and technically demanding studies, these memory stem T cells were shown to have plasticity and that a single naive T cell can reconstitute diverse effector and memory T-cell subsets in mice.31,32 Ongoing studies in human allotransplant patients indicate that infusions of very low numbers of CMV-specific T cells are able to provide protective immunity.33 Efficient systems for the growth of human T cells were developed based on principles of costimulation.34,35 When these culture systems were employed in patients with HIV infection, efficient engraftment and long-term persistence of T cells was demonstrated after ACT.36,37 CD4+ T cells rendered CCR5-deficient by zinc finger nuclease (ZFN) editing also have been shown to persist for at least 5 years after ACT in patients with chronic HIV infection, consistent with the existence of central memory T cells in humans with long life spans.38

3  APPROACHES TO ENGINEER LYMPHOCYTES USING SYNTHETIC BIOLOGY In contrast to promising results in patients with chronic infection, ACT with natural T cells was not generally shown to be beneficial in cancer patients. A major reason for this is that tolerance to tumor antigens is induced by a variety of mechanisms, including deletion of tumor reactive cells and the induction of adaptive resistance mediated by checkpoint molecules such as CTLA-4 and PD1.39

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Synthetic biology, an emerging discipline aimed at reprogramming living organisms through the combined use of genetics, engineering principles, and systems and computational analysis, is able to enhance T-cell behavior.40–42 Cells are inherently capable of carrying out complex computations and responses, and CTLs of the immune system in particular are composed of cells poised to kill tumor cells after through careful assessment of targets. Approaches to genetically engineered lymphocytes have been reviewed previously.5 Using the principles of gene transfer it is possible to change “at will” the specificity of T cells using CAR and TCR of known specificity and affinity. Additional strategies have involved the overexpression in T cells of prosurvival signals such as telomerase,43 antiapoptotic genes,44 and the downregulation of proapoptotic molecules such as Fas.45 Yet another approach to enhance T-cell survival involves the expression of dominant-negative receptors for inhibitory molecules such as dominant-negative receptors for TGF-beta.46 Over the past decade a number of tools including ZFN, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system (CRISPER/ Cas9) have been developed to permit gene-specific disruption and site-specific insertion of novel DNA sequences.47 To date only ZFNs have been used for human trials.38 However, in vitro these tools are enabling the discovery of genes that increase the function by T cells by loss of function or gain of function mutations. For example, Zhou and coworkers48 reported a novel RNA-interference based screen conducted in vivo using ACT with transduced TCR transgenic T cells, with enrichment of shRNAs occurring in T cells whose numbers in the tumor bed increased upon knock down of relevant genes. Others used a CRISPR library approach for the in vitro analysis of LPS signaling in dendritic cells.49 Both screens identified known as well as unknown targets that affected the response being assessed. Using CRISPR/Cas9 technology, Konermann and coworkers reported a discovery method testing selective genome-wide gene activation to achieve desired phenotypes.50 Together, these new tools are overcoming a key challenge facing the cancer immunology field in the discovery of the most suitable targets to synthetically “hack” T cell to enhance T-cell antitumor functions. The diverse approaches to effectively engineer T cells combined with recently acquired mechanistic insights into T-cell biology and tumor immunity have converged to the point where the rational engineering of potent antitumor T-cell immunity is a practical and clinically testable reality. The majority of clinical approaches have employed T cells engineered to stably express transgenes via virus-based transduction. Virus-mediated gene transfer approaches typically employ vectors that are derived from gamma retroviruses or more recently lentiviruses, principally because of their ability to integrate into the host genome and drive long-term transgene expression, as well as for their low intrinsic immunogenicity. Gammaretrovirus-based transduction requires replicating cells for viral integration into genomic DNA, whereas lentiviral vectors

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can also integrate into nondividing cells; lentiviral vectors also appear to be less susceptible to silencing by host restriction factors and can deliver larger DNA sequences than retroviruses.51 Although virus-based approaches result in reasonably efficient transduction of primary T cells, they have considerable limitations in terms of cost to manufacture clinical-grade material, the total size of DNA that can be included in the virus vectors, and the potential, principally for retroviruses, for the integration events to result in insertional oncogenesis. A new virus-based system that has not yet entered clinical trials is based on foamy virus vectors, which possess favorable integration properties and are not pathogenic in humans.52 Nonvirus-based approaches benefit from lower manufacturing cost and are in principle less immunogenic than viral approaches. Although such approaches are theoretically safer because they are not dependent on viral elements integrating into host DNA, their safety record is shorter than that of virus-based vectors. Nonviral approaches to introduce transgenes into T cells involve the utilization of transposon elements such as sleeping beauty and piggybac as well as ZFN, TALEN, and CRISPR/Cas9 based-technologies, which allow for the ability to engineer T-cell populations with transgene insertions into specific chromosomal loci or that are biallelically disrupted for specific genes.53–57 Such technologies offer significant potential to engineer T cells in a manner that allows for the ability to interrupt or otherwise modulate expression of particular proteins that may be deleterious to therapeutic function.

