Difficulty: Advanced
Prerequisites: Parts 1 and 2 of these notes. Familiarity with T cell and NK cell biology, MHC restriction, antibody variable regions (scFv), retroviral/lentiviral gene transfer, and basic concepts of gene editing (zinc finger nucleases, shRNA).
Where Parts 1 and 2 focused on engineered proteins, this section covers engineered cells. The central idea is that immune cells can be taken from a patient, modified ex vivo, and returned to the patient to provide long-lasting, sometimes curative, therapy. Cell surface engineering attaches molecules or nanoparticles to immune cell membranes. HSC gene therapy corrects genetic defects in hematopoietic stem cells to cure primary immunodeficiencies or confer HIV resistance. Adoptive immunotherapy redirects the killing machinery of T cells or NK cells toward tumour targets using engineered receptors (TCRs or CARs). This is where synthetic immunology moves from treating symptoms to potentially curing disease.
Immune cells can be engineered at the surface (attaching cytokine-loaded nanoparticles or co-stimulatory molecules), at the genome level (HSC gene therapy via gene addition, correction, or knockdown), or at the receptor level (adoptive immunotherapy with engineered TCRs or CARs). CAR T cell therapy has shown dramatic results in blood cancers but faces challenges in solid tumours, cost, persistence, and serious adverse effects like cytokine release syndrome.
Cell surface engineering
Strategies for attaching molecules or particles to the surface of immune cells to modulate their function or use them as delivery vehicles. Includes covalent linkage of nanoparticles carrying cytokines and attachment of co-stimulatory molecules via palmitoylated protein A.
Think of it as: customising the outside of an immune cell the way you might add accessories to a vehicle.
Hematopoietic stem cell (HSC) gene therapy
Ex vivo modification of a patient's own HSCs (isolated from bone marrow), followed by reinfusion. The modified HSCs reconstitute the dysfunctional cell lineage. Three approaches: gene addition, gene correction, gene knockdown.
In simple terms: fixing the factory (the stem cell) so every immune cell it makes from that point is functional.
Gene addition
Introduction of a wild-type gene into HSCs using retroviral or lentiviral vectors that integrate into the host genome. Used to cure primary immunodeficiencies (e.g. X-linked SCID, Wiskott-Aldrich syndrome, beta-thalassemia).
Gene correction
Using synthetic nucleases (zinc finger nucleases, TALENs, homing meganucleases) to introduce targeted double-strand breaks in the genome, enabling gene disruption, reconstitution, or editing. Example: mutating CCR5 to confer HIV resistance.
Gene knockdown
Using short hairpin RNAs (shRNAs) delivered by retroviral vectors to silence specific genes. The shRNA is processed by Dicer and the RISC complex to degrade target mRNA. Example: downregulating CCR5 to prevent HIV entry.
Zinc finger nucleases (ZFNs)
Synthetic proteins containing a nuclease domain and an engineered DNA-binding domain (zinc finger motifs). Used to introduce targeted double-strand breaks at specific genomic sites. The break is repaired by non-homologous end joining (NHEJ), often resulting in disruptive mutations.
Adoptive immunotherapy
Isolating immune cells from a patient, expanding and engineering them ex vivo to redirect their effector function toward a target (usually a tumour), then reinfusing them. The engineered cells traffic to disease sites, expand, and persist.
Chimeric antigen receptor (CAR) T cells
T cells transduced with a synthetic receptor (CAR) that combines an scFv (for antigen recognition) with transmembrane and intracellular signalling domains (CD3-zeta, plus co-stimulatory domains like CD28, 4-1BB, OX40 in later generations). CARs recognise non-MHC-restricted surface antigens, unlike TCRs.
In simple terms: a T cell given a custom-built antenna that lets it lock onto a tumour cell without needing MHC presentation.
AND-gate CAR
A logic-gated CAR system requiring two antigens to be present on the same cell for T cell activation. A truncated CAR (no signalling) binds antigen A with high affinity; a functional CAR binds antigen B with low affinity. Only when both antigens are present does signalling occur. Increases specificity, reduces off-target toxicity.
Inhibitory CAR (iCAR)
A CAR with the signalling domain of an immunoinhibitory receptor (CTLA-4 or PD-1) instead of an activating one. If the target cell expresses a "do not kill" antigen, the iCAR blocks activation. Enables discrimination between tumour and normal tissue.
Cytokine release syndrome
When lymphocytes targeted for destruction by therapeutic antibodies or adoptive immunotherapy are activated prior to apoptosis, they release cytokines en masse. This causes a systemic inflammatory response with symptoms resembling severe infection.
