• 4 structure levels: primary → secondary (α-helix/β-sheet) → tertiary (3D fold) → quaternary (subunits)
• Functions: enzyme, hormone, signal, transport, receptor, immune defense, structural
• Eukaryotic synthesis: transcription → mRNA processing (cap/polyA/splice) → translation → post-translational modification (PTM)

1. Plasmid manufacturing — vector selection + gene insertion
2. Transformation — into host (E. coli / yeast / CHO) + selection
3. Expression — incubation + induction
4. Purification — column binding, elution, size-exclusion chromatography

• Hybridoma tech (1975): fuse antibody-producing B cell + immortal myeloma cell → unlimited mAb
• Structure: IgG = 2 heavy + 2 light chains, Y-shaped, ~150 kDa (2×50 + 2×25)
• CDRs (6 total: 3 heavy + 3 light) = antigen-binding site (paratope) → specificity
• Fc region functions: bind Fc receptors → ADCC/phagocytosis; activate complement (C1q); placental transfer + long half-life via FcRn

• Nanobody — camelid single-domain, 12–15 kDa; great penetration/stability but short half-life (kidney-cleared), no Fc effector function
• Humanization — swap mouse framework for human (reduces anti-drug antibodies)
• Peptibody — peptide + Fc fusion; e.g. Romiplostim (Nplate, thrombocytopenia)
• ADC — antibody + linker + cytotoxic payload; e.g. Mylotarg. Steps: bind → internalize → linker cleave → drug kills cell
• Bispecific — binds two antigens. IgG-like (has Fc, stable) vs non-IgG (scFv, no Fc). Knob-into-hole solves random chain pairing

Proteins are the true gene products — they interact with nearly everything in the body, generating and regulating all kinds of physiological responses, from catalysing reactions to signalling and defending against pathogens. Five reasons the lecture gives:

A functional protein is precisely twisted, folded, and coiled into a molecule of unique shape (tertiary and quaternary structure). That 3D shape — and its ability to change conformation — is what enables the protein to carry out its specific function. Denaturation = loss of shape = loss of function.

Pancrelipase (Creon, Pancreaze) — treats exocrine pancreatic insufficiency by replacing digestive enzymes: lipase, protease, and amylase.

Enzyme as treatment rather than replacement — Asparaginase (Elspar): used in acute lymphoblastic leukaemia (ALL) and some lymphomas (especially paediatric) by breaking down asparagine, which leukaemia cells need to survive. Some asparaginases are PEGylated to improve pharmacokinetics — the same half-life-extension logic as lipidation in L1.

Hormones in the menstrual cycle: oestrogen, progesterone, luteinising hormone, FSH and its receptor. Hormones in IVF: GnRH antagonists + human chorionic gonadotropin (hCG) — note hCG is a 237-amino-acid glycoprotein heterodimer. (This ties directly into the glycosylation material in L3.)

A protein-based vaccine contains specific proteins or peptides derived from a pathogen. The sequence is the exam favourite — follow it in order:

1. APCs (antigen-presenting cells — dendritic cells, macrophages) recognise the foreign proteins.
2. APCs engulf the proteins, process them, and display fragments on their surface using MHC molecules.
3. APCs migrate to nearby lymph nodes and present fragments to naive T cells.
4. Helper T cells that recognise the fragments become activated and proliferate.
5. Activated helper T cells secrete cytokines that stimulate B cells specifically.
6. Stimulated B cells differentiate into plasma cells, which produce antibodies specific to the vaccine proteins.
7. Some activated B cells become memory B cells for rapid future response.
8. Helper T cells also promote cytotoxic T cells, which directly kill infected cells displaying the pathogen's proteins.
9. Circulating antibodies neutralise the pathogen on future exposure; memory B and T cells mount a rapid, strong, specific response → long-lasting protection.

• Nomenclature: monoclonal antibody names often end in "-mab"; "-tu" denotes tumours. (e.g. rituximab, cetuximab.)
• Limitations: development and production can be complex and expensive; use may be associated with side effects such as infusion reactions and immunogenicity.
• Applications beyond therapy: laboratory research (protein function, cell signalling, disease mechanisms; antibody-based assays and imaging); diagnostics (ELISA, immunohistochemistry, flow cytometry); therapeutics (cancer, autoimmune disorders, infectious diseases).

• Disulfide bonds are cleaved by reducing agents — DTT, TCEP, mercaptoethanol → generates two bands on SDS-PAGE (heavy chain and light chain).
• Estimated size: 50×2 + 25×2 = 150 kDa. This is the experimental proof behind the 150 kDa figure.
• V = variable region · H = heavy chain · L = light chain · C = constant region.
• CDRs (CDR1, CDR2, CDR3 in each variable region) → 6 per antibody. They form the paratope, which interacts directly with the antigen's epitope. CDR sequence variability is what allows antibodies to recognise a vast array of antigens.