4  DESIGN OF CAR T CELLS CARs are synthetic, engineered receptors that can target surface molecules in their native conformation.58,59 Unlike TCRs, CARs engage molecular structures independent of antigen processing by the target cell and independent of MHC; recognition features that are desirable as an approach to overcome resistance mechanisms found in tumors and chronic infection. CARs typically engage the target via a single-chain variable fragment (scFv) derived from an antibody, although natural ligands have also been used.60 Individual scFv’s targeting a surface molecule are derived either from murine or humanized antibodies, or synthesized and identified by screening of phage display libraries.61 Unlike TCRs, where a narrow range of affinity dictates the activation and specificity of the T cell, CARs typically have a much higher and perhaps broader range of affinities that will engage the target without necessarily encountering crossreactivity issues. Varying the affinity can increase the therapeutic index of CAR T cells, by increasing the discrimination of normal tissue expression from levels of surrogate antigen expressed on tumors.62,63 The length, flexibility, and origin of the hinge domain is also an important variable in the design of CARs.62,64,65 A major challenge to the field is that it is currently necessary to empirically test these design variables as there are no general rules guiding CAR design for selected target molecules.

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FIGURE 30.2  CAR design. In the natural immune system, B cells display antibodies on their surface and T cells have TCRs on the surface. A synthetic CAR T cell is a chimera of a B- and T cell, displaying both an antibody fragment (scFv) and an endogenous TCR.

The “generations” of CARs typically refer to the number and composition intracellular signaling domains (Fig. 30.2). “First-generation” CARs include only CD3zeta as an intracellular signaling domain whereas second-generation CARs also include costimulatory domains. Most investigators are currently using the hinge and transmembrane domains of CD8alpha or CD28. Hinge domains derived from Fc regions have also been investigated and modified in length,62 and in some instances these hinges have also been reported to engage Fc receptors and activate innate immune cells.65

5  LESSONS FROM CLINICAL TRIALS ON PATIENTS WITH CHRONIC INFECTION Adoptive immunotherapy began in humans shortly after the discovery and production of IL-2 (Table 30.1). The initial trials in cancer patients were largely unsuccessful66; the reasons for the failure of the initial trials have only recently become apparent and include quantitative and qualitative deficiencies in the repertoire of the infused TCRs, infusion of cells that had reached replicative senescence, and susceptibility to checkpoint pathways in vivo that prevented responses. However, in patients with chronic viral infections, ACT was shown to have promise in early trials as ACT with natural CTL clones specific for cytomegalovirus and Epstein–Barr virus infection were shown to be effective in immunosuppressed individuals.67,68 Substantial data exist to indicate that CD8+ T cells can affect the outcome and viral load in HIV-1 infection. Naturally occurring gag-specific CTL responses are inversely associated with viremia.69 In contrast, adoptive CTL therapy for HIV/AIDS, though demonstrating safety and promising engraftment and trafficking of cells to sites of viral replication, has not been clinically effective,70 and is accompanied by rapid emergence of viral escape mutants.71 Mathematical modeling suggests that adoptive transfer of CTLs should augment HIV-1 immunity and control viral replication, but only

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TABLE 30.1 Potential Indications for Various Forms of ACT Therapies Indication

Cell type

References

Metastatic melanoma

Tumor infiltrating lymphocytes

[76]

B-cell lineage tumors

CD19 CAR T cells

[77]

Synovial cell sarcoma Multiple myeloma

NY-ESO-1 TCR T cells

[78] [79]

Lymphopenia/immune reconstitution

Polyclonal T cells

[36]

Donor lymphocyte infusions

Allogeneic T cells:

[80]

Type 1 diabetes

Autologous Tregs

[81]

Prevention of organ transplant rejection

Inducible and natural Tregs

[82]

Graft versus host disease

Cord blood derived donor Tregs

[83]

HIV: increase immunity

CTL clones

[84]

HIV: increase immunity

CAR T

[85]

HIV: increase resistance

CCR5-deficient T cells

[38]

CMV: therapy for drug-resistant virus

CTLs

[33,84]

EBV: therapy for drug-resistant virus

CTLs

[68]

when the replicative capacity of the genetically modified CTLs is preserved and functional CD4+ T cells are present.72,73 Thus, an attractive strategy is the use of genetically enhanced TCRs to facilitate immune-mediated control of viral replication.74 Ultimately, a two-pronged approach involving gene-modified T cells comprising CD4+ T-cell protection and CTL augmentation therapy might be optimal.75

6  CLINICAL TRIALS WITH CAR T CELLS DIRECTED AGAINST B-CELL MALIGNANCIES Maus and coworkers reviewed the literature and listed all CAR T-cell trials up to late in 2013.86 A search of the clinicaltrials.gov data base reveals that there are 88 trials testing CAR T cells that are currently registered.a Geographically most of the trials are being conducted in the United States, although China is now the second most prevalent site of investigation with CAR T cells. Although the earliest trials of CAR T-cell therapy were performed in patients with solid tumors,87,88 these trials were disappointing. It is actually in trials of patients with B-cell malignancies that the most exciting results have recently been obtained.77,89–92 a. Search terms “chimeric antigen receptor,” Dec. 10, 2015.

