By Vince Giuliano
There is current excitement about a new approach to using a person’s own immune system to fight and defeat otherwise incurable cancers: Adoptive immunotherapy, a technology that is currently the subject of multiple clinical trials. Here, I cover selected current research in this area and summarize where we are.
Adoptive immunotherapy, in its simplest terms is “a form of immunotherapy used in the treatment of cancer in which an individual’s own white blood cells are coupled with a naturally produced growth factor to enhance their cancer-fighting capacity(ref).” There are multiple strategies for pursuing adoptive immunotherapy including a classical approach using IL-2 that can be effective but involves very high toxicity(ref) and a newer approach which is based on modifying chimeric antigen receptors on T cells so that they recognize CD-19 markers on tumor cells.
Background on stem cell therapies
The use of stem cell therapies to treat leukemias and selected congenital disorders is far from new. The November 2011 publication Pediatric Hematopoietic Stem Cell Transplantation traces the history back 50 years. “Hematopoietic stem cell transplantation was first performed more than 50 years ago. The earliest work in the field was performed using animal models in the mid 1950s. During the 1960s, the first few successful hematopoietic stem cell transplants used in the treatment of congenital immunodeficiency disorders and end-stage leukemia were reported. The success of these early attempts was compromised by high morbidity and mortality, in large part due to toxicity related to the chemotherapy (called conditioning) administered prior to bone marrow transplantation , post-transplant infectious complications, and graft versus host disease .” The newer approach described below being autologous (using patient’s own stem cells) avoids the immune response of graft versus host disease. And risk of infectious complications is also less.
CARS – Chimeric antigen receptors
Enhancing T cells with chimeric antigen receptors (CARs) has been of particular interest with respect to developing therapies for otherwise-refractory leukemias. “Using gene transfer technologies, T cells can be genetically modified to stably express antibody binding domains on their surface that confer novel antigen specificities that are major histocompatibility complex (MHC)–independent. Chimeric antigen receptors (CARs) are an application of this approach that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-ζ chain or FcγRI protein into a single chimeric protein(ref).” See ref and ref for additional background.
The potential advantages of a CAR-based therapy are outlined in the 2010 publication Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells. “Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.”
The 2010 publication Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptordescribes the general technology. “Infusions of antigen-specific T cells have yielded therapeutic responses in patients with pathogens and tumors. To broaden the clinical application of adoptive immunotherapy against malignancies, investigators have developed robust systems for the genetic modification and characterization of T cells expressing introduced chimeric antigen receptors (CARs) to redirect specificity. Human trials are under way in patients with aggressive malignancies to test the hypothesis that manipulating the recipient and reprogramming T cells before adoptive transfer may improve their therapeutic effect. — These examples of personalized medicine infuse T cells designed to meet patients’ needs by redirecting their specificity to target molecular determinants on the underlying malignancy. The generation of clinical grade CAR+ T cells is an example of bench-to-bedside translational science that has been accomplished using investigator-initiated trials operating largely without industry support. The next-generation trials will deliver designer T cells with improved homing, CAR-mediated signaling, and replicative potential, as investigators move from the bedside to the bench and back again.”
The August 2011 publication T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemiareports: “Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non–cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR+ T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex–independent approach for the effective treatment of B cell malignancies.” – “In most cancers, tumor-specific antigens are not yet well defined, but in B cell malignancies, CD19 is an attractive tumor target. Expression of CD19 is restricted to normal and malignant B cells (5), and CD19 is a widely accepted target to safely test CARs. Although CARs can trigger T cell activation in a manner similar to an endogenous T cell receptor, a major impediment to the clinical application of this technology to date has been the limited in vivo expansion of CAR+ T cells, rapid disappearance of the cells after infusion, and disappointing clinical activity (4, 6).”
Another August 2011 publication on the same research Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia reports “We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in T cells [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×105 cells per kilogram of body weight) of autologous chimeric antigen receptor–modified T cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at high levels for 6 months in the blood and bone marrow and continued to express the chimeric antigen receptor. A specific immune response was detected in the bone marrow, accompanied by loss of normal B cells and leukemia cells that express CD19. Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was an expected chronic toxic effect.”
An important issue in T cell cancer therapies is getting the T Cells to find their ways to the cancer, and this has been a subject of investigation. The 2011 publicationExpression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T cells Expressing a Mesothelin-Specific Chimeric Antibody Receptor reports “Purpose: Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects. — Experimental Design: The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors. — Results: CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity. — Conclusions: CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo.”
CARs and clinical trials
Among the dozen open clinical trials using CARs for leukemias is one related to using CD19 CAR and another to using a CD-30 CAR for non-Hodgkin’s Lymphoma (NHL) and Chronic Lymphocytic leukemia (B-CLL)(ref)(ref).
