- Email: [email protected]
A New Agent in the Strategy to Cure AIDS
© The American Society of Gene & Cell Therapy
commentary would still be necessary, but with agents such as fludarabine replacing alkylators, acute and late effects would be minimized. Even for patients undergoing allogeneic transplantation for malignancies with these nonchemotherapeutic approaches, one could imagine being able to focus solely on rejection and more targeted anticancer therapy while minimizing the toxic effects of the standard conditioning regimens. Finally, for malignancies that express CD45 and/ or anti-c-Kit, it might be possible that these novel approaches could replace alkylating chemotherapy and radiation altogether. REFERENCES
1. Chhabra, A, Ring, AM, Weiskopf, K, Schnorr, PJ, Gordon, S, Le AC et al. (2016). Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Sci Transl Med 8: 351ra105. 2. Palchaudhuri, R, Saez, B, Hoggatt, J, Schajnovitz, A, Sykes, DB, Tate, TA et al. (2016). Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing
immunotoxin. Nat Biotechnol 34: 738–745. 3. Dvorak, CC, Hassan, A, Slatter, MA, Hönig, M, Lankester, AC, Buckley, RH et al. (2014). Comparison of outcomes of hematopoietic stem cell transplantation without chemotherapy conditioning by using matched sibling and unrelated donors for treatment of severe combined immunodeficiency. J Allergy Clin Immunol 134: 935–943. 4. Pai, SY, Logan, BR, Griffith, LM, Buckley, RH, Parrott, RE, Dvorak, CC et al. (2014). Transplantation outcomes for severe combined immunodeficiency, 2000–2009. N Engl J Med 371: 434–446. 5. Hoggatt, J, Kfoury, Y and Scadden, DT (2016). Hematopoietic stem cell niche in health and disease. Annu Rev Pathol 11: 555–581. 6. Czechowicz, A, Kraft, D, Weissman, IL and Bhattacharya, D (2007). Efficient transplantation via antibodybased clearance of hematopoietic stem cell niches. Science 318: 1296–1299. 7. Xiao, TZ, Singh, K, Dunn, E, Ramachandran, R and Cowan, MJ (2012). T cell and B cell immunity can be reconstituted with mismatched hematopoietic stem cell transplantation without alkylator therapy in Artemis-deficient mice using anti-natural killer cell antibody and photochemically treated sensitized donor T cells. Biol Blood Marrow Transplant 18: 200–209. 8. Straathof, KC, Rao, K, Eyrich, M, Hale, G, Bird, P, Berrie, E et al. (2009). Haemopoietic stem-cell transplantation with antibody-based minimal-intensity conditioning: a phase 1/2 study. Lancet 374: 912–920. 9. Burtner, CR, Chandrasekaran, D, Santos, EB, Beard,
See page 1913
A New Agent in the Strategy to Cure AIDS John A Zaia1 doi:10.1038/mt.2016.194
I
n recent years the field of immunotherapy has expanded to include therapies never before imagined. Since recombinant DNA (rDNA) technology was first applied to the production of active antigen-specific antibodies more than 30 years ago,1 there has been a revolution in the development of antibody-based therapies. At present, in the field of cancer alone, 22 monoclonal antibodies are approved for clinical use in the United States.2 In this issue of Molecular Therapy, Yang and colleagues3 describe the development of a bispecific reagent that binds both an HIV-1-specific T-cell Center for Gene Therapy, Hematologic Malignancies and Stem Cell Transplantation Institute, City of Hope, Duarte, California, USA 1
Correspondence: Center for Gene Therapy, Hematologic Malignancies and Stem Cell Transplantation Institute, 1500 East Duarte Road, City of Hope, Duarte, California 91010, USA. E-mail: [email protected]
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receptor and an effector T cell, raising the possibility for a new approach to control and perhaps cure HIV/AIDS. Bispecific antibody (BiAb) binding is not a new concept. Antigen-specific IgG itself has bispecific capability, mediating binding to effector cells or to complement for killing of antigen-expressing cells. The goal in utilizing BiAbs is the redirection of immune cells to an antigen target, and BiAbs were first made by chemical crosslinker or hybrid hybridoma methods more than three decades ago.4 The extensive history of BiAb research for T-cell redirection has been reviewed.5 New approaches to Tcell redirection include the so-called BiTE reagents and CAR T cells using a singlechain variable fragment (scFv) of antibody to redirect the T cells. The BiTE therapeutics “engage” the T cell using a BiAb that links CD3+ T cells to a specific antigen.6, 7 The first BiTE to be approved for clinical use in the United States is blinatumomab
10.
11.
12.
13.
14.
15.