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Many groups including those at Memorial Sloan Kettering, Seattle Children’s and the Fred Hutchinson Cancer Research Center, the National Cancer Institute, and others, have reported clinical responses in relapsed refractory leukemia and lymphoma. At our center, we have treated more than 200 patients with CAR T cells targeting B-cell malignancies including chronic lymphoid leukemia, acute lymphoid leukemia, non-Hodgkin’s lymphoma, and myeloma. B-cell malignancies are particularly amenable to be targeted using CAR T-cell therapy, due to the presence of the CD19 and CD20 antigens on most B-cell malignancies from the most immature B-cell acute lymphoblastic leukemia (B-ALL) to the most mature lymphomas93 and the fact that patients can tolerate prolonged periods of B-cell aplasia. Treatment with CAR T cells specific for CD19 has resulted in complete responses in B-ALL and chronic lymphocytic leukemia. However, one recurrent observation among trials in B-ALL is the emergence of CD19-negative blasts at relapse in a substantial minority (approximately 15%) of patients.91,92,94 Loss of CD19 is rarely observed in CLL, suggesting that CD19 escape is more difficult, or that CD19 negative precursor cells for CLL do not occur. Fewer published studies are currently available for comparison of response rates against mature B-cell neoplasms and non-Hodgkin lymphoma and no examples of relapse attributable to CD19-negative escape variants have yet been reported. Myeloma has been successfully targeted in a pilot trial with CD19 CAR T cells,95 an event that was unexpected since myeloma typically does not express CD19. After treatment with B cell–directed CAR T cells, the response rates vary by disease. In CLL we observe an approximate 60% overall response rate in relapsed/refractory patients.96 In contrast, in young adults and children with ALL who have already failed a stem-cell transplant, there is a remarkable 90% complete response rate.94,97 There are some late relapses, largely attributed to either loss of functional CAR T cells in the patient, or to an emergence of a leukemia clone that does not express the CD19 target antigen that is recognized by the CAR T cell.98 The emerging solution to this is to have another “CAR in the garage” targeting other markers on B cells, a strategy that we have just begun testing with CD22-specific CAR T cells.

7  TOXICITIES AND MANAGEMENT In most cases the toxicities associated with CAR T cells are on-target and reversible, a characteristic setting this treatment apart from nearly all previous cancer therapies comprised of cytotoxic chemotherapy and radiation therapy.99 These toxicities include B-cell aplasia, tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome.89,100 Hyperferritinemia and elevations in serum C-reactive protein are observed in patients with cytokine release syndrome and macrophage activation syndrome.91,97 The occurrence of B-cell aplasia and duration of persistence of CAR T cells in patients with leukemia is a predictive biomarker of response to therapy.94,96 Cytokine release

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syndrome has been reported by all groups after treatment of B-cell malignancies, and has also been observed after the treatment of multiple myeloma,95 which is typically considered a CD19 negative malignancy. Cytokine release syndrome is now effectively managed with tocilizumab and other reagents that block IL-6 signaling.91,101 The use of cytokine blockage may be preferable to immunosuppression with corticosteroids due to enhanced therapeutic effects and less systemic immunosuppression.102 CNS symptoms and signs consisting of expressive aphasia, confusion, and seizures have been observed after CD19 CAR therapy.103 This syndrome is reversible and the cause of these syndrome remains unexplained because there is no obvious correlation with symptoms and tumor in the CNS. This may be a drug class effect because the syndrome is also observed after treatment with blinatumomab, a CD19 directed bispecific single-chain antibody.104

8  CONCLUSIONS AND FUTURE CHALLENGES Adoptive immunotherapy using autologous T cells endowed with CARs has emerged as a powerful approach to treating cancer. However, a current limitation of this approach is that autologous CAR T cells must be generated on a bespoke basis. The development of automated culture technologies employing robotics will be required to scale out this therapy for general incorporation into the routine practice of medicine.105 Further, the potential development of third party cells through large-scale manufacturing of T cells deficient in expression of their TCR and other molecules will likely ameliorate this issue through the use of “off-the-shelf” CAR T cells.106 There are now dozens of cell therapy companies and it is likely that this emerging industry will solve issues of manufacturing feasibility.18 The major scientific issue in the field is whether engineered T cells will have substantial impact in adenocarcinoma and other solid tumors; at this point efficacy following adoptive transfer is limited and most routinely observed with natural tumor infiltrating lymphocytes and TCR T cells rather than CAR T cells.107 Finally, the ability to mass produce genetically modified T cells with desired specificities has the potential to enable “instant vaccines” as a form of immunotherapy that can be effective rapidly and in immunosuppressed patients, where standard vaccines are often ineffective.

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