Inhibitory effect (3 routes): (1) receptor blocking / signal interference — prevents natural ligands (growth factors) binding → inhibits proliferation/survival signalling; (2) neutralisation of ligands — binds circulating cytokines/growth factors directly; (3) inducing receptor downregulation — causes internalisation and degradation of receptors, reducing cell-surface receptor number.

• Diagnostics — ELISA, lateral flow assays (e.g. pregnancy tests), immunohistochemistry.
• Research tools — Western blotting, flow cytometry, immunoprecipitation.
• Biosensors — detecting pathogens, toxins, or analytes in environmental and clinical samples.

Single-domain antibody fragments from camelids (llamas, camels), retaining full-size binding ability at a fraction of the size: ~12–15 kDa vs 150 kDa.

Therapeutic antibodies are initially developed in animals (commonly mice) because they generate strong immune responses to human antigens — but animal antibodies are recognised as foreign, producing anti-antibody antibodies that reduce effectiveness and cause reactions. Humanisation replaces the non-human parts with human sequences while retaining target binding.

The peptide provides the biological activity; the Fc region provides stability and prolonged circulation. Example: Romiplostim (Nplate) — mimics thrombopoietin to stimulate platelet production in thrombocytopenia.

ADCs are targeted cancer therapies combining an antibody with a cytotoxic drug. Three components, each with a purpose:

1. Target binding — the antibody binds an antigen on the cancer cell surface.
2. Internalisation — the ADC-antigen complex is taken in by endocytosis.
3. Linker cleavage — inside the cell, the linker is cleaved, releasing the drug.
4. Cytotoxic action — the drug interferes with critical cellular processes (e.g. DNA replication).
5. Selective killing — targeted delivery minimises damage to healthy cells.
6. Cell death — accumulation of drug induces apoptosis or other forms of cell death.

Why bispecifics are so hard to make: structural similarity of the chains, random assembly, lack of a natural pairing mechanism, and complex engineering requirements. The fix: knob-into-hole mutations to force correct heavy-chain pairing.

Bioconjugation for protein modification. The reasoning starts from cyclic peptides, which offer: high stability against enzymatic degradation and a constrained conformation for target binding. Combining that with an antibody gives a peptidic bispecific antibody (pBsAb) with reduced immunogenicity.

Foundational work: Wong, C. T. T.; Rowlands, D. K.; Wong, C. H.; Li, H. Y.; Tam, J. P. Angew. Chem. Int. Ed. 2012, 124, 5718.

How it was verified (the experimental logic — useful for assignment questions):

• SDS-PAGE and LC-MS of the conjugation reaction under different conditions; stability experiment 3 days after conjugation.
• ELISA binding experiments of mAb and pBsAb against EGFR and SIRP-alpha.
• Confocal imaging and flow cytometry for selective binding against EGFR-overexpressing cells and macrophages.
• Macrophage-engaging assay: (1) seed cancer cells to fully cover the 96-well plate bottom → (2) add fluorescent-labelled macrophages (RAW264.7) with PBS, mAb, or pBsAb → (3) allow interaction for 10 min → (4) invert and wash the plates → (5) image plate bottoms under fluorescence microscope → (6) count macrophages.
• 3D tumour model: confocal Z-stack images of HT29 3D spheroids — testing spheroid infiltration.
• Blockage of the "don't eat me" signal: ELISA of the CD47–SIRP-α binding assay — blocking CD47 (the "don't eat me" signal) lets macrophages phagocytose the tumour cell. Plus serum stability testing.

The card's enzyme-replacement row lists Creon (pancrelipase) for "exocrine pancreatic insufficiency" but omits the specific disease the lecture ties it to. The source states:

The card's "Protein drug classes" table does not include a monoclonal-antibody row with a specific indication, and rituximab only appears as a nomenclature example. The source gives it an explicit therapeutic use:

The source (slide on mAb uses) shows Cetuximab as a highlighted therapeutic monoclonal antibody. The card mentions cetuximab only in passing under nomenclature. Its significance here is that it is an anti-EGFR antibody — the same target Dr. Wong's pBsAb later engages, making it the commercial precursor that frames the lecture's own bispecific work.

The card frames mAbs around specificity and unlimited supply but does not state the lecture's three-pronged advantage wording. The source is explicit:

• High specificity — bind a single epitope.
• Consistency — uniform product, unlike polyclonal mixes.
• Unlimited production — hybridoma / recombinant lines supply inexhaustible, identical antibody.