BC, Adair, JE, Hamlin, DK et al. (2015). 211Astatineconjugated monoclonal CD45 antibody-based nonmyeloablative conditioning for stem cell gene therapy. Hum Gene Ther 26: 399–406. Dvorak, CC, Horn, BN, Puck, JM, Czechowicz, A, Shizuru, JA, Ko, RM et al. (2014). A trial of plerixafor adjunctive therapy in allogeneic hematopoietic cell transplantation with minimal conditioning for severe combined immunodeficiency. Pediatr Transplant 18: 602–608. Peranteau, WH, Endo, M, Adibe, OO, Merchant, A, Zoltick, PW and Flake, AW (2006). CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation. Blood 108: 4268–4274. Goessling, W, Allen, RS, Guan, X, Jin, P, Uchida, N, Dovey, M et al. (2011). Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models. Cell Stem Cell 8: 445–458. Kuo, CY and Kohn, DB (2016). Gene therapy for the treatment of primary immune deficiencies. Curr Allergy Asthma Rep 16: 39. De Ravin, SS, Wu, X, Moir, S, Anaya-O’Brien, S, Kwatemaa, N, Littel, P et al. (2016). Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8: 335ra57. Bernardo, ME and Aiuti, A (2016). The role of conditioning in hematopoietic stem cell gene therapy. Hum Gene Ther; e-pub ahead of print 16 August 2016.
(Blincyto, Amgen). CAR T cells are genetically modified ex vivo to express a “chimeric antigen receptor” and are very promising cancer therapies that are still in development.8 An advantage of the BiTE and CAR strategies is that they bypass the human leukocyte antigen (HLA) restriction for antigen recognition. Thus, neither the HLA-specific epitope presentation nor the recognition by a native T-cell receptor (TCR) is required. A next generation of bispecific reagent has linked epitope-specific TCRs to an scFv specific for a cytotoxic cell to effect what has been termed “immune-mobilizing monoclonal TCRs,” called “ImmTACs” when against cancer9 and “ImmTAVs” for targeting virus antigens.3 ImmTACs have already entered clinical trials in melanoma patients (ClinicalTrials.gov identifier NCT02570308). The first in vitro use of an anti-HIV-1 ImmTAV is described in the report by Yang et al. as a TCR specific for HIV-1 p17 cloned and genetically linked to an scFv that binds to CD3+CD8+ T cells (CD8). The reagent has remarkable activity at picomolar concentrations, as has also been shown for ImmTACs.9 Yang et al. chose HIV-1 p17 as the target antigen, and these p17-epitope specific ImmTAVs were able to arm CD8 cells for killing of HIV-1-infected CD3+CD4+ T cells. When peripheral blood mononuclear cells from antiretroviral (ARV)-treated HIV-1-in-
www.moleculartherapy.org vol. 24 no. 11 november 2016
© The American Society of Gene & Cell Therapy
fected donors were activated in vitro to induce HIV-1 replication, the autologous ImmTAV-treated CD8 T cells reduced the number of HIV-1-infected cells by 60– 80% compared to the control ImmTAVtreated autologous CD8+ cells. Among the CD8+ T-cell subsets, the effector and effector memory T cells were more potent in reducing HIV-1 infection than were the central memory and naive CD8+ T cells. To study the in vitro effect of ImmTAVs on reactivated HIV-1, CD4+ T cells from HIV-1-infected donors were stimulated with a mitogen, a situation in which HIV-1 antigen could be seen in both the activated CD4 cells and in CD25–/CD69–/ HLA-DR–/CD4+ “resting” T cells. With CD8-ImmTAV treatment in vitro, both the p17-expressing activated and resting CD4 cells were killed, suggesting that the ImmTAV could be effective in a mixed-cell population as might occur when HIV-1 is being activated from latency in patients. This is important, because any future testing of the reagent in ARV-treated AIDS patients would target the latent virus reservoir, a situation of minimal HIV-1 antigen expression.10 Furthermore, by targeting an internal HIV-1 gene product, the approach could be effective without regard to surface expression of the HIV-1 envelope proteins, normally targeted by other immunotherapy strategies. The question, of course, is whether the ImmTAVs could act on latently infected cells from patients. To test this, CD4+ T cells from five HIV-1-infected donors receiving ARVs were cultured in vitro with the latency reactivation agents (LRAs) romidepsin and bryostatin. ImmTAVtreated CD8+ T cells from a healthy donor prevented HIV-1 reactivation in four of five challenges, but the CD8+ T cells treated with control ImmTAVs had no effect. The results are exciting and indicate that HIV-1 reactivation can be controlled by ImmTAVs, but it is a leap of faith to conclude that the ImmTAVs had an effect on the HIV-1 reservoir. It remains to be seen whether the antiviral function in this artificial system will occur when treating patients with chronic infection. Nevertheless, this supports a clinical strategy for testing of ImmTAVs based on use of LRAs. How might ImmTAVs have a role in the cure for HIV/AIDS? The ultimate goal of cure research is the control of active infection while eradicating the reservoir of latent infection. A strategy for achiev-
Molecular Therapy vol. 24 no. 11 november 2016
