Chimeric Antigen Receptor Design Research Articles

Chimeric Antigen Receptor T-cells- Gene Therapy of the Future for Malignant Diseases?

Chimeric antigen receptors are synthetic, genetically modified receptors of T-cells. The introduction of chimeric antigen receptors into autologous patient T-cells can redirect the lymphocytes to specific antigen targets on the surface of malignant cells. This has recently emerged as an intriguing therapy approach in both hematologic malignancies and later also in solid tumors. Various chimeric antigen receptor designs and manufacturing processes were developed and seem to have a strong impact on the activity of chimeric antigen receptor T-lymphocytes and thereby the therapy success. The individual variables are currently being tested in numerous clinical trials. In this review, I will briefly describe the principle, basic structure and construction of chimeric antigen receptor T-lymphocytes.

From T-cell Engineering to CAR therapy: Progress and Prospects

The genetic engineering of T cells provides a means to rapidly generate anti-tumor T cells for any cancer patient. This approach is predicated on gene transfer technology that enables the expression of receptors for antigen and other gene products in primary T cells. Tumor targeting may be achieved through the transfer of a physiological receptor for antigen, which is known as the T cell receptor (TCR), or synthetic fusion receptors, which we grouped under the general term of chimeric antigen receptor (CAR). CARs are recombinant receptors for antigen, which, in a single molecule, redirect T cell specificity and eventually enhance anti-tumor potency. Functional augmentation is achieved through the design of second generation CARs, which not only redirect cytotoxicity, but also reprogram T cell function and longevity through their costimulatory properties. The combined activating and costimulatory domains incorporated in second-generation CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19 CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date. Two decades ago, we selected CD19 as the prime target for developing our CAR technology and provided the first proof-of-principle that CD19-targeted human peripheral blood T cells could eradicate a broad range of B cell malignancies in immunodeficient mice (Brentjens RJ, Riviere I, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. 2003;9(3):279-86). CD19 has since become the poster child for CAR therapies. Complete remissions have been reported from several centers in patients with non-Hodgkin lymphoma, chronic lymphocytic leukemia and, most dramatically, acute lymphoblastic leukemia. Two types of second generation CARs, utilizing either CD28 or 4-1BB as their costimulatory signaling components, have been used in ALL patients. Both have yielded dramatic outcomes, in adults as well as in children. Our data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, while 4-1BB-based CARs direct a gradual T cell accumulation that may eventually overcome lesser functional potency. These distinct kinetic features can be exploited to further develop CAR T cell therapies for a variety of cancers. We have now modeled CD19 CAR therapy for ALL in a "stress test", wherein we purposefully lower the infused T cell doses to challenge the CAR therapy. We have compared novel CAR designs intended to recruit both CD28 and 4-1BB signaling. These quantitative analyses reveal striking disparities that hinge on subtle variations in the structural design of CARs and co-expressed costimulatory molecules. Remarkably, we find that some of the most effective engineering strategies activate and sustain the recruitment of the IFNβ pathway through the induction of IRF7, while lowering the induction of exhaustion markers relative to second generation CARs activating either CD28 or 4-1BB alone. The field is thus poised to move beyond the CD28 vs 4-1BB debate, which will be rendered obsolete by the emergence of superior CAR designs that coopt the use of costimulatory ligands, cytokines and/or checkpoint blockade inhibitors. A new field of immunopharmacology is emerging. Disclosures Sadelain: Juno Therapeutics: Consultancy, Equity Ownership, Other: Co-Founder, stockholder, Patents & Royalties: Licensed patents on CARs.

