Chimeric antigen receptor–T cell therapy manufacturing: modelling the effect of offshore production on aggregate cost of goods

Cell and gene therapies have demonstrated excellent clinical results across a range of indications with chimeric antigen receptor (CAR)–T cell therapies among the first to reach market. Although these therapies are currently manufactured using patient-derived cells, therapies using healthy donor cells are in development, potentially offering avenues toward process improvement and patient access. An allogeneic model could significantly reduce aggregate cost of goods (COGs), potentially improving market penetration of these life-saving treatments. Furthermore, the shift toward offshore production may help reduce manufacturing costs. In this article, we examine production costs of an allogeneic CAR-T cell process and the potential differential manufacturing costs between regions. Two offshore locations are compared with regions within the United States. The critical findings of this article identify the COGs challenges facing manufacturing of allogeneic CAR-T immunotherapies, how these may evolve as production is sent offshore and the wider implication this trend could have.     

Targeting γδ T cells for immunotherapy of HIV disease

Disruption of circulating γδ T-cell populations is an early and common outcome of HIV infection. T-cell receptor (TCR)-γ2δ2 cells (expressing the Vγ2 and Vδ2 chains of the γδ TCR) are depleted, even though they are minimally susceptible to direct HIV infection, and exemplify indirect cell depletion mechanisms that are important in the progression to AIDS. Among individuals with common or normally progressing HIV disease, the loss of TCR-γ2δ2 cells has a broad impact on viral immunity, control of opportunistic pathogens and resistance to malignant disease. Advanced HIV disease can result in complete loss of TCR-γ2δ2 cells that are not recovered even during antiretroviral therapy with complete virus suppression. However, normal levels of TCR-γ2δ2 were observed among natural virus suppressors (low or undetectable virus without antiretroviral therapy) irrespective of their MHC haplotype, consistent with their disease-free status. The pattern of loss and recovery of TCR-γ2δ2 cells revealed their unique features and functional capacities, and encourage the development of immune-based therapies to activate and expand this T-cell subset. New research has identified drugs that might reconstitute the TCR-γ2δ2 population, recover their functional contributions, and improve control of HIV replication and disease. Here, we review research on HIV and TCR-γδ T cells to highlight the consequences of depleting this subset and the unique features of TCR-γδ biology that argue in favor of clinical strategies to reconstitute this T-cell subset in individuals with HIV/AIDS.     

Role of α-fetoprotein in differentiation of regulatory T lymphocytes

The effect of native α-fetoprotein (AFP) on the expression of T-regulatory lymphocyte (Treg) markers by activated CD4⁺ lymphocytes with different proliferative status was studied. α-Fetoprotein did not affect the ratio of proliferating and non-proliferating activated CD4⁺ cells. In the study of Treg differentiation, it was found that AFP at concentrations of 50 and 100 μg/mL significantly inhibited the number of nonproliferating CD4⁺FOXP3⁺ and CD4⁺FOXP3⁺HELIOS⁺ lymphocytes without affecting the expression of Treg markers by proliferating CD4⁺ lymphocytes.     

Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor–T cell manufacture

Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)–T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.     

Extrathymic differentiation and antigen response of αβT lymphocytes in pregnancy

We studied reactivity of αβT-lymphocytes in CBA pregnant females toward male antigens and the presence of gene rearrangement in T-cells antigen receptor in peripheral lymphoid organs of mice in the case of three breeding variants: CBA × BALB/c (normal allogenic pregnancy), CBA × CBA (syngenetic pregnancy), and CBA × DBA/2 (prone to abortion combination). It was shown that proliferative response of αβT-lymphocytes in pregnant CBA females to male spleen cells was the most marked at normal allogenic pregnancy, the least marked at syngenic pregnancy, and was not observed at the combination CBA × DBA/2. In addition, cells of paraaortic lymphatic nodes (draining uterus) respond to male antigen reliably more effectively than lymphocytes in mesenterial and axillary lymphatic nodes. Simultaneous estimation of recombinase RAG-1, the key enzyme in rearrangement of T-receptor genes, revealed similar principles: predominant activity of recombinase in T-lymphocytes in paraaortal lymphatic nodes of CBA pregnant females. This points to the relationship between extrathymic rearrangement of antigen receptor genes and change in the antigen-detecting repertoire of these cells. The possible biological significance of the discovered phenomenon is discussed.     