commentary ing this goal has been proposed.11 In addition to better understanding the various elements of AIDS pathogenesis, the promise of a cure for AIDS will require restoration of the damaged immune system to have effective T cell–specific anti-HIV-1 function. Ironically, as CD4 T-cell counts improve after control of HIV-1 by ARVs, effective HIV-1-specific immunity actually diminishes for reasons related to lack of HIV-1 antigen stimulation, global immune activation, and T-cell exhaustion.12, 13 The attack on the HIV-1 reservoir will require some method for HIV-1 activation, perhaps with LRAs,14 and then destruction of the virally infected cell by the now-refurbished T-cell immunity. Several factors should be considered when trying to predict whether the ImmTAV approach will bolster the immune system of an HIV-1-infected person and eradicate infection. First, in natural HIV-1 infection, the virus is able to escape T-cell immunity by several means, but especially by direct mutation of a targeted epitope, by HIV-1 reduction of HLA expression necessary for antigen presentation, and by immune exhaustion.15 In the ARV-treated patient, in whom HIV-1 replication is minimal, new mutations are unlikely. The repository of HIV-1 variation for certain epitopes of interest can be examined in the proviral DNA of the patient, and so the ImmTAV could be chosen based on one or more epitopes known to be present in the HIV-1 reservoir. Ultimately, a combination therapy with multiple ImmTAVs could be the best way to avoid the mutation-based problem of immune escape. It will be important to better understand the proteins expressed after LRA treatment. But, because of its importance in HIV-1 replication and pathogenesis, HIV-1 p17 is probably a well-chosen target.16 Yang et al. should be congratulated for being able to target an internal antigen likely to be detected in newly activated HIV-1-infected cells. The investigators have shown not only that ImmTAVs can be effective when used in vitro with LRAs, but also that ARV-induced mitigation of recent infection allows for some level of cytotoxicity of infected cells in vitro. One cannot be sure based on this small experiment that the ImmTAV strategy will be able to search and destroy the entire HIV-1 reservoir, but at least it is active at low HIV1 antigen expression. The strategy can be faulted because of its requirement for HLA class I specificity.
Any cell that lacks HLA class I antigens should be immune to ImmTAVs. It might be necessary in a cure strategy to use not only LRAs but also agents that activate HLA expression. More importantly, the treatment must be “HLA compatible” for each patient, and, at a practical level, this suggests that easy scalability of the method will be a challenge, not only during clinical trials but also if/when the method is ready for application to a genetically diverse population of AIDS patients. Second, the treatment must be safe, and this will be of foremost importance knowing that redirected T-cell therapies have profound biological effects that are not for the faint of heart. Cytokine release syndrome (CRS) is a potentially lethal event associated with T-cell activation. Blinatumomab, a CD19:CD3 BiTE, and CD19:CD3 CARs have formidable antileukemia cytotoxic potencies, but both induce the release of cytokines with profound effects.17, 18 CRS can result in fever, headache, fatigue, nausea, hypotension, liver and neurologic abnormalities, and other organ-specific adverse events. With blinatumomab, encephalopathy occurs in as many as 60% of treated patients (cf. FDA blackbox warning). Considering that HIV-1 is an infection not only of the immune system but also of brain, this suggests that a T-cell redirection with ImmTAVs seeking areas of HIV-1 infection could have significant central nervous system toxicity. Given the experience with BiTEs and CARs, it will remain to be seen whether this new strategy for T-cell redirection will be tolerated in the setting of HIV/AIDS. In summary, a therapeutic method that would both activate HIV-1 from latency and destroy the cellular site of such reactivation is the promise of HIV/AIDS cure strategies.15 ImmTAVs are likely to enter the fray in the search for an HIV/ AIDS cure. Only clinical trials of this new reagent will determine whether they can deliver on such a promise. REFERENCES 1.
2.
3.
Cabilly, S, Riggs, AD, Pande, H, Shively, JE, Holmes, WE, Rey, M et al. (1984). Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli. Proc Natl Acad Sci USA 81: 3273–3277. Carvalho, S, Levi-Schaffer, F, Sela, M and Yarden, Y (2016). Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18. Br J Pharmacol 173: 1407–1424. Yang, H, Buisson, S, Bossi, G, Wallace, Z, Hancock, G, So, C et al. (2016). Elimination of latently HIV-infected cells from antiretroviral therapy–suppressed subjects by
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commentary
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5. 6.
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8.
engineered immune-mobilizing T-cell receptors. Mol Ther 24: 1913–1925. Staerz, UD and Bevan, MJ (1986). Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc Natl Acad Sci USA 83: 1453–1457. Lum, LG and Thakur, A (2011). Targeting T cells with bispecific antibodies for cancer therapy. BioDrugs 25: 365–379. Mack, M, Riethmuller, G and Kufer, P (1995). A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc Natl Acad Sci USA 92: 7021–7025. Loffler, A, Kufer, P, Lutterbuse, R, Zettl, F, Daniel, PT, Schwenkenbecher, JM et al. (2000). A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 95: 2098–2103. Gill, S and June, CH (2015). Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol Rev 263: 68–89.