Efficacy and Safety of Humanized Chimeric Antigen Receptor (CAR)-Modified T Cells Targeting CD19 in Children with Relapsed/Refractory ALL

Background Targeted immunotherapy with T cells expressing a chimeric antigen receptor (CAR) can produce dramatic anti-tumor responses. We previously demonstrated complete remissions (CRs), prolonged persistence, and sustained responses in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CAR-modified T cells (CTL019). However, a subset (~25%) of patients has limited persistence of CTL019, which can increase the risk of relapse. We hypothesized that repeat infusion may prolong persistence in some patients but would be ineffective in patients with immune-mediated rejection. As most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin, anti-mouse reactivity is one potential cause of immunogenicity that may be overcome by fully human or humanized CAR design. We now report on retreatment with murine or humanized CD19-directed CAR T cells. Aims We sought to 1) prolong persistence by reinfusion of murine CTL019 in children with r/r CD19+ ALL and evidence of poor persistence and 2) determine the safety and efficacy of CD19-directed CAR T cells with a humanized scFv domain in children with B cell recovery or CD19+ relapse after prior CAR T cell therapy. Method T cells collected from patients were transduced with a lentiviral vector encoding a CAR composed of murine or humanized anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/anti-CD28 beads, cryopreserved, and then infused. The humanized scFv was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Results Of 50 patients in CR at 1 month after CTL019 infusion (50/53 CR), 14 patients received a repeat infusion of murine CTL019 at 3 and/or 6 months after initial infusion and 11 are evaluable for response. Indications were B cell recovery (n=4), CD19+ minimal residual disease (MRD, n=2), or undetectable CTL019 in peripheral blood by flow cytometry (≤0.1% CTL019+ of CD3+, n=5). Minimal toxicities were observed and included fever and fatigue. CTL019 reinfusion induced B cell aplasia for a second time in 1 of 4 children treated for B cell recovery. Two of these patients later relapsed, 1 with CD19+ and 1 with CD19- disease. Of 2 patients with CD19+ MRD, 1 had no response and subsequently experienced a CD19+ relapse and the second became MRD-negative but had B cell recovery. All 5 children reinfused for poor CTL019 persistence demonstrated continued B cell aplasia 3-15 months after repeat infusion with detectable CTL019 in peripheral blood by flow cytometry (0.2-2.8% CTL019+ of CD3+) in 4 patients. Of this group, 4 patients remain in remission 12-21 months after initial infusion, and 1 experienced a CD19- relapse. To overcome the potential for anti-murine immune-mediated rejection, patients previously treated with CAR-modified T cells were eligible for a phase 1 study of humanized CD19-directed CAR T cells (CTL119). Five children were infused for B cell recovery (n=3) or CD19+ relapse (n=2). Cytokine release syndrome (CRS), seen in 2 patients, was limited to fever, headache, and nausea and did not require vasopressor or respiratory support. Three patients showed no response and progressed (n=1) or relapsed with CD19+ (n=1) or CD19- (n=1) ALL. Responses were demonstrated in 2 patients: 1) MRD-negative CR in a patient treated for CD19+ relapse and 2) reduction in MRD and return of B cell aplasia in a patient treated for CD19+ MRD and B cell recovery, with subsequent progression to CD19+ relapse. Both responding patients were previously resistant to murine CTL019 reinfusion, suggesting a possible immune-mediated rejection mechanism. Ongoing studies are investigating anti-CAR responses. Conclusion Repeat infusion of murine CTL019 may prolong B cell aplasia in patients with early evidence of poor persistence and in a fraction of patients with B cell recovery. In the first demonstration of humanized anti-CD19 CAR efficacy, retreatment with CTL119 expressing a humanized scFv induced remission of ALL refractory to prior CD19-directed murine CAR T cell therapy, suggesting that immune-mediated rejection may be an important mechanism of resistance in the subset of patients with rapid CAR cell loss and loss of B cell aplasia. Further investigation into anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy, Research Funding. Off Label Use: CTL019 and CTL119 for relapsed/refractory ALL. Rheingold:Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. Aplenc:Sigma Tau: Consultancy. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding. Brogdon:Novartis: Employment. Loew:Novartis: Employment. Zheng:Novartis: Patents & Royalties. Levine:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding.

