From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
that there is an unexpected twist to this story. They make full use of the green fluorescent protein (GFP) that they have inserted into the Gata1 gene to serve as a reporter for gene activation. As expected, they observe that, in the context of a wild-type Gata1 reporter transgene, expression of GFP is suppressed in the HSPC compartment. In contrast, Gata1 reporter transgenes lacking the intervening 3.2-kb DNA fragment but retaining the 3 positive regulatory elements express GFP abundantly in this compartment. This striking difference in GFP expression is most graphically illustrated in Figure 3, panels D and F, of their article (compare wild-type transgene, G1B-GFP, with the mutant transgene, MG-GFP). This result demonstrates that the intervening 3.2-kb DNA fragment is required for suppression of the Gata1 gene in the HSPC compartment. But how is this achieved? A survey of epigenetic modifications has revealed that the Gata1 locus is heavily methylated in the HSPC compartment.5 DNA methylation, occurring at cytosine residues in the dinucleotide sequence 59CG-39, is a very stable modification which is strongly associated with repression of gene activity. Takai et al analyze DNA methylation of the transgenes in HSPCs. In the case of the wild-type transgene, they find high methylation levels at the Gata1 promoter and regulatory elements. In contrast, DNA methylation was much reduced in the mutant transgene lacking the intervening 3.2-kb DNA fragment. This fragment is therefore required for high DNA methylation levels of critical Gata1 regulatory elements in HSPCs. Hypermethylated DNA attracts repressor proteins including DNA methyltransferase 1 (DNMT1). DNMT1 ensures that DNA methylation patterns are faithfully passed on to the daughter cells after cell division, thus locking the Gata1 gene stably in an inactive configuration. The authors provide evidence that under these conditions GATA2 is unable to bind to the Gata1 regulatory elements, providing a mechanistic explanation for the inability of GATA2 to activate the Gata1 gene in HSPCs (see figure). To proceed to lineage commitment and differentiation would require demethylation of the Gata1 locus, and it remains to be investigated how this is achieved. This could be through a passive process, involving failure to maintain methylation after DNA replication.
3392
Interestingly, Takai et al characterize a binding site for E2F transcription factors which provides a potential lead. In quiescent cells, E2F factors form a repressive complex including the Retinoblastoma protein and DNMT1.6 When the cells enter the cell cycle, the Retinoblastoma protein is phosphorylated and the repressive complex is released, allowing E2F to become active. If the repressive complex is not reformed after cell division, DNMT1 would fail to maintain DNA methylation and the methylation marks would be lost. Alternatively, demethylation might be an active process, for which a number of different mechanisms have been proposed7 including oxidation of the methyl groups by TET proteins, modification by AID/APOBEC enzymes, and base excision DNA repair. Aberrant DNA methylation is a hallmark of many cancers including hematologic malignancies, and DNA methylation inhibitors are used to treat patients with myelodysplastic syndromes. The results described by Takai et al suggest that restoration of expression of genes required for differentiation, such as GATA1, contributes to the therapeutic efficacy of DNA methylation inhibitors. Finally, the current study relies on the use of transgenes, which are analyzed in the presence of the endogenous Gata1 locus. An important next step would be to assess the role of the elements identified in the context of the endogenous locus. Although this used to be a mammoth task, the flurry of recent