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Liddy, N, Bossi, G, Adams, KJ, Lissina, A, Mahon, TM, Hassan, NJ et al. (2012). Monoclonal TCR-redirected tumor cell killing. Nat Med 18: 980–987. Bruner, KM, Murray, AJ, Pollack, RA, Soliman, MG, Laskey, SB, Capoferri, AA et al. (2016). Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 22: 1043–1049. Deeks, SG, Lewin, SR, Ross, AL, Ananworanich, J, Benkirane, M, Cannon, P et al. (2016). International AIDS Society global scientific strategy: towards an HIV cure 2016. Nat Med 22: 839–850. Day, CL, Kaufmann, DE, Kiepiela, P, Brown, JA, Moodley, ES, Reddy, S et al. (2006). PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443: 350–354. Lederman, MM, Calabrese, L, Funderburg, NT, Clagett, B, Medvik, K, Bonilla, H et al. (2011). Immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory CD4 cells. J Infect Dis 204: 1217–1226.
See page 2021
The Toughest Nut to Crack: Will We Ever Have a Preventive and Effective HIV-1 Vaccine? Zwi N Berneman1 doi:10.1038/mt.2016.195
I
n this issue of Molecular Therapy, Negri et al. report that a simian immunodeficiency virus (SIV)-based lentiviral vector encoding the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) under the control of an internal cytomegalovirus promoter elicited antibody and T-cell responses in rhesus macaques.1 The antibodies elicited can block the binding of HIV-1 Env to its CD4 target molecule and show antibody-dependent cell-mediated cytotoxicity (ADCC) in all animals tested after vaccine boosting. The lentiviral vector used was integrasedefective, an important safety measure that should minimize integration in the host genome. The expression of the transgene was mediated by episomal plasmidlike forms of the recombinant vector. The main importance of this article is that the authors have devised a vaccination tool that leads to the production of antibodies that lasts longer than that achieved in other reported SIV/HIV vaccination studies. Antwerp University Hospital and University of Antwerp, Antwerp, Belgium 1
Correspondence: Zwi N Berneman, Antwerp University Hospital, Edegem, Belgium. E-mail: [email protected]
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These responses last for at least one year and can be significantly augmented by a booster vaccination. Moreover, these anti-Env antibodies are of particular interest because some of their properties have been associated with protection against HIV-1, i.e., (i) they have ADCC activity; (ii) they are directed against the variable region 2 (V2) of Env; (iii) they are present in mucosal secretions; and (iv) they bind Env from different HIV-1 clades and block the CD4-binding site of Env. To put the findings of Negri et al. into perspective, one needs to consider the long, arduous, and largely frustrating history of HIV-1 vaccine development. The AIDS epidemic is still progressing through the world, but a much sought-after preventive HIV-1 vaccine remains elusive. Recently Bob Gallo summarized the problems in developing an effective HIV vaccine and recalled Albert Sabin opining some 30 years ago that an HIV vaccine would not be possible.2 The only HIV-1 vaccine trial showing any efficacy in humans, the ALVAC-prime AIDSVAX-boost RV144 trial, only reduced the infection rate by 31.2%.3 The protection afforded in this
14. Margolis, DM, Garcia, JV, Hazuda, DJ and Haynes, BF (2016). Latency reversal and viral clearance to cure HIV1. Science 353: aaf6517. 15. Lederman, MM, Cannon, PM, Currier, JS, June, CH, Kiem, HP, Kuritzkes, DR et al. (2016). A cure for HIV infection: “not in my lifetime” or “just around the corner”? Pathog Immun 1: 154–164. 16. Fiorentini, S, Marini, E, Caracciolo, S and Caruso, A (2006). Functions of the HIV-1 matrix protein p17. New Microbiol 29: 1–10. 17. Grupp, SA, Kalos, M, Barrett, D, Aplenc, R, Porter, DL, Rheingold, SR et al. (2013). Chimeric antigen receptor– modified T cells for acute lymphoid leukemia. N Engl J Med 368: 1509–1518. 18. Klinger, M, Brandl, C, Zugmaier, G, Hijazi, Y, Bargou, RC, Topp, MS et al. (2012). Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell–engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood 119: 6226–6233.
