Bispecific antibody activated T cells: A newly developed T cells with enhanced proliferation ability and cytotoxicity.

Adoptive cell therapy using ex vivo expanded lymphocytes has shown remarkable efficacy in tumor immunotherapy recently. Among various transfused immune cells, T lymphocytes are the most widely used since they are critical mediators of the immune system and have the capacity to kill tumor cells. However, there are drawbacks in the expanded T cells for transfusion including limited cytotoxicity, limited proliferation and lack of specificity. To improve the quality of these ex vivo expanded T cells, we have designed a new method to expand a group of T cells which are named bispecific antibodies activated T cells. It is the first time that such cells are induced by introducing the bispecific antibody drug (blinatumomab) and feeder cells (normal B cells and irradiated B cell originated lymphoma cells) to the traditional T cells culture system. Culture of freshly isolated human peripheral blood mononuclear cells in this newly designed cell culture system enabled these expanded T cells that (a) displayed a robust proliferation ability; (b) showed fully activated phenotype and enhanced cytokines production; (c) had a low proportion of CD4+CD25+ T regulatory cells and (d) exhibited strengthened cytotoxicity at relatively low effector: target ratios. This work further confirmed the feasibility of rapid induction and expansion of large amounts of human T cells in vitro by using bispecific antibodies and feeder cells. This strategy could also be used for other immune cells rapid expansion and help to improve the quality of these expanded immune cells for adoptive transfusion.

Natural Killer Cell Expansion with Autologous Feeder Layer and Anti-CD3 Antibody for Immune Cell Therapy of Hepatocellular Carcinoma.

BACKGROUND: one of the promising approaches for treatment of some cancers is adoptive cell therapy using natural killer (NK) cells. Various methods have been investigated for ex vivo expansion of NK cells in large-scale, but most of them involved cancer or genetically modified cells as feeder layer and also some of them have the risk of T cell contamination and graft-versus-host disease (GVHD). METHOD: In this study, irradiated autologous peripheral blood mononuclear cells (PBMCs) as feeder layer with an anti-CD3 monoclonal antibody (mAb) were used. For activation and expansion of NK cells, human recombinant IL2 and IL15 were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC in compared to iNKC were analyzed by CCK-8 assay and degranulation of NK cells after co-culturing was assayed by measuring CD107a expression. Enzyme-Linked Immunosorbent Assay (ELISA) assay was used for the ability of NK cells to secretion of IFN-γ (interferon-γ) and TNF-α (Tumor Necrosis Factor-α) after co-culture with HCC cells. Real Time PCR analysis was used for expression of human Perforin and Granzyme B genes in the NK cells exposed to target HepG2 cells. RESULT: This method strongly expanded highly purified NK cells with powerful cytotoxicity against HCC cells. The expanded NK cells showed high level of expression of degranulation marker and human Perforin and Granzyme B genes, and also was secreted larger amounts of TNF-α and IFN-γ compared with fresh isolated NK cells. CONCLUSION: we proposed an effective method for expansion of cytotoxic NK cells using irradiated autologous PBMC as feeder layer for more successful transfer of allogeneic NK cell in immuno cell therapy of HCC.

Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer.

Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells.

Natural killer (NK) cells can be expanded upon activation by proliferative cytokines (such as IL-2 and IL-15). The NK cell expansion can be greatly enhanced by proteins from feeder cells such as tumor cell lines or PBMCs. Therefore, coculture systems of irradiated feeder cells and NK cells in media containing IL-2 and IL-15 have been developed to generate large numbers of NK cells, although NK cell expansion protocol using anti-CD3 antibody (OKT-3) without feeder cells has also been developed. Commonly used feeder cell lines are RPMI8866, Epstein-Barr lymphoblastoid cell line (EBV-LCL), and K562. Stimulation with NK-sensitive K562 cells is known to augment NK cell proliferation to IL-2, IL-15, and IL-21 in combination.Recently, remarkable NK cell-expansion rates are achieved when genetically engineered (GE) feeder cells are used. Dr. Dario Campana's group found that membrane-bound IL-15 and 4-1BBL, coexpressed by K562 cells, acted synergistically to augment K562-specific NK stimulatory capacity, resulting in vigorous expansion of peripheral blood CD56(+) CD3(-) NK cells without concomitant growth of T lymphocytes. Here, we describe an in vitro expansion method of human NK cells among PBMCs by coculturing with GE_K562 cells.

Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21.

Natural killer (NK) cells have gained significant attention for adoptive immunotherapy of cancer due to their well-documented antitumor function. In order to evaluate the therapeutic efficacy of NK cell adoptive immunotherapy in preclinical models with a potential for clinical translation, there is a need for a reliable platform for ex vivo expansion of NK cells. Numerous methods are reported in literature using cytokines and feeder cells to activate and expand human NK cells, and many of these methods are limited by low-fold expansion, cytokine dependency of expanded NK cells or expansion-related senescence. In this chapter, a robust NK cell expansion protocol is described using K562 cell line gene modified to express membrane bound IL21 (K562 mb.IL21). We had previously demonstrated that this platform enables the highest fold expansion of NK cells reported in the literature to date (>47,000-folds in 21 days), and produces highly activated and pure NK cells without signs of senescence, as determined by telomere shortening.

Large-Scale Culture and Genetic Modification of Human Natural Killer Cells for Cellular Therapy.

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.

Gene Modification of Human Natural Killer Cells Using a Retroviral Vector.

As part of the innate immune system, natural killer (NK) cells are regarded as promising effector cells for adoptive cell therapy approaches to treat patients with cancer. In some cases, genetic modification of the NK cells may be considered but such manipulation has to be integrated into the expansion method to allow the generation of clinically relevant numbers of gene-modified NK cells. Therefore, an efficient gene transfer procedure is needed.Our group developed a retrovirus-based transduction protocol capable of robust expansion of gene-modified NK cells with a high rate of transgene expression. Actively dividing cells is a prerequisite for efficient gene transfer when using a retroviral vector. In the procedure presented here, strong activation of the NK cells was provided by a combination of IL-15 and the K-562 feeder cells. Beside the interest in developing a simple procedure compliant with good manufacturing practice (GMP) for the production of therapeutic products, this approach also provides a valuable means of generating genetically modified primary NK cells for future preclinical studies.

Modification of Expanded NK Cells with Chimeric Antigen Receptor mRNA for Adoptive Cellular Therapy.

NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors and cells infected by viruses. The regulation of NK activation vs inhibition is regulated by the expression of a variety of NK receptors (NKRs) and specific NKRs' ligands expressed on their targets. However, factors limiting NK therapy include small numbers of active NK cells in unexpanded peripheral blood and lack of specific tumor targeting. Chimeric antigen receptors (CAR) usually include a single-chain Fv variable fragment from a monoclonal antibody, a transmembrane hinge region, and a signaling domain such as CD28, CD3-zeta, 4-1BB (CD137), or 2B4 (CD244) endodimers. Redirecting NK cells with a CAR will circumvent the limitations of the lack of NK targeting specificity. This chapter focuses on the methods to expand human NK cells from peripheral blood by co-culturing with feeder cells and to modify the expanded NK cells efficiently with the in vitro transcribed CAR mRNA by electroporation and to test the functionality of the CAR-modified expanded NK cells for use in adoptive cellular immunotherapy.