Antibody Therapy Flashcards
Describe the quarternary structure of antibodies.
Antibodies are comprised of four peptide chains, two 440AA heavy chains and two 220AA light chains. The glycosylated heavy chains are separated by short linking sequences before the light chains j bind.
Each antibody domain is a 110AA immunoglobulin fold domain, a common and very stable motif constructed of two beta sandwich fold inclined at around 30˚to one another and packed together.
Each domain consists of a pair of β-sheets; one with 3 strands and one with 5 strands. A single covalent disulphide bridge holds the 2 sheets together, which results in a rigid and very stable domain.
How are monoclonal antibodies produced?
Kohler and Milstein (1975) took immobilized B-cells from mice and fused them with myeloma cells (cancerous blood cells) to form Hybridomas secreting monoclonal antibodies.
They showed that each antibody had rearranged the germline genes to form individual clones to neutralize cancerous blood factors – monoclonal antibodies are created to target one specific antigen.
What are the crystallisable fragments of antibodies?
Fc - the constant region formed by the heavy chains
Fab(ulous) - a single arm of the Y which is antigen binding, comprising a full immunoglobulin domain made up of a light chain and half of what was the heavy chain.
Fv - a half of the Fab which contains the CDR and so determines the paratope.
What determines antibody specificity?
The variable regions of both light and heavy chains (VL and VH) contain three hypervariable regions referred to as complementarity determining regions (CDRs), which directly contact a portion of the antigen’s surface during binding.
As such, the variable region of each arm (Fv) contains 6 CDRs – 3 from the light chain and 3 from the heavy chain
Glycosylation in the Fc fragment is also important for specificity.
How can antibodies directly lead to cell destruction?
Via complement dependent cytotoxicity (CDC), Antibody-dependent cell cytotoxicity (ADCC), triggering apoptosis or Fc-dependent phagocytosis.
These imply that cancer could be treated with antibodies raised against the tumour cells.
What is the complement pathway?
The complement cascade is initiated when multiple IgGs (or one or more IgMs) binds to a cell. This allows them to recruit c1q, which activates and recruits a bunch of proteins eventually leading to formation of a Membrane Attack Complex (MAC).
The MAC leads to cell destruction by creating a large membrane pore, effectively puncturing the cell allowing release of the cell contents and eventual death.
What is Antibody-dependent cell cytotoxicity?
ADCC is is initiated by the recognition of IgG-coated cells by Fcγ-Receptors, which are expressed by effector immune cells such as NK cells, macrophages, and neutrophils.
Crosslinking these receptors initiates signalling through immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs).
This begins many immune responses, but primarily cell lysis mediated by the delivery of perforin and granzymes to the cell by the immune effector cell.
What is Fc-dependent phagocytosis?
IgGs can stimulate various FcRs on immune effector cells to induce phagocytosis of the cell to which they are bound, leading to lysosomal degradation.
How were monoclonal antibodies first used in human therapy?
Initially, monoclonal antibodies were raised by injecting the antigen into a mouse, then isolating the mouse antibodies and using them as a boost to/replacement for the human antibodies.
However, this caused the human to raise human anti-mouse antibodies (HAMA) against them, thus causing a dangerous immune response.
How are genetic mouse antibodies made less immunogenic?
Chimeric antibodies (-xi-) were first used in which the mouse raised Fabs or Fvs were spliced onto human Fc domains. Using much more sequence than this leads to a loss of specificity, as structural regions throughout the antibody can have indirect effects on the paratope.
Antibodies are now often humanised (-zu-), with ~90% of the antibody being made with human sequence - essentially grafting the mouse-raised CDRs onto human antibodies but with strategical structural edits to preserve specificity.
What was the first chimeric antibody approved for treatment?
Rituximab, constructed from mouse Fv grafted onto otherwise human antibodies, was given FDA approval in 1997.
It targets CD20, a protein expressed only on B-cells - thus leading to their selective destruction. This is used as a treatment for B-cell lymphoma, destroying both malignant and normal B-cells which causes a period of B-cell depletion before normal ones are regenerated.
What effect does rituximab have on B cells?
It kills them through ADCC, CDC and by inducing apoptosis.
The relative importance of each of these mechanisms of action are not fully understood. They may act independently or in concert. What is clear from clinical studied and practise is that Rituxan is highly effective in depleting B-cell populations and in shrinking B-cell lymphomas.
What was the first humanised antibody approved for treatment?
Trastuzumab was constructed with CDRs from mouse and human IgG1, approved by the FDA in 1998 for treatment of breast cancer.
How does trastuzumab target breast cancer?
Trastuzumab is anti-HER2 (human epidermal growth factor receptor 2), an EGF family RTK that is a prominent oncogene in many epithelial tumours. It is mutated in 25% of breast cancer, and in those who do have HER2 mutations have significantly more aggressive cancers.
Trastuzumab targets the ligand binding domain, competetively inhibiting the receptor, and also stimulates ADCC and possibly other immune responses.
What is avastin?
Anti-VEGF antibody to target tumour-associated vasculature. An antibody humanised with IgG1 c-regions. First anti-angiogenesis therapy to increase survival in cancer patients, approved in 2004.