Think of it as: the immune cells firing one last volley of alarm signals before they die, overwhelming the body.
Tumour lysis syndrome
When tumour cells attacked by lymphocytes break down rapidly, releasing their intracellular contents into the blood. Breakdown products can accumulate to levels that cause renal failure.
Goal: attach molecules or particles to immune cell surfaces to modulate function or use cells as delivery vehicles
Cytokine nanoparticle approach: autocrine factors (IL-15, IL-21) packaged into lipid nanoparticles, tethered to CD8+ T cell surfaces by covalent linkage. Low doses that had no effect systemically achieved massive in vivo T cell expansion and enhanced melanoma killing. Same approach used to enhance HSC engraftment via local release of glycogen synthase kinase-3-beta inhibitor.
Co-stimulatory molecule approach: palmitoylated protein A added to T cell surface, then incubated with B7 fused to Fc of IgG1. This ectopic B7 expression on CD8+ T cells enhanced anti-tumour potency in vivo.
Macrophages as delivery vehicles: loaded with nanocarriers containing antiviral drugs, exploiting macrophages' inherent ability to migrate to hypoxic, necrotic, and inflamed tissues. Reduced HIV-infected cell counts in humanized mice.
Immune cells as viral carriers: T cells and DCs used to deliver oncolytic viruses to tumours. T cells carry viruses nonspecifically on their surface; DCs phagocytose viruses, protecting them from neutralising antibodies.
Challenges: constitutive membrane turnover and internalisation reduce expression; risk of interfering with membrane fluidity or cellular functions.
HSCs isolated from patient bone marrow, transduced with retroviral/lentiviral particles, reinfused. Can cure disease rather than merely treat symptoms.
Gene addition
Most commonly attempted approach
First success (2000): autologous transplantation of bone marrow cells carrying wild-type IL-2R-gamma in a mouse Moloney leukemia virus vector cured X-linked SCID. However, 5 of 20 patients developed leukemia due to insertional activation of the LMO2 oncogene.
Safety shift to lentiviral vectors: long terminal repeat (LTR) lacks a robust enhancer (reduced risk of activating neighbouring genes), can transduce non-dividing cells, easily pseudotyped. Self-inactivating lentivirus designs now standard.
Lentiviral successes: cured one patient with beta-thalassemia (beta-globin cDNA into haematopoietic precursors), cured Wiskott-Aldrich syndrome patients (self-inactivating lentivirus expressing WAS from its own promoter).
Gene correction
Uses zinc finger nucleases (ZFNs), homing meganucleases, or TALENs to introduce targeted double-strand breaks
The break is repaired by non-homologous end joining (NHEJ), potentially disrupting, reconstituting, or editing the gene
Key example: mutating CCR5 (the HIV co-receptor) via ZFNs to generate the CCR5-delta-32 allele, rendering T cells resistant to HIV infection
HSCs transduced with CCR5-targeting ZFNs and engrafted into immunocompromised mice showed lower viral titres after HIV challenge
First human trial: autologous ZFN-transduced CD4+ T cells infused into 12 HIV-infected patients. Safe. In one patient, viral load dropped to undetectable levels.
Gene knockdown
shRNAs delivered by retroviral vectors silence target genes
shRNA is processed by Dicer, then the RISC complex uses it to degrade complementary mRNA
Strategy for HIV: transduce autologous CD4+ T cells and CD34+ HSCs with lentiviral vectors encoding CCR5-directed shRNA plus a small-protein HIV fusion inhibitor
Immune cells isolated, expanded, engineered ex vivo to redirect effector function toward a target, then reinfused. Primarily used for cancer.
Engineered T cells: TCR-based approaches
T cells genetically engineered to express high-affinity TCRs for tumour antigens
Delivery: retroviruses (most common), transposon systems, or mRNA electroporation
TCR sources:
Cloned from a cancer patient who responded well to adoptive cell transfer
Isolated from humanized mice primed to recognise tumour antigens
Replaced by a CAR (see below)
CAR T cells
CARs combine scFvs (from antibody variable regions) with transmembrane and intracellular signalling domains (CD3-zeta + co-stimulatory domains like CD28, 4-1BB, OX40)
Key advantage: CARs recognise non-MHC-restricted surface antigens (unlike TCRs, which require MHC presentation of processed intracellular peptides)
CD19-specific CAR T cells produced complete and lasting remission in some leukemia/lymphoma patients, though CD19-negative relapse has been observed
AND-gate CARs: require two antigens on the same cell. Truncated CAR (no signalling) binds antigen A; functional but low-affinity CAR binds antigen B. Only dual expression triggers killing. Increases specificity for tumour cells vs normal tissue.