CAR T cells transform to trucks: chimeric antigen receptor–redirected T cells engineered to deliver inducible IL-12 modulate the tumour stroma to combat cancer

Adoptive T cell therapy recently achieved impressive efficacy in early-phase clinical trials; this significantly raises the profile of immunotherapy in the fight against cancer. A broad variety of tumour cells can specifically be targeted by patients' T cells, which are redirected in an antibody-defined, major histocompatibility complex-unrestricted fashion by endowing them with a chimeric antigen receptor (CAR). Despite promising results for some haematologic malignancies, the stroma of large, established tumours, the broad plethora of infiltrating repressor cells, and cancer cell variants that had lost the target antigen limit their therapeutic efficacy in the long term. This article reviews a newly described strategy for overcoming some of these shortcomings by engineering CAR T cells with inducible or constitutive release of IL-12. Once redirected, these T cells are activated, and released IL-12 accumulates in the tumour lesion where it promotes tumour destruction by at least two mechanisms: (1) induction of an innate immune cell response towards those cancer cells which are invisible to redirected T cells and (2) triggering programmatic changes in immune-suppressive cells. Given the enormous complexity of both tumour progression and immune attack, the upcoming strategies using CAR-redirected T cells for local delivery of immune-modulating payloads exhibited remarkable efficacy in pre-clinical models, suggesting their evaluation in clinical trials.     

Chimeric antigen receptor T cells shape myeloid cell function within the tumor microenvironment through IFN-γ and GM-CSF

The infiltration of suppressive myeloid cells into the tumor microenvironment restrains anti-tumor immunity. However, cytokines may alter the function of myeloid lineage cells to support tumor rejection, regulating the balance between pro- and anti-tumor immunity. In this study, it is shown that effector cytokines secreted by adoptively transferred T cells expressing a chimeric Ag receptor (CAR) shape the function of myeloid cells to promote endogenous immunity and tumor destruction. Mice bearing the ovarian ID8 tumor were treated with T cells transduced with a chimeric NKG2D receptor. GM-CSF secreted by the adoptively transferred T cells recruited peripheral F4/80(lo)Ly-6C(+) myeloid cells to the tumor microenvironment in a CCR2-dependent fashion. T cell IFN-γ and GM-CSF activated local, tumor-associated macrophages, decreased expression of regulatory factors, increased IL-12p40 production, and augmented Ag processing and presentation by host macrophages to Ag-specific T cells. In addition, T cell-derived IFN-γ, but not GM-CSF, induced the production of NO by F4/80(hi) macrophages and enhanced their lysis of tumor cells. The ability of CAR T cell therapy to eliminate tumor was moderately impaired when inducible NO synthase was inhibited and greatly impaired in the absence of peritoneal macrophages after depletion with clodronate encapsulated liposomes. This study demonstrates that the activation of host macrophages by CAR T cell-derived cytokines transformed the tumor microenvironment from immunosuppressive to immunostimulatory and contributed to inhibition of ovarian tumor growth.     

Influenza A virus—a model for viral antigen presentation to cytotoxic T lymphocytes

Human influenza A virus is characterized by its high degree of variability and by its ability to cause frequent epidemics of disease. Most of the variation occurs in the two surface glycoproteins of the virus, against which protective antibodies are directed. In contrast, the strong MHC class I-restricted CTL response to infection with virus is predominantly specific for internal viral proteins which are relatively well conserved, and is cross-reactive between different strains of influenza A virus. However, the natural evolution of influenza viruses is largely driven by selection with antibody, with no firm evidence of selection by CTL. In normal individuals influenza virus produces an acute, localized infection, and this in part may reflect an inability to escape the CTL response.     

T regulatory cells: aid or hindrance in the clearance of disease?