Expanding the Ligand Binding Repertoire of Chimeric Antigen Receptors Using Lamprey Variable Lymphocyte Receptors

Although chimeric antigen receptors (CARs) are yielding promising clinical results, the technology remains limited by the availability of conjugate cancer cell target antigens. As a means to increase both the identification of cancer cell target antigens and simultaneously generate unique single chain binding domain structures we utilized the immune system of jawless vertebrates. Sea lamprey possess an adaptive immune system primarily characterized by variable lymphocyte receptors (VLRs) as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins (Ig). VLRs have a fundamentally different structure and geometry than Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures with their variable region consisting of multiple assembled repeating sequences termed leucine rich repeats. These repeats can be directly inserted onto a CAR scaffold by genetic engineering. To test this platform technology, a yeast display method previously described (Tasumi et al., PNAS 2009; Xu et al., Humana press 2011) was used as a means of assaying and selecting appropriate VLRs. VLRs meeting the set criteria are sequenced and cloned into a lentiviral vector (LV) CAR transgene cassette plasmid. We constructed a CAR containing a well characterized VLR specific for the B cell receptor of a murine B cell leukemia (BCL) cell line. The CAR design incorporates the anti-BCL-VLR, Myc tag, CD28 transmembrane domain, and the intracellular CD3ζ signaling domain. SIN VLR-CAR LV was produced at high titer (~1x108) and used to transduce Jurkat cells. Transduced Jurkat cells showed successful CAR protein expression confirmed via Western Blot as well as persistent surface CAR expression for over 2 months with cell viability remaining over 85%. To determine whether the VLR was capable of signaling through the CAR, transduced Jurkat cells were incubated with the BCL cell line expressing the target B cell receptor. Using this assay, we demonstrated potent T cell activation via the VLR-CAR. CAR expressing T-cells demonstrated activation in ~80% of the cells. Furthermore, NK-92 cells expressing the VLR-CAR demonstrated an ability to recognize and kill BCL cells at target to effector ratios as low as 1:1, with 30% target cell killing. Little to no killing was observed with a control B cell line. Additionally, we created a CAR using a published VLR sequence targeting the human surface antigen CD5 (Yu et al., J. Immunol Methods, 2012). CD5 is primarily a T-cell marker, thus an anti-CD5-VLRCAR could be potentially used to target T-cell malignancies. Jurkat cells were transduced with high titer SIN VLR-CAR LV at various MOIs. Since Jurkat cells express CD5 on their surface, we tested the different transduced groups for self-activation at several time points. The corresponding transduced copy number was determined using quantitative PCR. The degree of activation directly correlated with the LV copy number, with highest activation being seen when the MOI approach 50, with >60% highly activated cells. No activation was observed in the GFP control groups. Additionally, we engineered three different CAR constructs consisting of two CD5 VLRs connected by either a helical linker with a 180° rotation, a helical linker with a 360° rotation, or a non-rigid linker allowing for flexibility among the two VLRs. Transduced Jurkat cells expressing the double CD5-VLR CARs indicated, via flow cytometry, that linking two VLRs does not provide any benefit in terms of activation compared to the single CD5-VLR-CAR. Current testing includes the use of NK-92 cells expressing the anti-CD5-VLR-CAR against T cell leukemia cell lines. Collectively, these results show that VLRs can be used to increase the repertoire of CAR binding motifs. Furthermore, these data suggest that VLRs provide both a unique and effective method for activating CAR-T cells and can expand the number and variety of antigens that may be targeted. DisclosuresDoering:Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding.

Development of HER2-specific chimeric antigen receptor T cells for the treatment of breast-to-brain metastasis

Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells has demonstrated robust and durable clinical efficacy in patients with CD19+ B cell malignancies. Broader application of this approach to brain and other advanced solid tumors is an immediate goal for the field and is presently under intense investigation. In 2014, 40,000 individuals in the U.S. alone succumbed to breast cancer, primarily as a result of metastatic disease. Approximately 30 percent of breast cancer patients carry an amplification of the HER2 gene and/or HER2 over-expression, which confers a particularly poor prognosis. Among the most common sites of HER2-positive breast cancer metastases is the brain. For patients with breast cancer that has metastasized to the brain, the 1-year survival rate is a dismal 20 percent. Despite clinical successes in both preventing relapse and treating systemic disease with HER2-targeted therapies, the currently available agents are only modestly effective in managing brain metastasis. Our group has demonstrated safety and transient anti-tumor responses in two U.S. FDA-authorized Phase I clinical trials evaluating local intracranial adoptive transfer of CAR T cells in glioma patients. Our current project builds on this clinical experience with locally administered CAR T cells that specifically target HER2 for the treatment of breast-to-brain metastasis. Our lab has initiated design and testing of CAR T cell therapy targeting HER2 based on an scFv derived from trastuzumab. We anticipate that local intracranial delivery will enhance therapeutic response, while reducing the likelihood of off-tumor systemic toxicities as previously observed. Importantly, HER2 expression in normal brain tissue is limited, supporting HER2 as a therapeutic target in brain metastasis. Our innovative CAR T cell platform focuses on engineering central memory T cells (Tcm) for therapeutic application, with the intent of improving persistence of T cells after infusion, a critical parameter correlated with ultimate therapeutic success. Herein, we have evaluated several HER2-CAR constructs incorporating either the CD28 or 4-1BB co-stimulatory domain using both in vitro T cell functional assays as well as orthotopic patient-derived xenograft models of breast-to-brain metastasis. While HER2-28z and HER2-BBz CAR T cells similarly kill HER2+ breast cancer cells in vitro, BBz CARs demonstrate lower induction of the exhaustion marker PD-1 and proliferate better compare with 28z CARs. Both HER2-CARs similarly lead to tumor eradication and prolonged survival. Based on these data, we have successfully developed HER2-specific CAR T cells, and plan to clinically develop these CARs for the treatment of HER2+ metastatic disease.

Smart CARs engineered for cancer immunotherapy.

Chimeric antigen receptors (CARs) are synthetic immunoreceptors, which can redirect T cells to selectively kill tumor cells, and as 'living drugs' have the potential to generate long-term antitumor immunity. Given their recent clinical successes for the treatment of refractory B-cell malignancies, there is a strong push toward advancing this immunotherapy to other hematological diseases and solid cancers. Here, we summarize the current state of the field, highlighting key variables for the optimal application of CAR T cells for cancer immunotherapy. Advances in CAR T-cell therapy have highlighted intrinsic CAR design and T-cell manufacturing methods as critical components for maximal therapeutic success. Similarly, addressing the unique extrinsic challenges of each tumor type, including overcoming the immunosuppressive tumor microenvironment and tumor heterogeneity, and mitigating potential toxicity, will dominate the next wave of CAR T-cell development. CAR T-cell therapeutic optimization, including intrinsic and extrinsic factors, is critical to developing effective CAR T-cell therapies for cancer. The excitement of CAR T-cell immunotherapy has just begun, and will continue with new insights revealed in laboratory research and in ongoing clinical investigations.

Chimeric Antigen Receptor T-Cells: New Approaches to Improve Their Efficacy and Reduce Toxicity.

The durable remission of B-cell leukemia and lymphoma following chimeric antigen receptor (CAR) T-cell therapy has brought this new form of adoptive immunotherapy to center stage with the expectation that CAR T-cell therapy may provide similar efficacy in other hematologic and solid cancers. Herein, we review recent advances in the areas of CAR design that improve CAR T-cell proliferation, engraftment, and efficacy, as well as clinical application strategies that are designed to improve clinical efficacy while reducing the risk of toxicity and broaden patient access to this promising form of cancer immunotherapy.

Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.

Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration.

T cell engineering as therapy for cancer and HIV: our synthetic future.

It is now well established that the immune system can control and eliminate cancer cells. Adoptive T cell transfer has the potential to overcome the significant limitations associated with vaccine-based strategies in patients who are often immune compromised. Application of the emerging discipline of synthetic biology to cancer, which combines elements of genetic engineering and molecular biology to create new biological structures with enhanced functionalities, is the subject of this overview. Various chimeric antigen receptor designs, manufacturing processes and study populations, among other variables, have been tested and reported in recent clinical trials. Many questions remain in the field of engineered T cells, but the encouraging response rates pave a wide road for future investigation into fields as diverse as cancer and chronic infections.

