CAR-T cell therapy in India requires a paradigm shift in training, education and health care processes.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of some kinds of cancers. Hundreds of companies and academic institutions are collaborating to develop gene-modified cell therapies using novel targets, different cell types, and manufacturing processes of autologous and allogenic cell therapies. The individualized, custom-made autologous CAR-T cell production platform remains a significant limiting factor for its large-scale clinical application. In this respect, the advances in standardization and automation of the process can have considerable impact on cost reduction. Development of off-the-shelf, ready-to-use universal killer cells can enable scaling up. Despite the wide use of this cell therapy in the United States, Europe and China, its development is limited in developing countries in Southeast Asia, Africa and Latin America. In this review, we focus on good manufacturing practices-compliant manufacturing requirements, operational logistics, and regulatory processes that need to be considered for high-quality gene-modified cell therapies from an Indian perspective. We also list the potential strategies to overcome challenges associated with translation to affordability and scalability.

Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes.

Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.

Gene-Edited Interleukin CAR-T Cells Therapy in the Treatment of Malignancies: Present and Future.

In recent years, chimeric antigen receptor T cells (CAR-T cells) have been faced with the problems of weak proliferation and poor persistence in the treatment of some malignancies. Researchers have been trying to perfect the function of CAR-T by genetically modifying its structure. In addition to the participation of T cell receptor (TCR) and costimulatory signals, immune cytokines also exert a decisive role in the activation and proliferation of T cells. Therefore, genetic engineering strategies were used to generate cytokines to enhance tumor killing function of CAR-T cells. When CAR-T cells are in contact with target tumor tissue, the proliferation ability and persistence of T cells can be improved by structurally or inductively releasing immunoregulatory molecules to the tumor region. There are a large number of CAR-T cells studies on gene-edited cytokines, and the most common cytokines involved are interleukins (IL-7, IL-12, IL-15, IL-18, IL-21, IL-23). Methods for the construction of gene-edited interleukin CAR-T cells include co-expression of single interleukin, two interleukin, interleukin combined with other cytokines, interleukin receptors, interleukin subunits, and fusion inverted cytokine receptors (ICR). Preclinical and clinical trials have yielded positive results, and many more are under way. By reading a large number of literatures, we summarized the functional characteristics of some members of the interleukin family related to tumor immunotherapy, and described the research status of gene-edited interleukin CAR-T cells in the treatment of malignant tumors. The objective is to explore the optimized strategy of gene edited interleukin-CAR-T cell function.

The Yin and Yang of Type 1 Regulatory T Cells: From Discovery to Clinical Application.

Regulatory T cells are essential players of peripheral tolerance and suppression of inflammatory immune responses. Type 1 regulatory T (Tr1) cells are FoxP3- regulatory T cells induced in the periphery under tolerogenic conditions. Tr1 cells are identified as LAG3+CD49b+ mature CD4+ T cells that promote peripheral tolerance through secretion of IL-10 and TGF-ß in addition to exerting perforin- and granzyme B-mediated cytotoxicity against myeloid cells. After the initial challenges of isolation were overcome by surface marker identification, ex vivo expansion of antigen-specific Tr1 cells in the presence of tolerogenic dendritic cells (DCs) and IL-10 paved the way for their use in clinical trials. With one Tr1-enriched cell therapy product already in a Phase I clinical trial in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), Tr1 cell therapy demonstrates promising results so far in terms of efficacy and safety. In the current review, we identify developments in phenotypic and molecular characterization of Tr1 cells and discuss the potential of engineered Tr1-like cells for clinical applications of Tr1 cell therapies. More than 3 decades after their initial discovery, Tr1 cell therapy is now being used to prevent graft versus host disease (GvHD) in allo-HSCT and will be an alternative to immunosuppression to promote graft tolerance in solid organ transplantation in the near future.

CAR-NK Cells in the Treatment of Solid Tumors.

CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having unprecedented efficacy against hematological malignancies, the treatment is far from flawless. Its greatest drawbacks arise from a challenging and expensive production process, strict patient eligibility criteria and serious toxicity profile. One possible solution, supported by robust research, is the replacement of T lymphocytes with NK cells for CAR expression. NK cells seem to be an attractive vehicle for CAR expression as they can be derived from multiple sources and safely infused regardless of donor-patient matching, which greatly reduces the cost of the treatment. CAR-NK cells are known to be effective against hematological malignancies, and a growing number of preclinical findings indicate that they have activity against non-hematological neoplasms. Here, we present a thorough overview of the current state of knowledge regarding the use of CAR-NK cells in treating various solid tumors.

Applications of CRISPR Genome Editing to Advance the Next Generation of Adoptive Cell Therapies for Cancer.

Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. SIGNIFICANCE: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.

Induced pluripotent stem cell-derived natural killer cells gene-modified to express chimeric antigen receptor-targeting solid tumors.