1. Immunise a mouse with the target antigen (lecture example: HER2) → spleen fills with specific B cells
2. Harvest spleen B cells; mix with murine myeloma cells
3. Fuse with PEG (polyethylene glycol) or electrofusion
4. Select in HAT medium → only the fused hybridoma survives
5. Limit dilution (≈1 cell per well) → screen for the clone secreting only the wanted antibody

Papain/pepsin are proteases — they cut the backbone, splitting the molecule into domains; DTT/TCEP are reductants — they break disulfides, splitting it into chains.

1. Fab binds target antigen (lecture example: rituximab → CD20 on a B-lymphoma cell)
2. The bound antibody presents an exposed Fc
3. The NK cell recognises it via FcγRIIIa (CD16) and activates
4. It releases perforin + granzymes; perforin punches pores in the target membrane
5. Granzymes enter → caspase cascade → target apoptosis

1. Sequence the murine VH and VL genes from the hybridoma antibody
2. Identify the CDRs by Kabat or Chothia numbering → 3 heavy + 3 light
3. Pick the human germline framework with highest homology to the murine variable region
4. Graft the six murine CDRs into the corresponding positions
5. Back-mutation — restore framework residues to the murine sequence where they support CDR conformation (the Vernier zone)
6. Express and verify; affinity usually drops → iterative optimisation

Why humanised beats chimeric: fewer foreign sequences → less anti-drug antibody response → less neutralisation → better sustained efficacy, which is what makes long-term dosing of chronic disease possible.

• Nanobody source: camelids (camels, llamas, alpacas; the lecture also names shark) make heavy-chain-only antibodies (HCAb) with no light chains — binding sits in one heavy-chain variable domain, the VHH, at 12–15 kDa, about one-tenth of an IgG
• Advantages: tiny → deep penetration into solid tumours and across the blood–brain barrier; very stable (tolerates 60–80 °C, organic solvents); cheap to make in E. coli or yeast; reaches hidden epitopes such as a crevice in an enzyme active site
• Disadvantages: short half-life (hours; kidney-cleared); no Fc → no ADCC/CDC; camelid origin → immunogenicity risk
• Half-life rescue — two fixes: PEGylation of the VHH (raises hydrodynamic radius → slower renal clearance), or fusion to a human Fc (keeps the small targeting domain, gains FcRn recycling and ADCC)
• Peptibody = therapeutically active peptide + human IgG1 Fc — peptide supplies the activity, Fc the stability and prolonged circulation. The linker (lecture example (GGGGS)n) must let the peptide fold and reach its target without masking the Fc's own receptor binding; bioactivity testing must validate both halves

Exam distinction: mAb versus fusion protein — same pathways, different molecular classes. Etanercept is the designated fusion protein; adalimumab and infliximab are the monoclonal antibodies. Romiplostim's disease is ITP, not anaemia.

• Payload criterion: extreme potency — IC50 in the picomolar range — with a defined mechanism
• Bystander effect: the neutral, lipophilic MMAE diffuses out of the antigen-positive cell and kills neighbouring antigen-negative cancer cells — how an ADC covers tumour heterogeneity. The charged DM1 cannot cross the membrane, so no bystander effect
• DAR (drug-to-antibody ratio) = payload molecules per antibody. Enhertu (DS-8201) is the high-DAR case (≈8), cleavable linker, strong bystander effect, used to overcome T-DM1 (Kadcyla) resistance — Kadcyla being the non-cleavable, no-bystander design. Mylotarg, the first ADC (CD33, calicheamicin), was withdrawn for toxicity then re-approved at a lower dose

• Supply is throttled by manufacturing difficulty. Only 9 bispecifics approved against over 200 in trials — chain pairing is the bottleneck. Each fix is extra process development, purification and analytics, and scarce technical capability allows pricing at this level
• Columvi's figure is a course price, not an open-ended monthly bill — a fixed-duration treatment completed in about 8.5 months, designed to finish. Framing US$350,000 as a finite one-off course is what makes it defensible, which is why the lecture pairs the figure with the duration
• The third contrast: non-IgG formats such as blinatumomab have no Fc and no FcRn recycling — half-life collapses to hours, so administration becomes continuous intravenous infusion (a portable pump, about four weeks). A delivery-cost model, not a manufacturing-cost model