Chimeric Antigen Receptors for Cancer: Progress and Challenges

Chimeric antigen receptors (CARs) genetically link an antigen-binding domain with cell-signaling domains to redirect immune cell specificity toward antigens expressed on the surface of cancer cells. Progress in CAR engineering over the last two decades has elucidated fundamental principles impacting CAR potency, and today CARs can be readily generated toward essentially any cell surface target on cancer cells. Efficacy thus far has been most impressive using CD19-CAR expressing T cells to treat B cell lymphoblastic leukemia, although clear activity has also been observed using CD19-CARs in patients with chronic lymphocytic leukemic and B cell lymphoma. Much less data is available regarding CAR efficacy for solid tumors. Here, we summarize current concepts of CAR design, with a focus on the relationship between structure and function, a review of the clinical results reported thus far, and the challenges to be addressed in future studies.

Abstract B08: Invariant NKT cells as a platform for cancer immunotherapy with chimeric antigen receptors

Abstract Advances in the design of chimeric antigen receptors (CARs) have improved the anti-tumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells and their restriction by polymorphic HLA molecules limit their therapeutic potential and are associated with toxicity, especially in allogeneic settings. We proposed that CAR expression in Vα24-invariant NKT cells (NKTs) can build upon the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKTs were engineered to express a CAR against the GD2 ganglioside, which is highly expressed by neuroblastoma. We compared CAR constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB co-stimulatory endodomains. CAR expression rendered NKTs highly cytotoxic against neuroblastoma cells without affecting their TCR-induced proliferation and CD1d-dependent killing of M2-polarized macrophages. We observed a striking Th1-like polarization of NKTs by 4-1BB-containing CARs in vitro and in mice. Expression of both CD28 and 4-1BB endodomains in the CAR maximized persistence of NKTs in NSG mice with neuroblastoma xenografts. These NKTs, expressing CAR with two co-stimulatory endodomains localized to the tumor site more effectively than T cells with the same CAR (p < 0.001). Three weekly injections of 107 CAR NKTs prolonged survival of humanized (hu)NSG mice with metastatic neuroblastoma (75.2 ± 6 days) compared to a single injection (52.1 ± 4.3 days, p = 0.007) or untreated control (35.4 ± 1.2 days, p < 0.0001). A single injection of CAR T cells was associated with tumor regression early after treatment but resulted in lethal graft-versus-host disease (GvHD) within 5 weeks that prevented evaluation of the anti-tumor activity. In contrast, CAR NKTs did not induce GvHD, even after repeated injections. These results establish the potential of NKTs to serve as a safe and effective platform for CAR-directed cancer immunotherapy. Citation Format: Andras Heczey, Daofeng Liu, Gengwen Tian, Ekaterina Marinova, Xiuhua Gao, Linjie Guo, John Hicks, Gianpietro Dotti, Leonid Metelitsa. Invariant NKT cells as a platform for cancer immunotherapy with chimeric antigen receptors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B08.

Adoptive immunotherapy for acute leukemia: New insights in chimeric antigen receptors.

Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells (LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despite the introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90's, chimeric antigen receptors (CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding "living drug" specifically targeting the tumor-associated antigen, and ensure long-term anti-tumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia.

Abstract 3157: Novel GD2-specific chimeric antigen receptor-modified T cells targeting osteosarcoma