The August 2011 publication Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemiasreports a Phase I clinical trial result “We report the findings from the first 10 patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) or relapsed B-cell acute lymphoblastic leukemia (ALL) we have enrolled for treatment with autologous T cells modified to express 19-28z, a second-generation chimeric antigen (Ag) receptor specific to the B-cell lineage Ag CD19. Eight of the 9 treated patients tolerated 19-28z+ T-cell infusions well. Three of 4 evaluable patients with bulky CLL who received prior conditioning with cyclophosphamide exhibited either a significant reduction or a mixed response in lymphadenopathy without concomitant development of B-cell aplasia. In contrast, one patient with relapsed ALL who was treated in remission with a similar T-cell dose developed a predicted B-cell aplasia. The short-term persistence of infused T cells was enhanced by prior cyclophosphamide administration and inversely proportional to the peripheral blood tumor burden. Further analyses showed rapid trafficking of modified T cells to tumor and retained ex vivo cytotoxic potential of CD19-targeted T cells retrieved 8 days after infusion. We conclude that this adoptive T-cell approach is promising and more likely to show clinical benefit in the setting of prior conditioning chemotherapy and low tumor burden or minimal residual disease. These studies are registered at www.clinicaltrials.org as #NCT00466531 (CLL protocol) and #NCT01044069 (B-ALL protocol).”
Relevant to this research, a November 2011 publication Time to put the CAR-T before the horse reports “In this issue of Blood, Brentjens and colleagues report on the feasibility, tolerability, and persistence of autologous CD19-directed chimeric antigen receptor (CAR) T cells in patients with relapsed chronic lymphocytic leukemia (CLL) and B-cell acute lymphocytic leukemia (B-ALL).1 These diseases are generally considered incurable, unless treated with allogeneic stem cell transplantation, which is evidence of the efficacy of immune-mediated mechanisms in their treatment. Alternative immunotherapy approaches have been investigated, including antitumor vaccines and adoptive transfer of CD19 CAR-T cells reported in this paper. — The engineering of CAR-T cells is unique in that T cells are collected from a patient and genetically modified to express a receptor that will bind to a surface antigen expressed on the patient’s own tumor cells. After infusion, autologous CAR-T cells home to sites of disease and also persist over time (see figure). The earliest CARs consisted of an extracellular antigen recognition domain (typically a single chain Fv variable fragment from a monoclonal antibody) linked via a transmembrane domain to an intracellular signaling domain (usually the CD3ζ endodomain), resulting in the redirection of T-cell specificity toward target antigen-positive cells.2 While effective in lysing tumor cells in vitro, the clinical utility of these first-generation CAR-T cells was limited by their inability to sufficiently activate and sustain themselves in vivo. Second generation CAR-T cells, with the addition of costimulatory domains including CD28, 4-1BB, or OX40 to the intracellular portion, are engineered to enhance cytokine secretion and effector cell expansion, and prevent activation-induced apoptosis and immune suppression by tumor-related metabolites.3 — Here, Brentjens and colleagues report the use of their CD19-CD28z CAR construct in 10 patients with CLL (n = 8) or B-ALL (n = 2). CD19 is present on the malignant CLL and B-ALL cells, as well as healthy B cells, but not on hematopoietic stem cells, plasma cells, and other healthy tissue. CLL patients, with bulky disease that recurred after at least 1 prior chemotherapy regimen, were enrolled in a phase 1 trial. The first 3 patients were treated with 1.2 to 3.0 × 107 19-28z+ T cells/kg without lymphocyte-depleting conditioning; the second cohort of 5 patients was to receive the same dose of T cells after dose-escalating cyclophosphamide conditioning. However, after an unexpected death because of a sepsis-like syndrome immediately after T-cell infusion in the first patient in this cohort, all subsequent patients received 0.4 to 1.0 × 107 19-28z+ T cells/kg over 2 days. Of 2 ALL patients with relapsed disease, only 1, in a second complete remission, was treated with cyclophosphamide conditioning followed by infusion of 3 × 106 19-28z+ T cells/kg. All patients experienced transient fevers within 24 hours after T-cell infusion, which was otherwise well tolerated. There were no clinical responses in the first CLL cohort, but there was 1 patient with marked reduction in lymphadenopathy, and 2 patients with stable disease in the second cohort. The ALL patient, who was in a complete remission at the time of infusion, had persistent B-cell aplasia after T-cell infusion, and subsequently had a planned allogeneic stem cell transplantation. These results correlated with 19-28z+ T-cell persistence in bone marrow and blood: there were no CAR-T cells at 1 month after infusion in the first CLL cohort, compared with retention of CAR-T cells up to 6 weeks after infusion in the blood and marrow of patients treated in the second CLL and ALL cohorts. When these CAR-T cells were collected 8 days after infusion and cultured with antigen-expressing fibroblasts, they exhibited marked expansion and cytotoxic effects. Furthermore, these T cells were found to have infiltrated tumor beds in the 1 patient who died shortly after T-cell infusion.”