trial was correlated with the induction of antibodies directed against HIV-1 Env, which were capable of mediating ADCC.4, 5 However, the action of these antibodies was short-lived, and protection waned rapidly. This seems to be a general feature of vaccination efforts against HIV-1 and SIV.2 Another problem with SIV and HIV vaccines is the stimulation of CD4+ Tlymphocyte help for antibody generation, which also expands the pool of activated cells that are highly susceptible to HIV or SIV infection.6 This problem of “excessive” CD4+ T-cell stimulation was most probably the main reason that, in two HIV-1 vaccination studies employing recombinant adenovirus vectors, the infection rate actually increased in vaccinated individuals.7 Negri et al. most probably succeeded in increasing the duration of the antibody response, because they used a nonintegrating lentiviral construct delivered by intramuscular injection. This strategy allowed for a longer transgene expression (in the absence of cell division),8 stimulating antibody production. In a study comparing immunization of rhesus macaques with env DNA and/or Env protein, the durability of humoral antibody response was mainly influenced positively by the DNA administered by electroporation.9 The road is still long from the present publication to an effective, fully protective HIV-1 vaccine, which may remain elusive in the end. First, the authors will have to demonstrate some protection against viral challenge in monkeys. Reallife vaccination will probably need to include at least two boosters administered within a timespan shorter than one year because only after one booster did all six monkeys studied develop the critical an-
www.moleculartherapy.org vol. 24 no. 11 november 2016
commentary would still be necessary, but with agents such as fludarabine replacing alkylators, acute and late effects would be minimized. Even for patients undergoing allogeneic transplantation for malignancies with these nonchemotherapeutic approaches, one could imagine being able to focus solely on rejection and more targeted anticancer therapy while minimizing the toxic effects of the standard conditioning regimens. Finally, for malignancies that express CD45 and/ or anti-c-Kit, it might be possible that these novel approaches could replace alkylating chemotherapy and radiation altogether. REFERENCES
1. Chhabra, A, Ring, AM, Weiskopf, K, Schnorr, PJ, Gordon, S, Le AC et al. (2016). Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Sci Transl Med 8: 351ra105. 2. Palchaudhuri, R, Saez, B, Hoggatt, J, Schajnovitz, A, Sykes, DB, Tate, TA et al. (2016). Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing
immunotoxin. Nat Biotechnol 34: 738–745. 3. Dvorak, CC, Hassan, A, Slatter, MA, Hönig, M, Lankester, AC, Buckley, RH et al. (2014). Comparison of outcomes of hematopoietic stem cell transplantation without chemotherapy conditioning by using matched sibling and unrelated donors for treatment of severe combined immunodeficiency. J Allergy Clin Immunol 134: 935–943. 4. Pai, SY, Logan, BR, Griffith, LM, Buckley, RH, Parrott, RE, Dvorak, CC et al. (2014). Transplantation outcomes for severe combined immunodeficiency, 2000–2009. N Engl J Med 371: 434–446. 5. Hoggatt, J, Kfoury, Y and Scadden, DT (2016). Hematopoietic stem cell niche in health and disease. Annu Rev Pathol 11: 555–581. 6. Czechowicz, A, Kraft, D, Weissman, IL and Bhattacharya, D (2007). Efficient transplantation via antibodybased clearance of hematopoietic stem cell niches. Science 318: 1296–1299. 7. Xiao, TZ, Singh, K, Dunn, E, Ramachandran, R and Cowan, MJ (2012). T cell and B cell immunity can be reconstituted with mismatched hematopoietic stem cell transplantation without alkylator therapy in Artemis-deficient mice using anti-natural killer cell antibody and photochemically treated sensitized donor T cells. Biol Blood Marrow Transplant 18: 200–209. 8. Straathof, KC, Rao, K, Eyrich, M, Hale, G, Bird, P, Berrie, E et al. (2009). Haemopoietic stem-cell transplantation with antibody-based minimal-intensity conditioning: a phase 1/2 study. Lancet 374: 912–920. 9. Burtner, CR, Chandrasekaran, D, Santos, EB, Beard,
See page 1913
A New Agent in the Strategy to Cure AIDS John A Zaia1 doi:10.1038/mt.2016.194
I
n recent years the field of immunotherapy has expanded to include therapies never before imagined. Since recombinant DNA (rDNA) technology was first applied to the production of active antigen-specific antibodies more than 30 years ago,1 there has been a revolution in the development of antibody-based therapies. At present, in the field of cancer alone, 22 monoclonal antibodies are approved for clinical use in the United States.2 In this issue of Molecular Therapy, Yang and colleagues3 describe the development of a bispecific reagent that binds both an HIV-1-specific T-cell Center for Gene Therapy, Hematologic Malignancies and Stem Cell Transplantation Institute, City of Hope, Duarte, California, USA 1
Correspondence: Center for Gene Therapy, Hematologic Malignancies and Stem Cell Transplantation Institute, 1500 East Duarte Road, City of Hope, Duarte, California 91010, USA. E-mail: [email protected]
1894
receptor and an effector T cell, raising the possibility for a new approach to control and perhaps cure HIV/AIDS. Bispecific antibody (BiAb) binding is not a new concept. Antigen-specific IgG itself has bispecific capability, mediating binding to effector cells or to complement for killing of antigen-expressing cells. The goal in utilizing BiAbs is the redirection of immune cells to an antigen target, and BiAbs were first made by chemical crosslinker or hybrid hybridoma methods more than three decades ago.4 The extensive history of BiAb research for T-cell redirection has been reviewed.5 New approaches to Tcell redirection include the so-called BiTE reagents and CAR T cells using a singlechain variable fragment (scFv) of antibody to redirect the T cells. The BiTE therapeutics “engage” the T cell using a BiAb that links CD3+ T cells to a specific antigen.6, 7 The first BiTE to be approved for clinical use in the United States is blinatumomab
10.
11.
12.
13.
14.
15.