Abstract Despite intensive multi-modality therapies employed in the treatment of osteosarcoma (OS) the prognosis for children with high-risk disease remains suboptimal. Since standard systemic therapy for OS has remained unchanged in the past thirty years, with no new agents identified to complement conventional regimens, there has been a significant plateau in the survival of OS patients thus necessitating the development of more targeted and efficacious therapeutics. Chimeric antigen receptor (CAR) modified T cells can meet this need by utilizing the immune system's surveillance capacity and potent cytotoxic mechanisms against tumor specific antigen targets with exquisite specificity. Since OS highly expresses the GD2 antigen, a viable immunotherapeutic target, we sought to assess if a novel 4th generation CAR modified T cell could target GD2 and induce cytotoxicity against OS. We have engineered a newer generation of CAR T cells that harness the vital co-stimulatory domains imperative for T cell activation while embedding the safety features of suicide induction. The latter, an advanced safety design iCasp9, is based on AP1903-induced caspase 9 dimer formation to trigger apoptosis, which has been incorporated in our CAR design. The 4th generation lentiviral CAR (4S-CAR) encodes a combination of CD28/CD137/CD27 T cell co-stimulatory domains and CD3zeta domain fused with a mutated FKBP12 (F36V) and caspase 9. To illustrate the self-destructive function, 4S-CAR T cells were treated with AP1903 to induce iCasp9 dimerization. High efficiency of apoptosis was observed within 30 min, and in 2 hours, more than 80% CAR T cells died. After demonstrated the ability to screen patient OS samples for GD2 expression, we found that OS cells expressing high levels of GD2 were effectively targeted and killed by 4S-GD2-CAR-T cells. 4S-GD2 CAR modified T cells were shown to overexpress the exhaustion marker PD-1 when in contact with target tumor cells, and multiple OS expressing cell lines were shown to overexpress PD-L1, and the PD-L1 level increased after cocultured with 4S-GD2 CAR modified T cells. The combination effects of immune checkpoint inhibitors such as anti-PD-L1 and anti-PD-1 antibodies with CAR T cells will be further investigated. Anti-GD2 expressing CAR T cells provide a novel targeted approach to eliminate residual disease with consummate specificity. As cellular therapeutics using CAR T cells become further entrenched in the forefront against refractory malignancies, OS provides a new and compulsory niche to further test the feasibility, safety and potential efficacy of this promising new technology. Citation Format: Monrat Chulanetra, Elias Sayour, Lamis Eldjerou, Joanne Lagmay, Rowan Milner, William Slayton, Lung-Ji Chang. Novel GD2-specific chimeric antigen receptor-modified T cells targeting osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3157. doi:10.1158/1538-7445.AM2015-3157

Abstract 4702: 4-1BB costimulation ameliorates exhaustion and prolongs in vivo persistence of chimeric antigen receptor (CAR) expressing T cells

Abstract Published reports have demonstrated high response rates of CD19 chimeric antigen receptor (CAR) modified T cells against acute lymphoblastic leukemia. CARs incorporating either CD28 or 4-1BB costimulatory domains mediated significant anti-leukemic effects. However CD19-41BBz CAR T cells could be detected for months and induced prolonged B cell depletion, whereas most patients treated with CD19-28z CAR T cells had no detectable CAR+ T cells by day 28, and B cell recovery was routinely observed. We report that CAR T cells incorporating the CD28 versus 4-1BB costimulatory domains show differential susceptibilities to T cell exhaustion. Using the tonically signaling 14g2a-based GD2 CAR as a model system, we identify that GD2-28z CARs rapidly acquire an exhaustion phenotype, characterized by high level expression of PD1, TIM3 and LAG3, high expression of exhaustion related transcription factors Blimp1 and T-bet, poor proliferative capacity, poor cytokine production, and poor anti-tumor efficacy in vivo. Interestingly, second generation GD2-28z CARs induce greater exhaustion than first generation GD2-z CARs, implicating CD28 costimulation in the induction of exhaustion. In contrast, GD2-41BBz CARs express lower levels of PD1 and TIM3, show increased cytokine production, improved persistence in vivo, and improved anti-tumor efficacy against both osteosarcoma and neuroblastoma xenografts. Similarly, CD19-41BBz CARs have improved persistence and lower expression of exhaustion markers compared to CD19-28z CARs following leukemia challenge. Finally, CD22-BBz CARs show lower exhaustion marker expression, improved persistence, and improved anti-leukemia efficacy in immunodeficient mice compared to CD22-28z CARs. Gene expression experiments are underway to further delineate the mechanism by which 4-1BB signaling ameliorates exhaustion in the setting of chronic CAR signaling. Together, this data demonstrates that exhaustion is a critical factor limiting the efficacy of CAR expressing T cells. CD28 and 4-1BB have opposing effects on exhaustion in CAR T cells, with CD28 signaling augmenting the phenotype while 4-1BB signaling mitigating exhaustion. This data is the first to demonstrate differential effects of costimulatory pathways on T cell exhaustion, likely explains differential persistence observed in the clinical trials of CAR therapeutics, and provides important insights for CAR design for future clinical applications. Citation Format: Adrienne H. Long, Waleed M. Haso, Jillian P. Smith, Alec J. Walker, Terry J. Fry, Rimas J. Orentas, Crystal L. Mackall. 4-1BB costimulation ameliorates exhaustion and prolongs in vivo persistence of chimeric antigen receptor (CAR) expressing T cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2015-4702