Inhibitory CARs (iCARs): scFv fused to PD-1 or CTLA-4 signalling domains. If a "do not kill" antigen is present, the iCAR blocks activation by the activating CAR. Reversible discrimination between target and non-target cells.
IL-12 secreting CAR T cells: constitutive autocrine IL-12 secretion enabled tumour eradication without prior chemotherapy conditioning and conferred resistance to regulatory T cell inhibition.
Both CD8+ and CD4+ T cells are useful: CD8+ induces apoptosis directly; CD4+ secretes IL-2 and recruits/sustains tumour-specific CD8+ T cells. Th1 cells produce IFN-gamma; Th17 cells may promote long-lived anti-tumour immunity.
NK cell-based immunotherapy
NK cells primarily in blood, so most successful against haematopoietic malignancies
Autologous NK cells: safe but limited clinical success
Allogeneic NK cells: more successful (metastatic melanoma, renal cell carcinoma, Hodgkin's disease, AML)
Engineered NK cells: cytokine transgenes (IL-2, IL-15, SCF) and CAR expression (CD19, CD20, ErbB2, CD33, CEA, GD2) have shown enhanced killing in vitro and improved anti-tumour activity in vivo
Immunogenicity: anti-drug antibodies may neutralise therapeutics or cause anaphylaxis
Infection risk: dampening inflammation for autoimmune disease also dampens pathogen defence
Tumour antigen selection: cross-reactivity with normal tissues can be fatal
Cytokine release syndrome: lymphocytes release cytokines en masse before dying, causing systemic inflammation
Tumour lysis syndrome: rapid tumour cell death floods the blood with breakdown products, risking renal failure
Antigen escape: tumour cells can lose the targeted antigen and relapse
Cost and complexity: adoptive immunotherapy is extremely expensive and labour-intensive
Molecules or cells that bind multiple antigens with diverse affinities (combining high-affinity effector-cell binding with multiple low-affinity tumour-targeting modules)
Synthetic RNA regulatory systems to control T cell proliferation with small drugs
Pathogen-derived effector molecules engineered into T cells to tune response amplitude
Engineering T cells to differentiate into regulatory T cells
Prosthetic gene networks encapsulated in mammalian cells to sense and respond to pathological signals in vivo
Synthetic bacteria to resolve inflammation or produce therapeutic molecules in the gut
CAR T cell therapy targeting CD19 (e.g. tisagenlecleucel, axicabtagene ciloleucel) has transformed outcomes for patients with relapsed/refractory B cell ALL and certain lymphomas, achieving complete remissions where all other treatments had failed.
The CCR5-delta-32 mutation is the same naturally occurring variant that made the "Berlin patient" (Timothy Ray Brown) resistant to HIV after a bone marrow transplant. ZFN-based gene correction aims to replicate this effect without needing a matched donor.
"CAR T cells and TCR-engineered T cells are the same thing." They are not. TCR-engineered T cells express a modified T cell receptor that recognises intracellular antigens presented on MHC. CAR T cells carry a synthetic receptor built from antibody fragments that recognises surface antigens without MHC restriction. This is a fundamental distinction.
"Gene therapy always uses gene addition." Three approaches exist: addition (introducing a new gene), correction (editing an existing gene with nucleases), and knockdown (silencing a gene with shRNA). The choice depends on the disease.
"Adoptive immunotherapy is a one-shot cure." While it has the potential for long-lasting effects (immune cells can persist and expand), challenges include limited in vivo persistence, antigen escape, the immunosuppressive tumour microenvironment, and serious adverse effects.
"Only T cells can be used in adoptive immunotherapy." NK cells, macrophages, and dendritic cells are also being engineered for therapeutic use.
Be able to distinguish the three HSC gene therapy approaches (addition, correction, knockdown) and give one clinical example of each.
Understand the structure and function of a CAR: scFv + transmembrane domain + CD3-zeta + co-stimulatory domains. Know what makes third-generation CARs different (multiple co-stimulatory domains).
AND-gate CARs and inhibitory CARs are commonly tested as examples of how synthetic biology logic is applied to improve specificity.
Know the difference between CAR-based and TCR-based recognition: MHC-restricted (TCR) vs non-MHC-restricted surface antigen (CAR).
Cytokine release syndrome and tumour lysis syndrome are key adverse effects to know by name and mechanism.
The CCR5-delta-32 / HIV example is a flagship case for gene correction and is likely to appear in exam questions.