articles describing CRISPR/CAS9 as a very efficient tool for mammalian genome engineering8 makes this a realistic proposition. Such experiments will provide an increasingly detailed picture of the dynamic regulation of endogenous GATA factor expression during hematopoiesis. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Takai J, Moriguchi T, Suzuki M, Yu L, Ohneda K, Yamamoto M. The Gata1 59 region harbors distinct cisregulatory modules that direct gene activation in erythroid cells and gene inactivation in HSCs. Blood. 2013;122(20): 3450-3460. 2. Ferreira R, Wai A, Shimizu R, et al. Dynamic regulation of Gata factor levels is more important than their identity. Blood. 2007;109(12):5481-5490. 3. Ferreira R, Ohneda K, Yamamoto M, Philipsen S. GATA1 function, a paradigm for transcription factors in hematopoiesis. Mol Cell Biol. 2005;25(4):1215-1227. 4. Grass JA, Jing H, Kim SI, et al. Distinct functions of dispersed GATA factor complexes at an endogenous gene locus. Mol Cell Biol. 2006;26(19):7056-7067. 5. Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST, Weissman IL. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A. 2007;104(30):12371-12376. 6. Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet. 2000;25(3): 338-342. 7. Wu SC, Zhang Y. Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol. 2010;11(9):607-620. 8. Menke DB. Engineering subtle targeted mutations into the mouse genome. Genesis. 2013;51(9):605-618. © 2013 by The American Society of Hematology
l l l IMMUNOBIOLOGY
Comment on Casucci et al, page 3461
Risky business: target choice in adoptive cell therapy ----------------------------------------------------------------------------------------------------Richard A. Morgan1
1
NATIONAL INSTITUTES OF HEALTH
In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities.1
T
here has been an explosion of interest in field of adoptive cell transfer due to the recent clinical success reported by several groups in the treatment of B cell
malignancies.2-4 Durable clinical responses have been documented in several patients, including those heavily pretreated with standard chemotherapy, using T cells genetically
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
Target antigen types for adoptive cell therapy. T cells can be genetically modified with tumor-targeting receptors (either T-cell receptors or CARs) and administered to patients in adoptive cell therapy trials. The types of tumors and tumor antigens targeted by these modified T cells can have no or significant off-tumor/on-target toxicity dependent on antigen expression. For tumor-specific antigens such as EGFRvIII in glioblastoma (GBM), there will likely be no toxicity due to the lack on antigen expression in normal tissues. Some (but not all) CT antigens have gene expression patterns limited to cancer and the non–MHC-bearing germ cells in the testis (eg, NY-ESO-1 in melanoma). It may be clinically acceptable to target tumor antigens that are expressed in malignancies and nonessential tissues such as the example of CD19, which is expressed in chronic lymphocytic leukemia (CLL) but also in normal B cells. B-cell depletion can be managed by IV immunoglobulin (IVIG) administration. The majority of tumor antigens follow the pattern investigated by Casucci et al, where the CD44v6 antigen is expressed in the tumor (eg, MM) as well as in normal tissues. As a potential method to manage expected monocytopenia, the investigators suggest that a suicide gene could be used to eliminate T cells after the antitumor response is complete.
engineered to express chimeric antigen receptors (CARs) directed to the CD19 antigen. Choosing target antigens for adoptive cell therapy is tricky (see figure). Ideally, a tumor antigen would be tumor specific; that is, not expressed in any normal tissue. Although chromosomal translocations are well documented in a variety of malignancies, those rearrangements that reproducibly result in the exact same novel protein sequence are rare. An example of this type of tumor antigen is one of the rearrangements of the epidermal growth factor receptor (EGRF variant III, or EGFRvIII), which is commonly found in glioblastoma as well as some other cancers.5 Viral-associated cancers are also in this category (eg, human papillomavirus 16/17–associated cervical cancer). Some cancers with large numbers of environmentally induced mutations (ie, lung cancer and melanoma) likely have patient-specific mutations that induce highly specific immune responses.6