• Signal peptide: proteins are made with a signal peptide removed by proteolytic cleavage as the protein is routed to its destination — the same proteolytic-cleavage PTM the lecture lists (proinsulin → insulin). Recombinant production exploits this by engineering a signal-peptide sequence into the plasmid so the product is secreted rather than trapped in the cell
• Th1 vs Th2: once the naive CD4+ T cell's TCR engages the MHC class II–peptide complex and CD28 binds CD80/CD86 for the second signal, the cell activates, proliferates and polarises into a Th1 or Th2 helper — the choice sets the character of the downstream response
• Why boosters work: on first immunisation the naive B and T cell pool is small, so the titre is low and mainly low-affinity IgM. In the germinal centre, B cells undergo somatic hypermutation and class switching IgM → IgG (enzyme AID), then differentiate into plasma cells and long-lived memory B cells. A booster re-stimulates the memory B cells, which rapidly become plasma cells making large amounts of high-affinity IgG
• Key molecules: BCR · CD40L–CD40 · IL-4 / IL-21 · AID · CCR7 / CCL19 / CCL21 (DC migration) · CD80 / CD86 with CD28 · Alum adjuvant (adsorbs antigen for slow release, stimulates APCs)

• E. coli (prokaryote) — no glycosylation; used for simple proteins such as insulin and growth hormone
• Pichia pastoris (yeast) — simple glycosylation, high yield; also the system behind recombinant hepatitis B surface antigen
• CHO / HEK293 (mammalian) — complex, human-like glycosylation; the standard platform for monoclonal antibodies, because Fc glycosylation decides effector function and half-life
• Sf9 (insect cells) — used for virus-like particle (VLP) vaccines
• Plasmid elements: target gene as cDNA (no introns — bacteria cannot splice); promoter (T7 prokaryotic, CMV mammalian); selection marker (antibiotic resistance, e.g. ampicillin); signal peptide sequence for secretion; optional tag (His-tag for nickel columns, Fc-tag for protein A)
• Transformation: plasmid into the host by heat shock or electroporation
• Expression scale: shake flasks small; stirred-tank fermenters and single-use wave reactors large
• Induction: add an inducer — IPTG for E. coli; tetracycline in some eukaryotic systems
• Process control: temperature, pH, dissolved oxygen, nutrient feed. The feed defines fed-batch — nutrients added progressively through the run rather than charged once at the start, raising cell density and titre
• Purification: affinity chromatography (protein A to the Fc) first — > 95% purity in one step; then ion exchange (IEX, net charge), size exclusion (SEC, removes aggregates) and hydrophobic interaction (HIC, polishing)

• Primary: linear sequence, N- to C-terminus. Anfinsen's principle — the folding information resides in this sequence; the primary structure determines every level above it
• Secondary: local backbone arrangement only. α-helix — right-handed, 3.6 residues per turn, H-bond from the carbonyl oxygen at i to the amide hydrogen at i+4; β-sheet — strands parallel or antiparallel, held by inter-strand H-bonds
• Tertiary: the overall fold, stabilised by hydrophobic interactions, ionic bonds, hydrogen bonds and disulfide bonds (the only covalent force). It creates the functional site — enzyme active centre, antibody binding site
• Quaternary: independently folded chains assembled non-covalently — lecture examples haemoglobin, an α2β2 tetramer, and the antibody IgG (2 heavy + 2 light chains)
• Induced fit — proteins are not rigid: binding a substrate or ligand changes conformation, and that change is the mechanism — enzymes accelerate reactions this way; receptors change conformation to activate intracellular domains (e.g. a tyrosine kinase domain); channels switch between open and closed states to control ion flow
• Misfolding disease — β-amyloid in Alzheimer's: a wrongly folded protein loses its function and becomes toxic. So intervene either to stabilise the functional conformation or to block the pathogenic conformational change. Denaturation = loss of shape = loss of function — why nanobodies tolerating 60–80 °C are exceptional
• PEGylation attaches polyethylene glycol to a protein, raising its hydrodynamic radius so renal clearance slows: half-life lengthens, dosing intervals extend, immunogenicity falls
• Krystexxa (recombinant uricase) — converts uric acid into the far more soluble allantoin, given by intravenous infusion every 2 weeks for chronic gout. Its immunogenicity is intrinsic to its origin: a recombinant protein from a pig–bovine chimera, which can provoke neutralising antibodies — hence antihistamine and steroid pre-treatment before infusion

The two routes: de novo = build nucleotides from scratch (this is what aminopterin blocks, at DHFR); salvage = recycle pre-made bases, and this route requires HGPRT.

Read back against the fate table above and the whole selection becomes one sentence: aminopterin removes route one, so survival requires route two; route two requires HGPRT; the B cell supplies HGPRT and the myeloma supplies immortality — only the fusion has both.

Two properties do the work. Uniqueness means cutting with one of those enzymes opens the circle at exactly one place instead of shredding it. Variety means you can choose two different enzymes for the insert and the vector, forcing the insert in orientation-specific rather than random — a gene ligated in backwards would be transcribed into nonsense.

It sits inside the pipeline already covered above: plasmid (vector selection + gene insertion via MCS) → transformation → expression → purification. Modern cloning often replaces it with Gibson assembly or Golden Gate, but the MCS is still the term the exam uses.