True or False: CARs recognise antigens presented on MHC molecules. (False. CARs recognise non-MHC-restricted surface antigens directly. It is TCRs that require MHC presentation.)
Fill in the blank: In an AND-gate CAR system, T cell activation requires ______ antigens to be present on the same target cell. (two)
True or False: The first successful HSC gene therapy for X-linked SCID had no adverse effects. (False. Five of 20 patients developed leukemia due to insertional activation of the LMO2 oncogene.)
Fill in the blank: Cytokine release syndrome occurs when lymphocytes release ______ en masse before dying. (cytokines)
True or False: Allogeneic NK cells have shown better clinical results in cancer than autologous NK cells. (True.)
Q: Explain how a CAR differs structurally and functionally from a natural TCR, and state one advantage of each.
A: A CAR is a synthetic receptor composed of an scFv (antibody-derived antigen-binding fragment) fused to a transmembrane domain and intracellular signalling domains (CD3-zeta plus co-stimulatory domains). It recognises intact surface antigens without MHC restriction. A TCR is a natural heterodimer (alpha/beta chains) that recognises processed peptide antigens presented on MHC molecules. Advantage of CAR: MHC-independent recognition, so tumour cells cannot escape by downregulating MHC. Advantage of TCR: can recognise intracellular antigens (after processing and MHC presentation), accessing a much wider range of tumour-associated targets.
Q: Describe the three approaches to autologous HSC gene therapy and give one clinical example of each.
A: (1) Gene addition: introducing a wild-type gene via viral vectors. Example: introduction of IL-2R-gamma to cure X-linked SCID. (2) Gene correction: using zinc finger nucleases or similar tools to create targeted mutations. Example: disrupting CCR5 to confer HIV resistance. (3) Gene knockdown: using shRNAs to silence a target gene. Example: downregulating CCR5 expression in T cells and HSCs to treat HIV.
Q: What is the AND-gate CAR strategy, and why was it developed?
A: The AND-gate uses two CARs on the same T cell. A truncated CAR (no signalling capacity) binds antigen A with high affinity; a fully functional but low-affinity CAR binds antigen B. Only when both antigens are present on the same target cell does CAR-induced signalling occur. It was developed to increase the target-cell specificity of CAR T cells, reducing off-target toxicity against normal tissues that express only one of the two antigens.
Q: Name and explain two serious adverse effects of adoptive immunotherapy.
A: (1) Cytokine release syndrome: lymphocytes targeted for destruction release cytokines en masse before undergoing apoptosis, causing a systemic inflammatory response resembling severe infection. (2) Tumour lysis syndrome: rapid destruction of tumour cells by lymphocytes releases intracellular breakdown products into the blood, which can accumulate to the point of renal failure.
Q: Why was the focus of HSC gene therapy shifted from mouse Moloney retroviral vectors to lentiviral vectors?
A: Mouse Moloney retroviral vectors carry a strong enhancer in their LTR that can activate neighbouring genes upon integration, as demonstrated by the insertional activation of the LMO2 oncogene (causing leukemia in 5 of 20 SCID patients). Lentiviral vectors have an LTR that lacks this robust enhancer, reducing the risk of oncogene activation. They also transduce non-dividing cells and can be pseudotyped to infect a broad spectrum of cells.
CAR T cell biology connects to Parts 1 and 2 (antibody variable regions, scFv structure, Fc-mediated effector functions) and to basic T cell signalling (CD3-zeta, co-stimulation via CD28 and 4-1BB).
HSC gene therapy connects to molecular biology (retroviral integration, lentiviral vectors, gene editing with ZFNs/TALENs/CRISPR) and haematology (stem cell transplantation, engraftment).
The concept of immune checkpoint modulation (CTLA-4, PD-1) recurs here in inhibitory CARs, linking back to the checkpoint inhibitor antibodies covered in Part 1.
Cell surface engineering, nanoparticle drug delivery, HSC gene therapy, gene addition, gene correction, gene knockdown, zinc finger nucleases, ZFN, TALEN, lentiviral vector, retroviral vector, CCR5, CCR5-delta-32, HIV resistance, adoptive immunotherapy, adoptive cell transfer, CAR T cells, chimeric antigen receptor, scFv, CD3-zeta, CD28, 4-1BB, AND-gate CAR, inhibitory CAR, iCAR, tumour antigen escape, cytokine release syndrome, tumour lysis syndrome, X-linked SCID, Wiskott-Aldrich syndrome, beta-thalassemia, NK cell immunotherapy, sipuleucel-T, dendritic cell therapy, oncolytic virus delivery, synthetic gene circuit, prosthetic gene network