CD4+ CD25+ T regulatory cells (Tregs) are classified as a subset of T cells whose role is the suppression and regulation of immune responses to self and non-self. Since their discovery in the early 1970s, the role of CD4+ CD25+ Tregs in both autoimmune and infectious disease has continued to expand. This review examines the recent advances on the role CD4+ CD25+ Tregs may be playing in various diseases regarding progression or protection. In addition, advances made in the purification and manipulation of CD4+ CD25+ Tregs using new cell markers, techniques and antibodies are discussed. Ultimately, an overall understanding of the exact mechanism which CD4+ CD25+ Tregs implement during disease progression will enhance our ability to manipulate CD4+ CD25+ Tregs in a clinically beneficial manner.     

Signal regulatory protein α regulates the homeostasis of T lymphocytes in the spleen

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell-cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4(+) T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin β receptor, as well as the in vivo response to lymphotoxin β receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.     

Manufacturing development and clinical production of NKG2D chimeric antigen receptor–expressing T cells for autologous adoptive cell therapy

Background aims

Adoptive cell therapy employing natural killer group 2D (NKG2D) chimeric antigen receptor (CAR)-modified T cells has demonstrated preclinical efficacy in several model systems, including hematological and solid tumors. We present comprehensive data on manufacturing development and clinical production of autologous NKG2D CAR T cells for treatment of acute myeloid leukemia and multiple myeloma (ClinicalTrials.gov Identifier: NCT02203825). An NKG2D CAR was generated by fusing native full-length human NKG2D to the human CD3ζ cytoplasmic signaling domain. NKG2D naturally associates with native costimulatory molecule DAP10, effectively generating a second-generation CAR against multiple ligands upregulated during malignant transformation including MIC-A, MIC-B and the UL-16 binding proteins.

Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.     

Evolutionary history of copy-number-variable locus for the low-affinity Fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection

Both sequence variation and copy-number variation (CNV) of the genes encoding receptors for immunoglobulin G (Fcγ receptors) have been genetically and functionally associated with a number of autoimmune diseases. However, the molecular nature and evolutionary context of this variation is unknown. Here, we describe the structure of the CNV, estimate its mutation rate and diversity, and place it in the context of the known functional alloantigen variation of these genes. Deletion of Fcγ receptor IIIB, associated with systemic lupus erythematosus, is a result of independent nonallelic homologous recombination events with a frequency of approximately 0.1%. We also show that pathogen diversity, in particular helminth diversity, has played a critical role in shaping the functional variation at these genes both between mammalian species and between human populations. Positively selected amino acids are involved in the interaction with IgG and include some amino acids that are known polymorphic alloantigens in humans. This supports a genetic contribution to the hygiene hypothesis, which states that past evolution in the context of helminth diversity has left humans with an array of susceptibility alleles for autoimmune disease in the context of a helminth-free environment. This approach shows the link between pathogens and autoimmune disease at the genetic level and provides a strategy for interrogating the genetic variation underlying autoimmune-disease risk and infectious-disease susceptibility.     

Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Background aimsNaturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1–5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed.MethodsSeveral methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model.ResultsWith the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo–expanded Treg were CD4⁺25⁺FOXP3⁺127^lo and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro.ConclusionsIn our NOD-SCID IL-2Rγ^null xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo–expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.     

Chemokines: agents for the immunotherapy of cancer?

Chemokines, a superfamily of small cytokine-like molecules, regulate leukocyte transport in the body. In recent years, we have witnessed the transition of immunotherapeutic strategies from the laboratory to the bedside. Here, we review the role of chemokines in tumour biology and the development of the host's anti-tumour defence. We summarize the current knowledge of chemokine-receptor expression by relevant cellular components of the immune system and the role of their ligands in the organization of the antitumour immune response. Finally, we discuss recent findings which indicate that chemokines have therapeutic potential as adjuvants or treatments in antitumour immunotherapy, as well as remaining questions and perspectives for translating experimental evidence into clinical practice.     