See this diagram.
A December 2011 review article on CAR leukemia therapies describing the current situation is Chimeric Antigen Receptors in Cancer Immuno-Gene Therapy: Current Status and Future Directions. “The concept of chimeric antigen receptors (CARs) as molecules able to redirect T lymphocytes toward tumor cells is currently being exploited in the field of cancer immunotherapy. Despite promising preliminary results, some clinical trials evidenced limitations for this technology that must be overcome for more extensive application of CARs in tumor immunotherapy. We describe here the fundaments of these molecules in terms of structure, function, possible targets and pre-clinical and clinical applications. We also discuss strategies that can potentially overcome the limitations seen so far, paving the road to a wider application of this exciting new technology.”
Other recent relevant publications
The research literature with respect to CARs is vast and I have been highly selective here of citations. Additional recent publications of relevance include;
Several additional CAR-related publications now available online carry 2012 publication dates
The 2012 publication Engineered T cells for the adoptive therapy of B-cell chronic lymphocytic leukaemia summarizes the hope of this line of research: “B-cell chronic lymphocytic leukaemia (B-CLL) remains an incurable disease due to the high risk of relapse, even after complete remission, raising the need to control and eliminate residual tumor cells in long term. Adoptive T cell therapy with genetically engineered specificity is thought to fulfil expectations, and clinical trials for the treatment of CLL are initiated. Cytolytic T cells from patients are redirected towards CLL cells by ex vivo engineering with a chimeric antigen receptor (CAR) which binds to CD19 on CLL cells through an antibody-derived domain and triggers T cell activation through CD3ζ upon tumor cell engagement. Redirected T cells thereby target CLL cells in an MHC-unrestricted fashion, secret proinflammatory cytokines, and eliminate CD19(+) leukaemia cells with high efficiency. Cytolysis of autologous CLL cells by patient’s engineered T cells is effective, however, accompanied by lasting elimination of healthy CD19(+) B-cells. In this paper we discuss the potential of the strategy in the treatment of CLL, the currently ongoing trials, and the future challenges in the adoptive therapy with CAR-engineered T cells.
The 2012 publication Engineered T Cells for the Adoptive Therapy of B-Cell Chronic Lymphocytic Leukaemia reports: “B-cell chronic lymphocytic leukaemia (B-CLL) remains an incurable disease due to the high risk of relapse, even after complete remission, raising the need to control and eliminate residual tumor cells in long term. Adoptive T cell therapy with genetically engineered specificity is thought to fulfil expectations, and clinical trials for the treatment of CLL are initiated. Cytolytic T cells from patients are redirected towards CLL cells by ex vivo engineering with a chimeric antigen receptor (CAR) which binds to CD19 on CLL cells through an antibody-derived domain and triggers T cell activation through CD3ζ upon tumor cell engagement. Redirected T cells thereby target CLL cells in an MHC-unrestricted fashion, secret proinflammatory cytokines, and eliminate CD19+ leukaemia cells with high efficiency. Cytolysis of autologous CLL cells by patient’s engineered T cells is effective, however, accompanied by lasting elimination of healthy CD19+ B-cells. In this paper we discuss the potential of the strategy in the treatment of CLL, the currently ongoing trials, and the future challenges in the adoptive therapy with CAR-engineered T cells.”
Immunotoxin and antibody drug conjugate cancer therapies
A closely related cancer therapy is also based on targeting cancer cells via surface antigens, but with antibodies delivering deadly payloads. The October 2011 publication Treatment of Hematologic Malignancies with Immunotoxins and Antibody-Drug Conjugates reports “To enable antibodies to function as cytotoxic anticancer agents, they are modified either via attachment to protein toxins or highly potent, low-molecular-weight drugs. Such molecules, termed immunotoxins and antibody-drug conjugates, respectively, represent a second revolution in antibody-mediated cancer therapy. Thus, highly toxic compounds are delivered to the interior of cancer cells based on antibody specificity for cell-surface target antigens.” I wrote about such a stem cell payload approach back in May 2009 in the blog entry Trojan-horse stem cells might offer an important new cancer therapy.
It remains to be seen whether CARs therapies will fulfill the high hopes expressed in many of these publications. The clinical trials will tell us a lot but time will tell a lot more. We also don’t know which of these antigen-based therapies will predominate in the long run – CARs, immunotoxins and antibody drug conjugates, or whether they all will have a place. At the moment it is not clear whether adoptive gene therapy will take its place as a complement to or replacement for chemotherapy. A closely related topic, also of considerable excitement, is the development of cancer vaccines. I intend to review that area in another blog post.
The exciting news is that at last we see a real possibility for cure of deadly leukemias.