BC, Adair, JE, Hamlin, DK et al. (2015). 211Astatineconjugated monoclonal CD45 antibody-based nonmyeloablative conditioning for stem cell gene therapy. Hum Gene Ther 26: 399–406. Dvorak, CC, Horn, BN, Puck, JM, Czechowicz, A, Shizuru, JA, Ko, RM et al. (2014). A trial of plerixafor adjunctive therapy in allogeneic hematopoietic cell transplantation with minimal conditioning for severe combined immunodeficiency. Pediatr Transplant 18: 602–608. Peranteau, WH, Endo, M, Adibe, OO, Merchant, A, Zoltick, PW and Flake, AW (2006). CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation. Blood 108: 4268–4274. Goessling, W, Allen, RS, Guan, X, Jin, P, Uchida, N, Dovey, M et al. (2011). Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models. Cell Stem Cell 8: 445–458. Kuo, CY and Kohn, DB (2016). Gene therapy for the treatment of primary immune deficiencies. Curr Allergy Asthma Rep 16: 39. De Ravin, SS, Wu, X, Moir, S, Anaya-O’Brien, S, Kwatemaa, N, Littel, P et al. (2016). Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8: 335ra57. Bernardo, ME and Aiuti, A (2016). The role of conditioning in hematopoietic stem cell gene therapy. Hum Gene Ther; e-pub ahead of print 16 August 2016.
(Blincyto, Amgen). CAR T cells are genetically modified ex vivo to express a “chimeric antigen receptor” and are very promising cancer therapies that are still in development.8 An advantage of the BiTE and CAR strategies is that they bypass the human leukocyte antigen (HLA) restriction for antigen recognition. Thus, neither the HLA-specific epitope presentation nor the recognition by a native T-cell receptor (TCR) is required. A next generation of bispecific reagent has linked epitope-specific TCRs to an scFv specific for a cytotoxic cell to effect what has been termed “immune-mobilizing monoclonal TCRs,” called “ImmTACs” when against cancer9 and “ImmTAVs” for targeting virus antigens.3 ImmTACs have already entered clinical trials in melanoma patients (ClinicalTrials.gov identifier NCT02570308). The first in vitro use of an anti-HIV-1 ImmTAV is described in the report by Yang et al. as a TCR specific for HIV-1 p17 cloned and genetically linked to an scFv that binds to CD3+CD8+ T cells (CD8). The reagent has remarkable activity at picomolar concentrations, as has also been shown for ImmTACs.9 Yang et al. chose HIV-1 p17 as the target antigen, and these p17-epitope specific ImmTAVs were able to arm CD8 cells for killing of HIV-1-infected CD3+CD4+ T cells. When peripheral blood mononuclear cells from antiretroviral (ARV)-treated HIV-1-in-
www.moleculartherapy.org vol. 24 no. 11 november 2016
© The American Society of Gene & Cell Therapy
fected donors were activated in vitro to induce HIV-1 replication, the autologous ImmTAV-treated CD8 T cells reduced the number of HIV-1-infected cells by 60– 80% compared to the control ImmTAVtreated autologous CD8+ cells. Among the CD8+ T-cell subsets, the effector and effector memory T cells were more potent in reducing HIV-1 infection than were the central memory and naive CD8+ T cells. To study the in vitro effect of ImmTAVs on reactivated HIV-1, CD4+ T cells from HIV-1-infected donors were stimulated with a mitogen, a situation in which HIV-1 antigen could be seen in both the activated CD4 cells and in CD25–/CD69–/ HLA-DR–/CD4+ “resting” T cells. With CD8-ImmTAV treatment in vitro, both the p17-expressing activated and resting CD4 cells were killed, suggesting that the ImmTAV could be effective in a mixed-cell population as might occur when HIV-1 is being activated from latency in patients. This is important, because any future testing of the reagent in ARV-treated AIDS patients would target the latent virus reservoir, a situation of minimal HIV-1 antigen expression.10 Furthermore, by targeting an internal HIV-1 gene product, the approach could be effective without regard to surface expression of the HIV-1 envelope proteins, normally targeted by other immunotherapy strategies. The question, of course, is whether the ImmTAVs could act on latently infected cells from patients. To test this, CD4+ T cells from five HIV-1-infected donors receiving ARVs were cultured in vitro with the latency reactivation agents (LRAs) romidepsin and bryostatin. ImmTAVtreated CD8+ T cells from a healthy donor prevented HIV-1 reactivation in four of five challenges, but the CD8+ T cells treated with control ImmTAVs had no effect. The results are exciting and indicate that HIV-1 reactivation can be controlled by ImmTAVs, but it is a leap of faith to conclude that the ImmTAVs had an effect on the HIV-1 reservoir. It remains to be seen whether the antiviral function in this artificial system will occur when treating patients with chronic infection. Nevertheless, this supports a clinical strategy for testing of ImmTAVs based on use of LRAs. How might ImmTAVs have a role in the cure for HIV/AIDS? The ultimate goal of cure research is the control of active infection while eradicating the reservoir of latent infection. A strategy for achiev-
Molecular Therapy vol. 24 no. 11 november 2016
