Mammalian Synthetic Biology Flashcards
Why blood cells?
- Blood cells do not stay in the body for a long time. There is less tumorigenicity compared to other cells.
- Blood cells are easy to transfuse.
- Blood cells are easy to culture in vitro.
- Blood transfusion is a standard clinical procedure.
CAR-T therapy
It involves ex vivo engineering of the patient’s autologous T cells to equip them with receptors targeting specific antigens on cancer cells and subsequently infusing these genetically modified T cells back into patients to bring about cancer-directed cytotoxicity.
The patient will need to be counselled on the various steps:
1. The process of leukapheresis to obtain the T cells from the patient for manufacturing of CAR T cells
2. The possibility of receiving bridging therapy while waiting for the CAR T cells to arrive – this could comprise more immunochemotherapy, radiotherapy or a combination
3. The admission for lymphodepletion chemotherapy before the infusion of CAR T-cell therapy and the toxicities
4. The discharge planning and follow-up
CAR-T extracellular structure
- The extracellular portion of the chimeric antigen receptor (CAR) molecule is typically generated from a monoclonal antibody against the target.
- The variable heavy (VH) and variable light (VL) chains, also known as the single-chain variable fragment (scFv), from the antibody sequence are connected by a linker to form the antigen-specific region of the CAR molecule.
- The hinge or spacer region anchors the scFv to the transmembrane region that traverses the cell membrane. Intracellularly, the co-stimulatory domain and CD3ζ chain signal once the scFv portion of the CAR recognizes and binds tumour antigen.
CAR-T intracellular structure
- Co-stimulatory signals are dependent on the co-stimulation domain used: CD28 is dependent on PI3K, whereas 4-1BB requires tumour necrosis factor (TNF), receptor-associated factors (TRAFs), and nuclear factor-κB (NF-κB).
- The CD3ζ chain contains three immunoreceptor tyrosine-based activation motif (ITAM) domains that, upon phosphorylation (P), signal through ζ-associated protein of 70 kDa (ZAP70).
- Downstream signalling leads to T cell effector functions including release of perforin and granzyme, leading to cell death of the target tumour cell. IL-2, interleukin 2.
CAR-T action
- Upon recognition of tumour antigen, the antitumour response activated downstream in chimeric antigen receptor (CAR) T cells leads to activation of innate immune cells owing to secretion of inflammatory cytokines such as granulocyte–macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF) and interferon-γ (IFNγ).
- This leads to a self-amplifying inflammatory activation loop in macrophages causing release of interleukin-1 (IL-1) and IL-6. Therapeutic intervention at various stages of this response can mitigate neurotoxicity and cytokine release syndrome (CRS).
- Therapeutics targeting GM-CSF (lenzilumab), the IL-6 receptor (tocilizumab) and the IL-1 receptor (anakinra) have been used for this purpose clinically. The tyrosine kinase inhibitor dasatinib affects T cell signalling to reduce CRS, and metyrosine inhibits macrophage inflammatory activation to achieve a similar effect.
CAR-NK
- Natural killer (NK) cells have been pursued as the basis for the development of allogeneic products owing to their intrinsic antitumour activity.
- A novel method involves deriving NK cells from cord blood and transducing them with an anti-CD19 chimeric antigen receptor (CAR) vector that has been engineered to ectopically producing interleukin-15 (IL-15) to increase expansion and efficacy. These cord blood CAR NK cells are cytotoxic in vitro and have persistence and antitumour effects in vivo.
- Attempts to make CAR cells from haematopoietic stem cells have shown that CAR expression during early lymphoid development suppresses BCL11B, thereby suppressing T cell-associated genes. As a result, the CAR cells have NK cell-like properties, including NK cell receptor expression. These cells, termed CAR-induced killer (CARiK) cells, require a second-generation CAR design with co-stimulation to have strong anti-leukaemic effects.
future CAR
- Klichinsky et al. recently published a description of the first CAR macrophage (CAR-M), which demonstrated antigen-specific phagocytosis and pro-inflammatory M1 polarization in vitro.
- CARs are also being used to redirect immunosuppressive CD4+CD25+ regulatory T cells (Treg cells) as potential therapeutics in autoimmune disease and organ transplant. CTLA4, cytotoxic T lymphocyteassociated antigen 4; IFNγ, interferon-γ; TGFβ, transforming growth factor-β; TNF, tumour necrosis factor.
Increasing blood supply
- Erythropoietin is required for production of RBC, hematopoiesis
- enucleation required for RBC production
HSPCs
Pro:
1. Generated RBCs are same as normal red cells.
2. The red cells stay longer in the circulation as they are fresh.
3. CD34+ cells from one person are enough to generate one unit of blood.
Con:
1. Expensive (US 5,000 per unit).
2. The stem cell source is limited.
3. The risk of contamination.
ESCs, iPSCs
Pro:
1. the source of cells is unlimited
2. the risk of contamination is low.
Con:
1. Expensive(US$10,000 per unit)
2. Slow
3. Very few cells undergo enucleation
immortalised cell lines
Pro:
1. the source of cells is unlimited
2. the risk of contamination is low.
3. Cheaper
Con:
1. Slow
2. Retics
3. enucleation rate is low.
RBC applications
RBCs are attractive drug delivery cargoes
1. The lack of any genetic material
2. A long lifespan (~120 days in humans)
3. Accessibility to both macro- and micro-circulation
4. A large cell surface and volume
5. RBC transfusion is a standard clinical
procedure
- Genetically engineered RBC- specific transmembrane protein
- Genetically engineered RBCs can be labelled with functional antibodies
- Genetically engineered RBCs have a long half-life in the circulation
- broad immune stimulation
- immune modulation(boast host immunity)