Unfortunately, the majority of tumors do not express specific mutations with common genetic alterations that can be targeted by antitumor antigen receptor–modified T cells. A potentially attractive alternative to the cancer-specific antigens may be a class of antigens known as cancer-testis (CT) antigens.7 The CT antigens (also called cancer-germ line antigens) are generally (but not exclusively) expressed in the testis and many tumor types. In the case of T-cell receptor–modified T cells, the testis is not a target for immunologic attack because the CT-expressing germ cells do not express major histocompatibility complex molecules. An example of a CT antigen that has been successfully targeted in adoptive cell therapy clinical trials is the NY-ESO-1 protein (gene name CTAG1B).8 The CD19 antigen is an example of a tumor antigen that is expressed in tumors and nonessential tissues. In the successful clinical trials describing anti-CD19 CAR therapies, prolonged B-cell depletion
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
was observed in several patients, and this necessitated the use of IVIG therapy. The majority of tumor antigens are in a class of proteins that are overexpressed in tumors but also have normal tissue expression. The CD44v6 tumor antigen is an example of this class of tumor antigen. Casucci et al demonstrated that CD44v6 was expressed on the majority of acute myeloid leukemia (AML) and multiple myeloma (MM) samples analyzed. Furthermore their data suggest that CD44v6 may be necessary for the establishment of tumors because when the gene was specifically knocked out using short interfering RNA, tumor engraftment in immunocompromised mice was significantly reduced. To target the CD44v6 antigen, the authors developed a CAR based on the anti-CD44v6 monoclonal antibody bivatuzumab and linked this to T-cell signaling domains from the CD28 and CD3z proteins. Using retroviral vectors to transfer the CAR into primary human T cells yielded T cells that displayed the appropriate in vitro anti-CD44v6 effector functions and actively inhibited AML and MM tumor formation in xenograft models. Many investigators would publish their data at this point, but for those investigators serious about human applications, this is insufficient. Normal tissue expression of CD44v6 was investigated at the RNA and protein levels and found to be present at easily detectable levels in keratinocytes and circulating monocytes. Although keratinocytes were not recognized by anti-CD44v6 CAR-engineered T cells, monocytes were and monocytopenia was observed in animal models. This type of off-tumor/on-target toxicity will be an unavoidable consequence for many adoptive cell therapy approaches using receptormodified T cells, and whereas transient monocytopenia may be manageable, longterm monocytopenia is not. Therefore, the investigators added a suicide gene to their gene transfer system (specifically the nonimmunogenic and quick-acting iCasp9 system) and were able to show rapid elimination of gene-engineered T cells in in vivo models. The report by Casucci et al is an excellent example of the identification of a potentially valuable tumor antigen for adoptive cell therapy that combines a target that may be essential for tumorigenesis in multiple cancers, with a detailed plan for clinical
3393
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
development based on the knowledge of potential toxicities and potential means to limit those toxicities. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Casucci M, Nicolis di Robilant B, Falcone L, et al. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood. 2013;122(20):3461-3472. 2. Brentjens RJ, Davila ML, Riviere I, et al. CD19targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177):177ra38. 3. Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509-1518.
4. Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012; 119(12):2709-2720. 5. Li G, Wong AJ. EGF receptor variant III as a target antigen for tumor immunotherapy. Expert Rev Vaccines. 2008;7(7):977-985. 6. Robbins PF, Lu YC, El-Gamil M, et al. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med. 2013;19(6):747-752. 7. Caballero OL, Chen YT. Cancer/testis (CT) antigens: potential targets for immunotherapy. Cancer Sci. 2009; 100(11):2014-2021. 8. Robbins PF, Morgan RA, Feldman SA, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. 2011;29(7): 917-924.