Absence of β2 integrins impairs regulatory T cells and exacerbates CD4+ T cell-dependent autoimmune carditis

The immunopathogenic mechanisms mediating inflammation in multiorgan autoimmune diseases may vary between the different target tissues. We used the K/BxN TCR transgenic mouse model to investigate the contribution of CD4(+) T cells and β(2) integrins in the pathogenesis of autoimmune arthritis and endocarditis. Depletion of CD4(+) T cells following the onset of arthritis specifically prevented the development of cardiac valve inflammation. Genetic absence of β(2) integrins had no effect on the severity of arthritis and unexpectedly increased the extent of cardiovascular pathology. The exaggerated cardiac phenotype of the β(2) integrin-deficient K/BxN mice was accompanied by immune hyperactivation and was linked to a defect in regulatory T cells. These findings are consistent with a model in which the development of arthritis in K/BxN mice relies primarily on autoantibodies, whereas endocarditis depends on an additional contribution of effector T cells. Furthermore, strategies targeting β(2) integrins for the treatment of systemic autoimmune conditions need to consider not only the role of these molecules in leukocyte recruitment to sites of inflammation, but also their impact on the regulation of immunological tolerance.     

Manufacturing chimeric antigen receptor T cells from cryopreserved peripheral blood cells: time for a collect-and-freeze model?

Background aimsChimeric antigen receptor (CAR)-modified T-cell therapy has revolutionized outcomes for patients with relapsed/refractory B-cell malignancies. Despite the exciting results, several clinical and logistical challenges limit its wide applicability. First, the apheresis requirement restricts accessibility to institutions with the resources to collect and process peripheral blood mononuclear cells (PBMCs). Second, even when utilizing an apheresis product, failure to manufacture CAR T cells is a well-established problem in a significant subset. In heavily pre-treated patients, prior chemotherapy may impact T-cell quality and function, limiting the ability to manufacture a potent CAR T-cell product. Isolation and storage of T cells shortly after initial cancer diagnosis or earlier in life while an individual is still healthy are an alternative to using T cells from heavily pre-treated patients. The goal of this study was to determine if a CAR T-cell product could be manufactured from a small volume (50 mL) of healthy donor blood.MethodsCollaborators at Cell Vault collected 50 mL of whole peripheral venous blood from three healthy donors. PBMCs were isolated, cryopreserved and shipped to the Medical College of Wisconsin. PBMCs for each individual donor were thawed, and CAR T cells were manufactured using an 8-day process on the CliniMACS Prodigy device with a CD19 lentiviral vector.ResultsStarting doses of enriched T-cell numbers ranged from 4.0 × 10⁷ cells to 4.8 × 10⁷ cells, with a CD4/CD8 purity of 74–79% and an average CD4:CD8 ratio of 1.4. On the day of harvest, total CD3 cells in the culture expanded to 3.6–4.6 × 10⁹ cells, resulting in a 74- to 115-fold expansion, an average CD4:CD8 ratio of 2.9 and a CD3 frequency of greater than 99%. Resulting CD19 CAR expression varied from 19.2% to 48.1%, with corresponding final CD19+ CAR T-cell counts ranging from 7.82 × 10⁸ cells to 2.21 × 10⁹ cells. The final CAR T-cell products were phenotypically activated and non-exhausted and contained a differentiated population consisting of stem cell-like memory T cells.ConclusionsOverall, these data demonstrate the ability to successfully generate CAR T-cell products in just 8 days using cryopreserved healthy donor PBMCs isolated from only 50 mL of blood. Notably, numbers of CAR T cells were more than adequate for infusion of an 80-kg patient at dose levels used for products currently approved by the Food and Drug Administration. The authors offer proof of principle that cryopreservation of limited volumes of venous blood with an adequate starting T-cell count allows later successful manufacture of CAR T-cell therapy.     

Pathogenesis of endometriosis: natural immunity dysfunction or autoimmune disease?

Endometriosis is a chronic inflammatory disease, characterized by implantation and growth of endometrial tissue outside the uterine cavity. This disabling condition is considered one of the most frequent diseases in gynecology, affecting 15-20% of women in their reproductive life. Pelvic endometriosis, the most common form of the disease, is associated with increased secretion of pro-inflammatory cytokines, neo-angiogenesis, intrinsic anomalies of the refluxed endometrium and impaired function of cell-mediated natural immunity. Recently, endometriosis has also been considered to be an autoimmune disease, owing to the presence of autoantibodies, the association with other autoimmune diseases and recurrent immune-mediated abortion. These findings are in apparent contradiction with the reduced cell-mediated natural immunity observed during the disease. In this review, we focus on the multiple processes underlying the complex pathogenesis of endometriosis, with particular emphasis on the role played by the immune system with the induction of autoimmunity.