The use of allogeneic, pluripotent stem cell-derived immune cells for cancer immunotherapy has been the subject of recent research, including clinical trials. The use of pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, regarding efficacy, safety, and producibility. In this review, we have described the characteristics of natural killer (NK) cells from multiple cell sources, including pluripotent stem cells, the chimeric antigen receptor (CAR)-modification method and strategy for these NK cells, and the current and planned clinical trials of CAR-modified induced pluripotent stem cell-derived NK cells.

Biomaterials in Chimeric Antigen Receptor T-Cell Process Development.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the cancer treatment landscape, utilizing ex vivo modified autologous T cells to treat relapsed or refractory B-cell leukemias and lymphomas. However, the therapy's broader impact has been limited, in part, by a complicated, lengthy, and expensive production process. Accordingly, as CAR T-cell therapies are further advanced to treat other cancers, continual innovation in cell manufacturing will be critical to their successful clinical implementation. In this Account, we describe our research efforts using biomaterials to improve the three fundamental steps in CAR T-cell manufacturing: (1) isolation, (2) activation, and (3) genetic modification.Recognizing that clinical T-cell isolation reagents have high cost and supply constraints, we developed a synthetic DNA aptamer and complementary reversal agent technology that isolates label-free CD8+ T cells with high purity and yield from peripheral blood mononuclear cells. Encouragingly, CAR T cells manufactured from both antibody- and aptamer-isolated T cells were comparable in therapeutic potency. Discovery and design of other T-cell specific aptamers and corresponding reversal reagents could fully realize the potential of this approach, enabling inexpensive isolation of multiple distinct T-cell populations in a single isolation step.Current ex vivo T-cell activation materials do not accurately mimic in situ T-cell activation by antigen presenting cells (APCs). They cause unequal CD4+ and CD8+ T-cell expansion, necessitating separate production of CD4+ and CD8+ CAR T cells for therapies that call for balanced infusion compositions. To address these shortcomings, we designed a panel of biodegradable cell-templated silica microparticles with supported lipid bilayers that display stimulatory ligands for T-cell activation. High membrane fluidity, elongated shape, and rough surface topography, all properties of endogenous APCs, were found to be favorable parameters for activation, promoting unbiased and efficient CD4/CD8 T-cell expansion while not terminally differentiating the cells.Viral and electroporation-based gene delivery systems have various drawbacks. Viral vectors are expensive and have limited cargo sizes, whereas electroporation is highly cytotoxic. Thus, low-cost nonviral platforms that transfect T cells with low cytotoxicity and high efficiency are needed for CAR gene delivery. Our group thus synthesized a panel of cationic polymers with different architectures and evaluated their T-cell transfection ability. We identified a comb-shaped polymer formulation that transfected primary T cells with low cytotoxicity, although transfection efficiency was low compared to conventional methods. Analysis of intracellular and extracellular barriers to transfection revealed low uptake of polyplexes and high endosomal pH in T cells, alluding to biological and polymer properties that could be further improved.These innovations represent just a few recent developments in the biomaterials field for addressing CAR T-cell production needs. Together, these technologies and their future advancement will pave the way for economical and straightforward CAR T-cell manufacturing.

Targeting the tumor microenvironment and T cell metabolism for effective cancer immunotherapy.

The successful implementation of immunotherapies has provided new impetus in the fight against cancer. Antibody-mediated blockade of immune checkpoint molecules PD-1/PD-L1 and CTLA-4 has had a dramatic impact upon the treatment of previously intractable cancers such as malignant melanoma, while adoptive cell therapies using chimeric antigen receptor-bearing T cells have proven highly efficacious in B cell leukemia. Furthermore, significant progress has been made in understanding the mechanisms by which tumors evade or become resistant to these immunotherapies. In this regard, approaches to broaden the applicability and enhance the efficacy of immunotherapies increasingly include modulation of tumor and immune cell metabolism. In this mini-review, we highlight the most recent studies describing novel approaches and targets for the manipulation of the tumor microenvironment and T cell metabolism and describe how these approaches are being combined with current immunotherapies in preclinical studies.

Challenges of driving CD30-directed CAR-T cells to the clinic.