l l l IMMUNOBIOLOGY
Comment on Asgari et al, page 3473
The C3a receptor, caspase-1, and release of IL-1b ----------------------------------------------------------------------------------------------------Charles A. Dinarello1,2
1
UNIVERSITY OF COLORADO DENVER; 2UNIVERSITY MEDICAL CENTER NIJMEGEN
In this issue of Blood, Asgari et al report that engagement of the C3a receptor triggers interleukin-1b (IL-1b) processing and release via caspase-1 activation. The role of complement activation in IL-1 production has a long history; complement products function as “alarmins” during innate responses. For many years before the term “innate immune response” was coined, it was fully understood that a highly nonspecific event such as activation of complement would induce a highly nonspecific molecule such as IL-1; these 2 linked processes would then affect a highly specific event such antigen-driven lymphocyte activation, for example, polarization to a T helper 1 (Th1) or a Th17 response. In this issue, investigators link the generation of C3a to playing a role in the activation of caspase-1. A unique and unexpected finding of the study is that engagement of the C3a receptor results in phosphorylation of extracellular signalregulated kinase-1 and 2 (ERK-1/2), which promotes the efflux of adenosine triphosphate (ATP) from the macrophage. Release of ATP is a rate-limiting step for activating caspase-1, as extracellular ATP triggers the P2X7 purinergic receptor to initiate oligomerization of NLRP3.1 he study by Asgari and coworkers1 is highly relevant to autoinflammatory diseases and particularly to sterile inflammation. In providing background on IL-1 for their readers, the authors make a common but excusable error by linking IL-1b to “autoimmune” diseases. However, the diseases for which there has been unquestionable benefit for blocking IL-1b are not classic autoimmune diseases but rather “autoinflammatory” diseases. Unlike autoimmune diseases, in autoinflammatory
T
3394
diseases there is no role for a dysfunctional T cell.2,3 In fact, the largest trial of any anti-cytokine is the CANTOS trial in which 17 200 patients at high risk for a cardiovascular event are being studied with canakinumab, a monoclonal antibody that neutralizes IL-1b. Although IL-1b does play a role in the pathogenesis of autoimmune diseases, it is best to consider IL-1 as an adjuvant for T- and B-cell activation4 during antigen-specific responses; alone, IL-1 does not simulate the proliferation of T cells.
In the present study, the authors study IL-1b as a cofactor in immune responses, very much as IL-1 was studied as a cofactor for the production of IL-2. Engagement of the C3a receptor enhances the production of IL-17; that IL-1b enhances the production of IL-17 is hardly a new finding. If C3a enhances lipopolysaccharide (LPS)–induced secretion of IL-1b, it is no surprise that IL-17 is similarly increased by C3aR engagement. The production of IL-1b was studied from fresh human blood monocytes stimulated with LPS, and the authors correctly point out that fresh human monocytes will release IL-1b when stimulated with LPS whereas macrophages and dendritic cells require a second signal such as a high concentration of ATP. This important difference between circulating blood monocytes and a monocyte-derived macrophage or dendritic cells was first reported in a previous issue of Blood.5 So what is new? The study then turns to a clinical issue: acute rejection of renal transplants. Successful kidney transplants change the lives of many with end-stage kidney disease, liberating them from the yoke of chronic hemo- or peritoneal dialysis. As with all transplanted organs, suppression needs to be carefully adjusted and increased with the early signs of rejection. The study compared the presence of C3a using immunohistochemistry of paraffin sections of kidney biopsies from healthy subjects with those from transplants. Indeed, they found large numbers of CD41 lymphocytes expressing IL-17 in the vicinity of macrophages and tubular epithelial cells expressing C3a. The study concludes that “all the players” are present in acute renal rejection and, moreover, that C3a is an alarmin as a danger-associated molecular pattern. The article also considers a role for C3a in the generation of ATP; indeed, oxidized ATP reduced IL-1b secretion by 50%. Is this new? In terms of a role for C3aR engagement, yes, but not unexpected as nearly all studies in which one adds oxidized ATP to human monocytes stimulated with LPS reduce IL-1b secretion.5 However, the present study does make an unexpected observation and that is the role of the pannexin-1 hemichannel for the efflux of ATP by engagement of C3aR. One may recall another role for this channel in macrophages that produce IL-1b upon