commentary ing this goal has been proposed.11 In addition to better understanding the various elements of AIDS pathogenesis, the promise of a cure for AIDS will require restoration of the damaged immune system to have effective T cell–specific anti-HIV-1 function. Ironically, as CD4 T-cell counts improve after control of HIV-1 by ARVs, effective HIV-1-specific immunity actually diminishes for reasons related to lack of HIV-1 antigen stimulation, global immune activation, and T-cell exhaustion.12, 13 The attack on the HIV-1 reservoir will require some method for HIV-1 activation, perhaps with LRAs,14 and then destruction of the virally infected cell by the now-refurbished T-cell immunity. Several factors should be considered when trying to predict whether the ImmTAV approach will bolster the immune system of an HIV-1-infected person and eradicate infection. First, in natural HIV-1 infection, the virus is able to escape T-cell immunity by several means, but especially by direct mutation of a targeted epitope, by HIV-1 reduction of HLA expression necessary for antigen presentation, and by immune exhaustion.15 In the ARV-treated patient, in whom HIV-1 replication is minimal, new mutations are unlikely. The repository of HIV-1 variation for certain epitopes of interest can be examined in the proviral DNA of the patient, and so the ImmTAV could be chosen based on one or more epitopes known to be present in the HIV-1 reservoir. Ultimately, a combination therapy with multiple ImmTAVs could be the best way to avoid the mutation-based problem of immune escape. It will be important to better understand the proteins expressed after LRA treatment. But, because of its importance in HIV-1 replication and pathogenesis, HIV-1 p17 is probably a well-chosen target.16 Yang et al. should be congratulated for being able to target an internal antigen likely to be detected in newly activated HIV-1-infected cells. The investigators have shown not only that ImmTAVs can be effective when used in vitro with LRAs, but also that ARV-induced mitigation of recent infection allows for some level of cytotoxicity of infected cells in vitro. One cannot be sure based on this small experiment that the ImmTAV strategy will be able to search and destroy the entire HIV-1 reservoir, but at least it is active at low HIV1 antigen expression. The strategy can be faulted because of its requirement for HLA class I specificity.
Any cell that lacks HLA class I antigens should be immune to ImmTAVs. It might be necessary in a cure strategy to use not only LRAs but also agents that activate HLA expression. More importantly, the treatment must be “HLA compatible” for each patient, and, at a practical level, this suggests that easy scalability of the method will be a challenge, not only during clinical trials but also if/when the method is ready for application to a genetically diverse population of AIDS patients. Second, the treatment must be safe, and this will be of foremost importance knowing that redirected T-cell therapies have profound biological effects that are not for the faint of heart. Cytokine release syndrome (CRS) is a potentially lethal event associated with T-cell activation. Blinatumomab, a CD19:CD3 BiTE, and CD19:CD3 CARs have formidable antileukemia cytotoxic potencies, but both induce the release of cytokines with profound effects.17, 18 CRS can result in fever, headache, fatigue, nausea, hypotension, liver and neurologic abnormalities, and other organ-specific adverse events. With blinatumomab, encephalopathy occurs in as many as 60% of treated patients (cf. FDA blackbox warning). Considering that HIV-1 is an infection not only of the immune system but also of brain, this suggests that a T-cell redirection with ImmTAVs seeking areas of HIV-1 infection could have significant central nervous system toxicity. Given the experience with BiTEs and CARs, it will remain to be seen whether this new strategy for T-cell redirection will be tolerated in the setting of HIV/AIDS. In summary, a therapeutic method that would both activate HIV-1 from latency and destroy the cellular site of such reactivation is the promise of HIV/AIDS cure strategies.15 ImmTAVs are likely to enter the fray in the search for an HIV/ AIDS cure. Only clinical trials of this new reagent will determine whether they can deliver on such a promise. REFERENCES 1.
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Cabilly, S, Riggs, AD, Pande, H, Shively, JE, Holmes, WE, Rey, M et al. (1984). Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli. Proc Natl Acad Sci USA 81: 3273–3277. Carvalho, S, Levi-Schaffer, F, Sela, M and Yarden, Y (2016). Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18. Br J Pharmacol 173: 1407–1424. Yang, H, Buisson, S, Bossi, G, Wallace, Z, Hancock, G, So, C et al. (2016). Elimination of latently HIV-infected cells from antiretroviral therapy–suppressed subjects by
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engineered immune-mobilizing T-cell receptors. Mol Ther 24: 1913–1925. Staerz, UD and Bevan, MJ (1986). Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc Natl Acad Sci USA 83: 1453–1457. Lum, LG and Thakur, A (2011). Targeting T cells with bispecific antibodies for cancer therapy. BioDrugs 25: 365–379. Mack, M, Riethmuller, G and Kufer, P (1995). A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc Natl Acad Sci USA 92: 7021–7025. Loffler, A, Kufer, P, Lutterbuse, R, Zettl, F, Daniel, PT, Schwenkenbecher, JM et al. (2000). A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 95: 2098–2103. Gill, S and June, CH (2015). Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol Rev 263: 68–89.