Chimeric antigen receptor T (CAR-T) cells are a promising new treatment for patients with relapsed or refractory hematologic malignancies, including lymphoma. Given the success of CAR-T cells directed against CD19, new targets are being developed and tested, since not all lymphomas express CD19. CD30 is promising target as it is universally expressed in virtually all classical Hodgkin lymphomas, anaplastic large cell lymphomas, and in a proportion of other lymphoma types, including cutaneous T cell lymphomas and diffuse large B cell lymphomas. Preclinical studies with CD30-directed CAR-T cells support the feasibility of this approach. Recently, two clinical trials of CD30-directed CAR-T cells in relapsed/refractory CD30+ lymphomas, including Hodgkin lymphoma, have been reported with minimal toxicities noted and preliminary efficacy seen in a proportion of patients. However, improving the persistence and expansion of CAR-T cells is key to further enhancing the efficacy of this treatment approach. Future directions include optimizing the lymphodepletion regimen, enhancing migration to the tumor site, and combination with other immune regulators. Several ongoing and upcoming clinical trials of CD30-directed CAR-T cells are expected to further enhance this approach to treat patients with relapsed and refractory CD30+ lymphomas.

Preclinical Evaluation of Chimeric Antigen Receptor-Modified T Cells Specific to Epithelial Cell Adhesion Molecule for Treating Colorectal Cancer.

Chimeric antigen receptor-modified T cells (CAR-T cells) have emerged as a promising cancer immunotherapy for solid tumors. Epithelial cell adhesion molecule (EpCAM) is overexpressed in a variety of tumors and is recognized as a biomarker for circulating tumor cells and cancer stem cells, representing an attractive target for adoptive T-cell immunotherapy. This study generated third-generation CAR-T cells with redirected specificity to EpCAM (EpCAM CAR-T) by lentiviral vector. The study demonstrated that EpCAM CAR-T cells can elicit lytic cytotoxicity to target cells in an EpCAM-dependent manner and secrete cytotoxic cytokines, including interferon gamma and tumor necrosis factor alpha. Furthermore, adoptive transfer of EpCAM CAR-T cells significantly delayed tumor growth and formation in xenograft models. In addition, the safety evaluation showed that CAR-T cells have no systemic toxicity in mice. The data confirmed the antitumor ability and safety of CAR-T cells targeting EpCAM and may provide a new target for CAR-T cell therapies in treating solid tumors.

NKT cells - New players in CAR cell immunotherapy?

Low levels of peripheral blood natural killer T (NKT) cells in cancer patients and a favorable outcome associated with a high number of tumor-infiltrating NKT cells demonstrated in several studies indicated the important role of these immune cells in the antitumor response. With effective antitumor immunity via direct tumor lysis, cytokine modulation of effector cells and regulation of immunosuppressive cells, type I NKT cells display interesting features/properties for the rapidly developing chimeric antigen receptor (CAR) technology. Due to their restriction to the monomorphic HLA-like molecule CD1d, but not to the polymorphic human leukocyte antigen (HLA), NKT CAR cells show potential for enabling autologous and allogeneic/off-the-shelf cancer immunotherapy. Promising results were obtained in preclinical NKT CAR cell studies, but clinical trials have not yet been conducted. In this review, we summarize the biological features of NKT cells, their role in antitumor immunity and recent advances in the development of NKT CAR cells.

Chimeric Antigen Receptor T Cells: Extending Translation from Liquid to Solid Tumors.

Successful translation of chimeric antigen receptor (CAR) T cells designed to target and eradicate CD19+ lymphomas has emboldened scientists and physicians worldwide to explore the possibility of applying CAR T-cell technology to other tumor entities, including solid tumors. Next-generation strategies such as fourth-generation CARs (CAR T cells redirected for universal cytokine killing, also known as TRUCKs) designed to deliver immunomodulatory cytokines to the tumor microenvironment, dual CAR designs to improve tumor control, inclusion of suicide genes as safety switches, and precision genome editing are currently being investigated. One major ongoing goal is to determine how best to generate CAR T cells that modulate the tumor microenvironment, overcome tumor survival mechanisms, and thus allow broader applicability as universal allogeneic T-cell therapeutics. Development of state-of-the-art and beyond viral vector systems to deliver designer CARs coupled with targeted genome editing is expected to generate more effective off-the-shelf CAR T cells with activity against a greater number of cancer types and importantly solid tumors.

Anti-CD123 chimeric antigen receptor T-cells (CART): an evolving treatment strategy for hematological malignancies, and a potential ace-in-the-hole against antigen-negative relapse.

Chimeric antigen receptor-modified T-cells (CART) are a potent and targeted immunotherapy which have induced remissions in some patients with chemotherapy refractory or relapsed (RR) hematologic malignancies. Hundreds of patients have now been treated worldwide with anti-CD19 CART cells, with complete response rates of up to 90%. CART therapy has a unique toxicity profile, and unfortunately not all responses are durable. Treatment failure occurs via two main routes - by loss of the CART cell population, or relapse with antigen loss. Emerging data indicate that targeting an alternative antigen instead of, or as well as CD19, could improve CART cell efficacy and reduce antigen-negative relapse. Other strategies include the addition of other immune-based therapies. This review explores the rationale, pre-clinical data and currently investigative strategies underway for CART therapy targeting the myeloid and lymphoid stem/progenitor antigen CD123.