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition.

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

Retargeted human avidin-CAR T cells for adoptive immunotherapy of EGFRvIII expressing gliomas and their evaluation via optical imaging.

There has been significant progress in the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. However, the challenge of monitoring the therapy in real time has been continually ignored. To address this issue, we developed optical molecular imaging approaches to evaluate a recently reported novel CAR strategy for adoptive immunotherapy against glioma xenografts expressing EGFRvIII. We initially biotinylated a novel anti-EGFRvIII monoclonal antibody (biotin-4G1) to pre-target EGFRvIII+ gliomas and then redirect activated avidin-CAR expressing T cells against the pre-targeted biotin-4G1. By optical imaging study and bio-distribution analysis, we confirmed the specificity of pre-target and target and determined the optimal time for T cells adoptive transfer in vivo. The results showed this therapeutic strategy offered efficient therapy effect to EGFRvIII+ glioma-bearing mice and implied that optical imaging is a highly useful tool in aiding in the instruction of clinical CAR-T cells adoptive transfer in future.

215. Detail Analysis of Non-Specific Activation of CD19 CAR-T Cells Caused by CAR Design

Adoptive immunotherapy using the chimeric antigen receptor (CAR) gene-modified T cells is a promising strategy to treat patients with malignancy and autoimmune diseases. The CAR-T cells can target any cell surface antigens in a HLA independent manner, therefore CAR-T therapy is applicable to a broad range of patients irrespective of HLA phenotype. Typically CARs have higher affinities compared to TCRs, and T cell activation and cytotoxic effect on target cells mediated by CARs correlated with the affinity until it reach the thresholds. The latest results of CD19 CAR-T immunotherapy clinical trials have demonstrated impressive potential in a range of B-lymphoid malignancies. In spite of the recent great success, serious adverse events occurred after infusion of CAR-T cells including “on-target off-organ” activation, and cytokine release syndrome has been observed in a number of CAR-T cell therapies as a result of excessive T cell activation. To improve the efficacy and safety of CAR-T cells, selection of the target tumor-associated antigen, that are expressed only on tumor cells, and the suitable CAR constructs showing the optimal T cell activities are essential, thereby minimizing the risk of side effects.Previously, we have found CAR-T cells acquired to express T cell activation marker CD25 without specific antigen stimulation during in vitro culture. To validate of consequence of CD25-expression, we have analyzed the immune phenotype and antigen specific activities of CAR-T cells, and found the excess activation of CAR-T cells caused T cells differentiation with much less Naive T cell populations, resulting reduction of T cell activities against specific tumor cells. All CAR-T cells we have tested were activated without specific antigen stimulation, although the intensity of non-specific activation were correlated with the expression levels of CARs and depended on the scFvs and the extracellular-spacer domains.In this study, aiming to select the optimal design of CD19 CAR for effective and safe immunotherapy, using anti-CD19 antibody, clone FMC63, we performed detail analysis of non-specific activation of CD19 CAR-T cells caused by the design of scFv (e.g.the leader sequences, the order of VH and VL, the spacer sequences between VH and VL, and the extracellular-spacer domains). In vitro culture of CD19-CAR T cells without antigen specific stimulation induced elevation of T cell activation marker expression, such as CD25 and CD69 on CAR T cells, and reduction of Naive phenotype T cell populations (CCR7+/CD45RA+), as the level of non-specific activation of CAR-T cells was depended on the structure of scFvs (the order of VH and VL, and the spacer sequences) and the extracellular-spacer domains. Especially, the CAR-T cells with CD8 hinge domain were highly activated without specific activation, and the excessive non-specific activation lowered the cytokine secretion and the cytotoxic activities against CD19-expressing tumor cells. Although the low level activation of CAR-T cells may contribute the long term survival of T cells in vivo, it is desired to select the optimal antibodies and CAR constructs which shows high antigen-specific activities with low non-specific activities for the safe and effective CAR-T cell therapy.