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2013 122: 3392-3394 doi:10.1182/blood-2013-09-527622
Risky business: target choice in adoptive cell therapy Richard A. Morgan
Updated information and services can be found at: http://www.bloodjournal.org/content/122/20/3392.full.html Articles on similar topics can be found in the following Blood collections Free Research Articles (4041 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
that there is an unexpected twist to this story. They make full use of the green fluorescent protein (GFP) that they have inserted into the Gata1 gene to serve as a reporter for gene activation. As expected, they observe that, in the context of a wild-type Gata1 reporter transgene, expression of GFP is suppressed in the HSPC compartment. In contrast, Gata1 reporter transgenes lacking the intervening 3.2-kb DNA fragment but retaining the 3 positive regulatory elements express GFP abundantly in this compartment. This striking difference in GFP expression is most graphically illustrated in Figure 3, panels D and F, of their article (compare wild-type transgene, G1B-GFP, with the mutant transgene, MG-GFP). This result demonstrates that the intervening 3.2-kb DNA fragment is required for suppression of the Gata1 gene in the HSPC compartment. But how is this achieved? A survey of epigenetic modifications has revealed that the Gata1 locus is heavily methylated in the HSPC compartment.5 DNA methylation, occurring at cytosine residues in the dinucleotide sequence 59CG-39, is a very stable modification which is strongly associated with repression of gene activity. Takai et al analyze DNA methylation of the transgenes in HSPCs. In the case of the wild-type transgene, they find high methylation levels at the Gata1 promoter and regulatory elements. In contrast, DNA methylation was much reduced in the mutant transgene lacking the intervening 3.2-kb DNA fragment. This fragment is therefore required for high DNA methylation levels of critical Gata1 regulatory elements in HSPCs. Hypermethylated DNA attracts repressor proteins including DNA methyltransferase 1 (DNMT1). DNMT1 ensures that DNA methylation patterns are faithfully passed on to the daughter cells after cell division, thus locking the Gata1 gene stably in an inactive configuration. The authors provide evidence that under these conditions GATA2 is unable to bind to the Gata1 regulatory elements, providing a mechanistic explanation for the inability of GATA2 to activate the Gata1 gene in HSPCs (see figure). To proceed to lineage commitment and differentiation would require demethylation of the Gata1 locus, and it remains to be investigated how this is achieved. This could be through a passive process, involving failure to maintain methylation after DNA replication.
3392
Interestingly, Takai et al characterize a binding site for E2F transcription factors which provides a potential lead. In quiescent cells, E2F factors form a repressive complex including the Retinoblastoma protein and DNMT1.6 When the cells enter the cell cycle, the Retinoblastoma protein is phosphorylated and the repressive complex is released, allowing E2F to become active. If the repressive complex is not reformed after cell division, DNMT1 would fail to maintain DNA methylation and the methylation marks would be lost. Alternatively, demethylation might be an active process, for which a number of different mechanisms have been proposed7 including oxidation of the methyl groups by TET proteins, modification by AID/APOBEC enzymes, and base excision DNA repair. Aberrant DNA methylation is a hallmark of many cancers including hematologic malignancies, and DNA methylation inhibitors are used to treat patients with myelodysplastic syndromes. The results described by Takai et al suggest that restoration of expression of genes required for differentiation, such as GATA1, contributes to the therapeutic efficacy of DNA methylation inhibitors. Finally, the current study relies on the use of transgenes, which are analyzed in the presence of the endogenous Gata1 locus. An important next step would be to assess the role of the elements identified in the context of the endogenous locus. Although this used to be a mammoth task, the flurry of recent
articles describing CRISPR/CAS9 as a very efficient tool for mammalian genome engineering8 makes this a realistic proposition. Such experiments will provide an increasingly detailed picture of the dynamic regulation of endogenous GATA factor expression during hematopoiesis. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Takai J, Moriguchi T, Suzuki M, Yu L, Ohneda K, Yamamoto M. The Gata1 59 region harbors distinct cisregulatory modules that direct gene activation in erythroid cells and gene inactivation in HSCs. Blood. 2013;122(20): 3450-3460. 2. Ferreira R, Wai A, Shimizu R, et al. Dynamic regulation of Gata factor levels is more important than their identity. Blood. 2007;109(12):5481-5490. 3. Ferreira R, Ohneda K, Yamamoto M, Philipsen S. GATA1 function, a paradigm for transcription factors in hematopoiesis. Mol Cell Biol. 2005;25(4):1215-1227. 4. Grass JA, Jing H, Kim SI, et al. Distinct functions of dispersed GATA factor complexes at an endogenous gene locus. Mol Cell Biol. 2006;26(19):7056-7067. 5. Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST, Weissman IL. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A. 2007;104(30):12371-12376. 6. Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet. 2000;25(3): 338-342. 7. Wu SC, Zhang Y. Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol. 2010;11(9):607-620. 8. Menke DB. Engineering subtle targeted mutations into the mouse genome. Genesis. 2013;51(9):605-618. © 2013 by The American Society of Hematology
l l l IMMUNOBIOLOGY
Comment on Casucci et al, page 3461
Risky business: target choice in adoptive cell therapy ----------------------------------------------------------------------------------------------------Richard A. Morgan1
1
NATIONAL INSTITUTES OF HEALTH
In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities.1
T
here has been an explosion of interest in field of adoptive cell transfer due to the recent clinical success reported by several groups in the treatment of B cell
malignancies.2-4 Durable clinical responses have been documented in several patients, including those heavily pretreated with standard chemotherapy, using T cells genetically
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
Target antigen types for adoptive cell therapy. T cells can be genetically modified with tumor-targeting receptors (either T-cell receptors or CARs) and administered to patients in adoptive cell therapy trials. The types of tumors and tumor antigens targeted by these modified T cells can have no or significant off-tumor/on-target toxicity dependent on antigen expression. For tumor-specific antigens such as EGFRvIII in glioblastoma (GBM), there will likely be no toxicity due to the lack on antigen expression in normal tissues. Some (but not all) CT antigens have gene expression patterns limited to cancer and the non–MHC-bearing germ cells in the testis (eg, NY-ESO-1 in melanoma). It may be clinically acceptable to target tumor antigens that are expressed in malignancies and nonessential tissues such as the example of CD19, which is expressed in chronic lymphocytic leukemia (CLL) but also in normal B cells. B-cell depletion can be managed by IV immunoglobulin (IVIG) administration. The majority of tumor antigens follow the pattern investigated by Casucci et al, where the CD44v6 antigen is expressed in the tumor (eg, MM) as well as in normal tissues. As a potential method to manage expected monocytopenia, the investigators suggest that a suicide gene could be used to eliminate T cells after the antitumor response is complete.
engineered to express chimeric antigen receptors (CARs) directed to the CD19 antigen. Choosing target antigens for adoptive cell therapy is tricky (see figure). Ideally, a tumor antigen would be tumor specific; that is, not expressed in any normal tissue. Although chromosomal translocations are well documented in a variety of malignancies, those rearrangements that reproducibly result in the exact same novel protein sequence are rare. An example of this type of tumor antigen is one of the rearrangements of the epidermal growth factor receptor (EGRF variant III, or EGFRvIII), which is commonly found in glioblastoma as well as some other cancers.5 Viral-associated cancers are also in this category (eg, human papillomavirus 16/17–associated cervical cancer). Some cancers with large numbers of environmentally induced mutations (ie, lung cancer and melanoma) likely have patient-specific mutations that induce highly specific immune responses.6
Unfortunately, the majority of tumors do not express specific mutations with common genetic alterations that can be targeted by antitumor antigen receptor–modified T cells. A potentially attractive alternative to the cancer-specific antigens may be a class of antigens known as cancer-testis (CT) antigens.7 The CT antigens (also called cancer-germ line antigens) are generally (but not exclusively) expressed in the testis and many tumor types. In the case of T-cell receptor–modified T cells, the testis is not a target for immunologic attack because the CT-expressing germ cells do not express major histocompatibility complex molecules. An example of a CT antigen that has been successfully targeted in adoptive cell therapy clinical trials is the NY-ESO-1 protein (gene name CTAG1B).8 The CD19 antigen is an example of a tumor antigen that is expressed in tumors and nonessential tissues. In the successful clinical trials describing anti-CD19 CAR therapies, prolonged B-cell depletion
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
was observed in several patients, and this necessitated the use of IVIG therapy. The majority of tumor antigens are in a class of proteins that are overexpressed in tumors but also have normal tissue expression. The CD44v6 tumor antigen is an example of this class of tumor antigen. Casucci et al demonstrated that CD44v6 was expressed on the majority of acute myeloid leukemia (AML) and multiple myeloma (MM) samples analyzed. Furthermore their data suggest that CD44v6 may be necessary for the establishment of tumors because when the gene was specifically knocked out using short interfering RNA, tumor engraftment in immunocompromised mice was significantly reduced. To target the CD44v6 antigen, the authors developed a CAR based on the anti-CD44v6 monoclonal antibody bivatuzumab and linked this to T-cell signaling domains from the CD28 and CD3z proteins. Using retroviral vectors to transfer the CAR into primary human T cells yielded T cells that displayed the appropriate in vitro anti-CD44v6 effector functions and actively inhibited AML and MM tumor formation in xenograft models. Many investigators would publish their data at this point, but for those investigators serious about human applications, this is insufficient. Normal tissue expression of CD44v6 was investigated at the RNA and protein levels and found to be present at easily detectable levels in keratinocytes and circulating monocytes. Although keratinocytes were not recognized by anti-CD44v6 CAR-engineered T cells, monocytes were and monocytopenia was observed in animal models. This type of off-tumor/on-target toxicity will be an unavoidable consequence for many adoptive cell therapy approaches using receptormodified T cells, and whereas transient monocytopenia may be manageable, longterm monocytopenia is not. Therefore, the investigators added a suicide gene to their gene transfer system (specifically the nonimmunogenic and quick-acting iCasp9 system) and were able to show rapid elimination of gene-engineered T cells in in vivo models. The report by Casucci et al is an excellent example of the identification of a potentially valuable tumor antigen for adoptive cell therapy that combines a target that may be essential for tumorigenesis in multiple cancers, with a detailed plan for clinical
3393
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
development based on the knowledge of potential toxicities and potential means to limit those toxicities. Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Casucci M, Nicolis di Robilant B, Falcone L, et al. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood. 2013;122(20):3461-3472. 2. Brentjens RJ, Davila ML, Riviere I, et al. CD19targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177):177ra38. 3. Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509-1518.
4. Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012; 119(12):2709-2720. 5. Li G, Wong AJ. EGF receptor variant III as a target antigen for tumor immunotherapy. Expert Rev Vaccines. 2008;7(7):977-985. 6. Robbins PF, Lu YC, El-Gamil M, et al. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med. 2013;19(6):747-752. 7. Caballero OL, Chen YT. Cancer/testis (CT) antigens: potential targets for immunotherapy. Cancer Sci. 2009; 100(11):2014-2021. 8. Robbins PF, Morgan RA, Feldman SA, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. 2011;29(7): 917-924.
l l l IMMUNOBIOLOGY
Comment on Asgari et al, page 3473
The C3a receptor, caspase-1, and release of IL-1b ----------------------------------------------------------------------------------------------------Charles A. Dinarello1,2
1
UNIVERSITY OF COLORADO DENVER; 2UNIVERSITY MEDICAL CENTER NIJMEGEN
In this issue of Blood, Asgari et al report that engagement of the C3a receptor triggers interleukin-1b (IL-1b) processing and release via caspase-1 activation. The role of complement activation in IL-1 production has a long history; complement products function as “alarmins” during innate responses. For many years before the term “innate immune response” was coined, it was fully understood that a highly nonspecific event such as activation of complement would induce a highly nonspecific molecule such as IL-1; these 2 linked processes would then affect a highly specific event such antigen-driven lymphocyte activation, for example, polarization to a T helper 1 (Th1) or a Th17 response. In this issue, investigators link the generation of C3a to playing a role in the activation of caspase-1. A unique and unexpected finding of the study is that engagement of the C3a receptor results in phosphorylation of extracellular signalregulated kinase-1 and 2 (ERK-1/2), which promotes the efflux of adenosine triphosphate (ATP) from the macrophage. Release of ATP is a rate-limiting step for activating caspase-1, as extracellular ATP triggers the P2X7 purinergic receptor to initiate oligomerization of NLRP3.1 he study by Asgari and coworkers1 is highly relevant to autoinflammatory diseases and particularly to sterile inflammation. In providing background on IL-1 for their readers, the authors make a common but excusable error by linking IL-1b to “autoimmune” diseases. However, the diseases for which there has been unquestionable benefit for blocking IL-1b are not classic autoimmune diseases but rather “autoinflammatory” diseases. Unlike autoimmune diseases, in autoinflammatory
T
3394
diseases there is no role for a dysfunctional T cell.2,3 In fact, the largest trial of any anti-cytokine is the CANTOS trial in which 17 200 patients at high risk for a cardiovascular event are being studied with canakinumab, a monoclonal antibody that neutralizes IL-1b. Although IL-1b does play a role in the pathogenesis of autoimmune diseases, it is best to consider IL-1 as an adjuvant for T- and B-cell activation4 during antigen-specific responses; alone, IL-1 does not simulate the proliferation of T cells.
In the present study, the authors study IL-1b as a cofactor in immune responses, very much as IL-1 was studied as a cofactor for the production of IL-2. Engagement of the C3a receptor enhances the production of IL-17; that IL-1b enhances the production of IL-17 is hardly a new finding. If C3a enhances lipopolysaccharide (LPS)–induced secretion of IL-1b, it is no surprise that IL-17 is similarly increased by C3aR engagement. The production of IL-1b was studied from fresh human blood monocytes stimulated with LPS, and the authors correctly point out that fresh human monocytes will release IL-1b when stimulated with LPS whereas macrophages and dendritic cells require a second signal such as a high concentration of ATP. This important difference between circulating blood monocytes and a monocyte-derived macrophage or dendritic cells was first reported in a previous issue of Blood.5 So what is new? The study then turns to a clinical issue: acute rejection of renal transplants. Successful kidney transplants change the lives of many with end-stage kidney disease, liberating them from the yoke of chronic hemo- or peritoneal dialysis. As with all transplanted organs, suppression needs to be carefully adjusted and increased with the early signs of rejection. The study compared the presence of C3a using immunohistochemistry of paraffin sections of kidney biopsies from healthy subjects with those from transplants. Indeed, they found large numbers of CD41 lymphocytes expressing IL-17 in the vicinity of macrophages and tubular epithelial cells expressing C3a. The study concludes that “all the players” are present in acute renal rejection and, moreover, that C3a is an alarmin as a danger-associated molecular pattern. The article also considers a role for C3a in the generation of ATP; indeed, oxidized ATP reduced IL-1b secretion by 50%. Is this new? In terms of a role for C3aR engagement, yes, but not unexpected as nearly all studies in which one adds oxidized ATP to human monocytes stimulated with LPS reduce IL-1b secretion.5 However, the present study does make an unexpected observation and that is the role of the pannexin-1 hemichannel for the efflux of ATP by engagement of C3aR. One may recall another role for this channel in macrophages that produce IL-1b upon
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2013 122: 3392-3394 doi:10.1182/blood-2013-09-527622
Risky business: target choice in adoptive cell therapy Richard A. Morgan
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