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Liddy, N, Bossi, G, Adams, KJ, Lissina, A, Mahon, TM, Hassan, NJ et al. (2012). Monoclonal TCR-redirected tumor cell killing. Nat Med 18: 980–987. Bruner, KM, Murray, AJ, Pollack, RA, Soliman, MG, Laskey, SB, Capoferri, AA et al. (2016). Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 22: 1043–1049. Deeks, SG, Lewin, SR, Ross, AL, Ananworanich, J, Benkirane, M, Cannon, P et al. (2016). International AIDS Society global scientific strategy: towards an HIV cure 2016. Nat Med 22: 839–850. Day, CL, Kaufmann, DE, Kiepiela, P, Brown, JA, Moodley, ES, Reddy, S et al. (2006). PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443: 350–354. Lederman, MM, Calabrese, L, Funderburg, NT, Clagett, B, Medvik, K, Bonilla, H et al. (2011). Immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory CD4 cells. J Infect Dis 204: 1217–1226.
See page 2021
The Toughest Nut to Crack: Will We Ever Have a Preventive and Effective HIV-1 Vaccine? Zwi N Berneman1 doi:10.1038/mt.2016.195
I
n this issue of Molecular Therapy, Negri et al. report that a simian immunodeficiency virus (SIV)-based lentiviral vector encoding the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) under the control of an internal cytomegalovirus promoter elicited antibody and T-cell responses in rhesus macaques.1 The antibodies elicited can block the binding of HIV-1 Env to its CD4 target molecule and show antibody-dependent cell-mediated cytotoxicity (ADCC) in all animals tested after vaccine boosting. The lentiviral vector used was integrasedefective, an important safety measure that should minimize integration in the host genome. The expression of the transgene was mediated by episomal plasmidlike forms of the recombinant vector. The main importance of this article is that the authors have devised a vaccination tool that leads to the production of antibodies that lasts longer than that achieved in other reported SIV/HIV vaccination studies. Antwerp University Hospital and University of Antwerp, Antwerp, Belgium 1
Correspondence: Zwi N Berneman, Antwerp University Hospital, Edegem, Belgium. E-mail: [email protected]
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These responses last for at least one year and can be significantly augmented by a booster vaccination. Moreover, these anti-Env antibodies are of particular interest because some of their properties have been associated with protection against HIV-1, i.e., (i) they have ADCC activity; (ii) they are directed against the variable region 2 (V2) of Env; (iii) they are present in mucosal secretions; and (iv) they bind Env from different HIV-1 clades and block the CD4-binding site of Env. To put the findings of Negri et al. into perspective, one needs to consider the long, arduous, and largely frustrating history of HIV-1 vaccine development. The AIDS epidemic is still progressing through the world, but a much sought-after preventive HIV-1 vaccine remains elusive. Recently Bob Gallo summarized the problems in developing an effective HIV vaccine and recalled Albert Sabin opining some 30 years ago that an HIV vaccine would not be possible.2 The only HIV-1 vaccine trial showing any efficacy in humans, the ALVAC-prime AIDSVAX-boost RV144 trial, only reduced the infection rate by 31.2%.3 The protection afforded in this
14. Margolis, DM, Garcia, JV, Hazuda, DJ and Haynes, BF (2016). Latency reversal and viral clearance to cure HIV1. Science 353: aaf6517. 15. Lederman, MM, Cannon, PM, Currier, JS, June, CH, Kiem, HP, Kuritzkes, DR et al. (2016). A cure for HIV infection: “not in my lifetime” or “just around the corner”? Pathog Immun 1: 154–164. 16. Fiorentini, S, Marini, E, Caracciolo, S and Caruso, A (2006). Functions of the HIV-1 matrix protein p17. New Microbiol 29: 1–10. 17. Grupp, SA, Kalos, M, Barrett, D, Aplenc, R, Porter, DL, Rheingold, SR et al. (2013). Chimeric antigen receptor– modified T cells for acute lymphoid leukemia. N Engl J Med 368: 1509–1518. 18. Klinger, M, Brandl, C, Zugmaier, G, Hijazi, Y, Bargou, RC, Topp, MS et al. (2012). Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell–engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood 119: 6226–6233.
trial was correlated with the induction of antibodies directed against HIV-1 Env, which were capable of mediating ADCC.4, 5 However, the action of these antibodies was short-lived, and protection waned rapidly. This seems to be a general feature of vaccination efforts against HIV-1 and SIV.2 Another problem with SIV and HIV vaccines is the stimulation of CD4+ Tlymphocyte help for antibody generation, which also expands the pool of activated cells that are highly susceptible to HIV or SIV infection.6 This problem of “excessive” CD4+ T-cell stimulation was most probably the main reason that, in two HIV-1 vaccination studies employing recombinant adenovirus vectors, the infection rate actually increased in vaccinated individuals.7 Negri et al. most probably succeeded in increasing the duration of the antibody response, because they used a nonintegrating lentiviral construct delivered by intramuscular injection. This strategy allowed for a longer transgene expression (in the absence of cell division),8 stimulating antibody production. In a study comparing immunization of rhesus macaques with env DNA and/or Env protein, the durability of humoral antibody response was mainly influenced positively by the DNA administered by electroporation.9 The road is still long from the present publication to an effective, fully protective HIV-1 vaccine, which may remain elusive in the end. First, the authors will have to demonstrate some protection against viral challenge in monkeys. Reallife vaccination will probably need to include at least two boosters administered within a timespan shorter than one year because only after one booster did all six monkeys studied develop the critical an-
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