224. Development of Safer & Optimized CAR-T Cells Using Lentiviral Vectors

Chimeric antigen receptors (CAR) modified T cells have shown very promising clinical results in hematological malignancies. CAR are usually composed of a scFv antigen binding domain linked to a spacer and intracellular signaling domains. CAR introduction into T cells leads to cells with optimal effector functions and with a new antigen binding ability. This strategy is well suited to skirt immune escape mechanisms.Despite their efficacy, CAR-T cells still show toxicity due to a cytokine storm, even if it is manageable. Improving the safety while maintaining or even improving the anti-tumor efficacy of CAR-T cells is therefore a crucial therapeutic challenge.Based on our proprietary lentiviral vector technology, we are developing a CAR-T cell platform aiming at producing innovative CAR-T cells. The development conducted by Theravectys are: -Development of proprietary lentiviral vectors coding for CAR of the second (containing the CD3ζ and the 4-1BB cosignaling domains) and third generations (containing the CD3ζ, the CD28 and the 4-1BB domains) directed against CD19 (for CD19+ leukemias and lymphomas), LMP-1 and -2 (for EBV-induced leukemias). Our lentiviral vectors will allow optimal expression of CAR therefore leading to a better efficacy of CAR-T cells. Hematological malignancies will be used as a benchmark.-Development of an inducible RUSH (Retention Using Selective Hook) system which is based on the streptavidin anchored to the membrane of the endoplasmic reticulum (ER) through a hook, and on the SBP (streptavidin binding protein) introduced into the CAR structure. The interaction between the streptavidin-hook and the SBP-CAR allows the CAR retention inside the ER. The addition of biotin displaces the equilibrium of binding of streptavidin towards biotin instead of SBP, thus leading to the release of the CAR from the ER and its expression to the cytoplasmic membrane. View Large Image | Download PowerPoint SlideThe release of the CAR will stop with drug exhaustion (or antagonist) and remaining cells can be easily reactivated through reintroduction of biotin. This system will increase safety of CAR-T cells. A CD19-CAR RUSH will be used as a proof-of-concept of the RUSH system.Most of the scFv used to date are of murine origin. Neutralizing antibodies against these murine scFv can limit the efficacy of CAR. As an alternative, we are screening and engineering specific humanized camelid nanobodies to be used as binding domains since they are highly homologous to the human VH domain of antibodies and they display high antigen binding capacities. The proof-of-concept will be made with a second generation CAR containing a nanobody directed against Her2 as a binding domain.Theravectys’ technological platforms allows flexibility and reactivity in the CAR design, production and evaluation, thus leading to the fast generation of safe and optimized CAR-T cells.

Chimeric antigen receptor T cells for cancer immunotherapy.

Article Tools UNDERSTANDING THE PATHWAY Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2014.60.3449 Journal of Clinical Oncology - published online before print April 20, 2015 PMID: 25897155 Chimeric Antigen Receptor T Cells for Cancer Immunotherapy Kevin J. CurranxKevin J. CurranSearch for articles by this author , Renier J. BrentjensxRenier J. BrentjensSearch for articles by this author Show More Memorial Sloan Kettering Cancer Center, New York, NY https://doi.org/10.1200/JCO.2014.60.3449 First Page Full Text PDF Figures and Tables © 2015 by American Society of Clinical Oncology

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials.