Immunopharmacology Flashcards
What is a biologic?
A biologic or biopharmaceutical is a medical product whose synthesis, extraction or manufacture involves living sources, such as human, animal or microbiological sources.
This includes:
- Protein based therapeutics
- Gene and cellular therapies, stem cells and transplantation
- Vaccines
- Blood products for transfusion
- Diagnostic reagents (e.g. allergens for allergy tests)
Protein based therapeutics can include:
-Peptide and protein hormones and growth factors
-Antibodies
-Engineered proteins, e.g. receptor binding site domains
An example of a biologic is insulin, first isolated from the bovine or porcine pancreas in the 1920s. Insulin was then the first medicine to be synthesised by recombinant DNA technology in the 1980s.
Insulin can replace the endogenous hormone, but it has to be administered via injection rather than orally. The stability of the formulation and the risk of immunogenicity are also disadvantages.
Pros and cons of protein therapeutics
An advantage of protein therapeutics is that it may be difficult to make a small molecule for the target, for example for receptors with large, complex binding sites or targets with unknown binding sites (no known endogenous ligand, e.g. HER2). You do not necessarily need to know the active site sequence to target the receptor.
There is a potential for higher affinity and selectivity - e.g. specifically targeting a mutant isoform.
There is a potential for a diverse molecular mechanism of action. Examples include interacting with the messenger molecule rather than the target or utilising immune-directed cytotoxicity (e.g. cancer treatment).
- can get your antibody to bind both an epitope on the cancer cell and one on an immune cell to bring these in close proximity
- can attach your cytotoxic drug to a cancer-cell specific antibody to reduce systemic side effects
A disadvantage of protein therapeutics is a possible lack of efficacy and pharmacokinetic challenges, e.g. administration and delivery to the target tissue and tissue access (e.g. BBB).
There may be species variation in protein sequences, which may cause a risk of lack of efficacy or immunogenicity with non-human sequences.
Manufacture of protein therapeutics can have issues involving complexity, reproducibility and purity of the synthetic process. Reproducibility is an issue particularly with polyclonal antibodies - these target many amino acids on the same protein, so how do we make sure they are the same every time?
TGN1412
Theralizumab (TGN1412) was an anti-CD28 antibody. It activated T-cells without activation of the T cell receptor via antigen-presenting cells, acting as a superagonist.
It is a humanised antibody, thought to be of use in autoimmune diseases in which regulatory T cells are low e.g. rheumatoid arthritis.
In a phase 1 trial, 6 volunteers received a dose 500 times lower than that found safe in animals. There was an immediate adverse reaction involving a cytokine storm. 4 of the volunteers had multiple organ malfunctions, and some lost fingers.
Animals may not be a good predictor of immune responses in humans, particularly with an antibody against the human form of the receptor. However, they did pick up the formation of antibodies against the therapeutic, so there was some indication of immunogenicity, but this was ignored.
Pre-clinical testing of TGN1412 was carried out in cynomolgus macaques (primates), at doses of up to 50 mg/kg for 1 week. This was well tolerated with no signs of toxicity or systemic immune stimulation. However, the assumption of 100% CD28 receptor homology between the primate species used in preclinical studies and humans was not supported by citations in the trial documentation, and later reviews revealed sequence differences of up to 4%.
Toxicology testing is difficult to perform for biologics. ICH guidelines state that immunogenicity of a biological product in an animal model is not predictive of immunogenicity in humans.
In vitro testing methods can also be used to aid immunogenicity testing.
In in vitro tests, no immuno-stimulatory response was seen when TGN1412 was simply mixed in solution with PBMCs. It only stimulated a pro-inflammatory cytokine response if appropriately presented to PBMCs, e.g. by immobilisation onto a plastic surface
Therefore, an appropriate in vitro test could have actually identified/allerted the trial coordinator of a possible immunotoxicity problem before animal testing was even carried out.
Cytokine storms or cytokine release syndrome could occur with any biologic when this acts as an allergen leading to activation of the immune system. Drugs for treating this response should be readily available when biologics are administered to patients.
Development of Therapeutic Antibodies
An antibody consists of a Fab (fragment antigen-binding) region, the part of an antibody that binds to antigens, including the Fv region responsible for antigen recognition.
The Fc region will bind to receptors on immune cells - e.g. in IgE antibodies, this binds to mast cells. This can be changed to bind to your immune cell of interest.
Antibodies are typically raised against an antigen by immunization with a protein
In the development of polyclonal antibodies, many different IgG molecules with a high affinity for the antigen are purified from serum after immunization. The antibodies have different variable regions against different sequences of the protein of interest.
In the development of monoclonal antibodies, IgG producing B cells are isolated from the immunized mouse and cloned, producing identical IgG molecules. These are more commonly used as therapeutics, after an antibody of interest is identified from the panel of polyclonal antibodies.
Current therapeutic antibodies are monoclonal, signified by -mab, e.g. trastuzumab and infliximab.
The ending also varies based on how the antibody was derived:
- Murine = momab
- Chimeric - an antibody in which the variable region has a mouse sequence but this is attached to a human antibody (human Fc region) = ximab
- Humanised - variable regions on the mouse antibody have been inserted into the human antibody sequence = zumab
- Human = umab
Developing therapeutic antibodies - different methods
To produce a mouse monoclonal antibody, inject a mouse with the antigen and isolate the B cells. A fused cell line is then created. This is an immortal line that produces antibodies, created by fusing tumour cells with the antibody-forming cells; this is a hybridoma. Polyclonal antibodies are obtained at the end. To obtain monoclonal antibodies, these are purified, the antibody of interest is selected and clonal expansion is used.
To create a chimeric antibody, you can alter the mouse antibody following purification. The mouse target sequence is attached to a human Ab.
To produce a humanised mAb, a similar approach is used. Following purification, the mouse binding sequence is integrated into a human Ab using a CDR graft.
To obtain human Abs, transgenic mice are used instead.
Mouse antibodies are more likely to produce an immune response. Humanised antibodies may still be recognised as foreign as it is not one that your own body has produced, but it is less likely.
Human antibodies can also be produced using a phage library. A phage antibody library is constructed using B cells from an immunised human donor or patient with a particular disease. The mRNA is extracted and expressed in bacteriophages, which creates a library of different antibodies. The library is then screened with immobilised antigens to identify an appropriate Ab. This is called biopanning.
Finally, antibodies can be obtained directly from humans. For example, to obtain human antibodies against Covid, you isolate Abs from a patient exposed to SARS-Cov2, purify these and administer them to other patients.
Antibody mechanism of action
Antibodies can work as receptor antagonists and enzyme inhibitors. The antibody binds to its target (receptor, enzyme, transporter, channel) and prevents activation by the ligand, having a similar action to an antagonist. Certuximab binds to and antagonises EGFRs, limiting proliferation of tumour cells.
Antibodies can also antagonise the stimulating growth factor or messenger. Examples include anti-VEGFA therapies like Bevacizumab and anti TNF-a like Infliximab. These bind to and neutralise the ligand, preventing it from activating the receptor.
We can also utilise antibodies for antibody-directed cell cytotoxicity. For this, we bind a cytotoxic drug to the antibody.
The antibody binds an epitope on the tumour cell. This allows selective targeting of the drug to the tumour site.
Antibody mechanism of action - Death receptors
Antibodies have been trialled as agonists for the death receptors (DR) for tumour apoptosis.
E.g. CDX-1140 is an agonist antibody at CD40, a receptor expressed on antigen presenting cells (APCs). This activates T cells and can drive T cell dependent tumour regression.
TRAIL Receptor Agonists (TRAs) activate DRs to stimulate apoptosis, which is impaired in cancer.
Activation of DRs on tumour cells activates pro-apoptotic signalling, leading to cell death. Antibodies cross-linking 2 DRs mimic activation by the ligand.
Conatumumab is a DR5-directed antibody.
Recombinant TRAIL peptides are also being tested.
Bispecific and Trispecific Antibodies
Bispecific and trispecific antibodies target 2 or 3 different antigens.
An example is catumaxomab, which targets CD3 epitopes in T cells and EpCAM epitopes in tumours in patients with malignant ascites.
The Fc region is changed so that the antibody binds macrophages which can phagocytose the tumour cell.
Two different types of immune cell are brought in close proximity to the tumour cell.
This is a trispecific antibody as three different epitopes are being targeted.
This increases selectivity, targeting the antibody to the tumour site and bringing two immune cells in close proximity to the tumour cell.
Autoimmune diseases
Autoimmune diseases occur when antibodies form against ‘self’ epitopes, which leads to tissue damage
Genetic factors also contribute to autoimmune diseases
Autoimmune conditions can be precipitated by pregnancy, infection, diet or the environment.
Autoantigens are present in everyone, but not everyone develops autoantibodies against these and not everyone that has autoantibodies develops an autoimmune disease.
Self tolerance prevents autoantigens activating the immune system
In autoimmune disease, tolerance is lost leading to attack of self by the immune system. We do not know why this happens.
Hashimoto’s disease
Hashimoto’s disease involves hypothyroidism.
Antibodies form against the thyroid gland. This becomes inflamed and damaged.
Symptoms include fatigue, an enlarged thyroid, weight gain, cold body temperature.
Women are 7x more likely to get it.
Age also plays a role in risk - it mostly occurs in middle-aged adults
It is a hereditary condition.
Treated with levothyroxine to combat hypothyroidism.
Grave’s disease
This is almost the opposite of Hashimoto’s, as autoantibodies stimulate the thyroid to produce thyroxine, causing hyperthyroidism.
Treated by inhibiting production of thyroid hormones, e.g. radioactive iodine
Type 1 diabetes
A mix of autoantibodies can be found in type 1 diabetes including:
- antibodies to glutamic acid decarboxylase (GAD-65) - found in ~80% patients
- antibodies against proteins on β cells (~70%)
- insulin autoantibodies (~50-60%)
Patients can have multiple types of autoantibodies at the same time
Treated with insulin
Systemic Lupus Erythematosus (SLE)
Anti-dsDNA antibodies are present in 80% of SLE patients.
Symptoms include joint pain, extreme tiredness and skin rashes, especially when exposed to the sun.
Severe symptoms include inflammation of the lungs, heart and kidneys.
Treated with immunosuppressants.
Crohn’s Disease and Ulcerative Colitis
Crohn’s disease and ulcerative colitis are chronic inflammatory bowel diseases that COULD be of autoimmune origin.
Crohn’s can affect any part of the GIT and cause transmural inflammation, i.e. affecting the entire wall of the GIT.
Ulcerative colitis affects the mucosal layer of the colon, causing the formation of ulcers.
Both can cause very severe diarrhoea, and Crohn’s can impair the absorption of nutrients.
They are possibly triggered by changes in colonic bacteria.
In ulcerative colitis, autoantibodies are found in 80-90% of patients.
In Crohn’s disease, antibodies against microtubules from gut bacteria are found.
Data suggests that UC may have an autoimmune component, whereas Crohn’s may be related to bacteria.
Fecal donations could be used as a treatment option to restore the balance of gut bacteria.
Immunosuppressants can still be used for the treatment of these conditions, even if we are unsure that there is an autoimmune involvement, as an overactive immune system is still involved in their pathology.
Rheumatoid vs Osteoarthritis
Osteoarthritis is characterised by cartilage loss, most commonly affecting the knees, hips and small hand joints due to ‘wear and tear’.
There is a link to being overweight.
Pain in OA is worsened by movement and eased by rest. It is also usually worse at the end of the day. Pain commonly affects the hands, knees, spine and hips.
Treatment options include pain management, intra-articular steroid injections (offered in moderate-severe pain but can further damage cartilage), NSAIDs or COXIBs, and surgery for joint replacement.
Rheumatoid Arthritis (RA) is a chronic inflammatory disorder caused by antibodies against citrullinated proteins including fibrin, vimentin, fibronectin and collagenase, which are structural proteins. Antibodies against these leads to their degradation, causing joint damage.
- Macrophages cause cartilage degradation but also activate osteoclasts to cause bone erosion.
Structural proteins in the skin are also affected, leading to the formation of nodules.
Signs include joint damage, muscle wastage and deformity. Symptoms include pain, stiffness and joint swelling.
Lab tests would indicate increased WBC count, increased erythrocyte sedimentation rate (ESR), anaemia and the presence of rheumatoid factor (antibodies to IgG).
- The membranes of RBCs are also affected, becoming more rigid, so sedimentation becomes faster. This can be used for diagnosis.
- This also leads to anaemia, as the RBCs are less able to squeeze through blood vessels and deliver oxygen.
Epidemiological risk factors include:
- age - peak age is 65-75 years
- gender - premenopausal f>m, post m=f
- post-partum
- stress
- smoking
Pain in RA behaves differently to that in OA, as it improves with movement, is worse on waking and affects small joints more.
Rheumatoid disease is a systemic disease. Emphasis is placed on the joints, but it does also affect:
- Eyes - inflammation occurs in 50% of patients
- Skin (nodules)
- Vasculitis - blood vessels are destroyed by inflammation
- Lungs
- Salivary glands (reduced)
- Pericarditis (inflammation of pericardium)
The aims of treatment are to relieve pain and modify the disease process, for example by preventing joint destruction or preserving/improving functional stability.
Symptom relief include NSAIDs, PPIs and other analgesics.
To slow down progression, we can use:
- Disease-modifying anti-rheumatic drugs (DMARDs) - used long-term
- Steroids
- Biologicals
Steroids
Can be administered via the intramuscular, intraarticular or intravenous route.
Used short-term to reduce inflammation in exacerbations, but long-term disease-modifying drugs are used instead.
Examples of steroids used include oral prednisolone, which has anti-inflammatory and immunosuppressive actions. It is used to induce remission, particularly in more severe disease cases.
In inflammatory bowel disease, enemas are used for more distal or rectal inflammation e.g. Predfoam.
Steroids alter gene transcription by causing dimerization and nucleus translocation of glucocorticoid receptors, which then interact with genomic response elements.
Anti-inflammatory genes (annexin A1, beta-AR, IkB, MKP1, IL-10/12) are activated and inflammatory genes (IL-2/3/6, TNF-a, chemokines, iNOS, COX-2, endothelin-1) are repressed.
Annexin A1 (lipocortin) is upregulated. it acts through formyl peptide receptors (FPRs) to inhibit the release of histamine from mast cells and also cytoplasmic phospholipase A2 (PLA2) and therefore synthesis of inflammatory prostaglandins and leukotrienes.
DMARDs
Disease-Modifying Anti-Rheumatic Drugs (DMARDs) take 3 months to work and have a small TI.
They act by inhibiting the immune system to slow the course of disease, but they have no analgesic activity.
Examples of DMARDs include methotrexate,
azathioprine/mercaptopurine, sulfasalazine and leflunomide.
Methotrexate (MTX) - DMARD
Methotrexate (MTX) is a dihydrofolate reductase (DHF reductase) inhibitor, inhibiting thymine (pyrimidine) synthesis. It has a similar structure to DHF, so it can compete with it.
Most folic acid in the body is recycled, but we need 400 micrograms per day.
Tetrahydrofolate (THF) is converted to methylene THF.
Thymidine synthase uses methylene THF to convert deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), which is required for thymine synthesis. Dihydrofolate is also formed as a result.
DHF reductase converts dihydrofolate back into tetrahydrofolate, recycling it and enabling the pathway to restart.
Methotrexate inhibits DHF reductase, therefore inhibiting folic acid recycling and depleting stores. This inhibits thymine synthesis.
There is not enough thymine to incorporate into DNA, so DNA and mRNA turnover are inhibited.
Cell proliferation and protein synthesis are inhibited, inhibiting the proliferation of immune cells.
MTX is given as a weekly dose, which is individual to the patient and can range from 7.5 to 25 mg.
MTX toxicity includes nausea, post-dose ‘flu’, hepatotoxicity, lung alveoliis/fibrosis, GIT effects, renal toxicity, and blood disorders as turnover of cells involved in blood coagulation is decreased.
Folic acid (folate/vitamin B9) administration reduces side effects. Strangely, therapeutic effect is not affected, but we do not know why. The folic acid is also taken weekly but on a different day - this might be about balance and maintaining a low baseline level.
As folic acid administration does not interfere with therapeutic effect MTX may have a more complex mechanism of action beyond DHF reductase inhibition.
Azathioprine/Mercaptopurine - DMARD
Azathioprine is converted to mercaptopurine and inhibits purine and hence DNA synthesis.
Mercaptopurine is produced by glutathione conjugation of azathioprine.
It is then converted to the cytotoxic compound 6-TIMP by HPRT.
6-TIMP inhibits amidophosphoribosyltransferase and therefore purine (A,G) synthesis. Immune cell production decreases.
Mercaptopurine is further metabolised by a number of pathways including thiopurine methyltransferase (TPMT).
Some patients have low TPMT activity (11%) and some virtually no TPMT activity (0.3%), which creates a risk of toxicity due to excess formation of cytotoxic nucleotide analogues through alternative metabolism pathways.
High TPMT activity can decrease drug efficacy.
Patients are tested for TPMT activity before being put on azathioprine or mercaptopurine. If activity is high or low, the patient could be put on a different drug or dose adjustment could be used. If the patient has no enzyme activity, they would not be put on this drug.
Cancer
The DMARDs 6-mercaptopurine and methotrexate are also used to treat cancer as they inhibit cell proliferation. They are classed as “anti-metabolites”.
Fluorouracil is also an anti-metabolite used in cancer. It is converted to FdUMP, which competes with dUMP for the thymidylate synthase enzyme, inhibiting thymine production. I therefore acts in the same pathway as MTX but at a different site.
Leflunomide - DMARD
Leflunomide (Arava) is an immunosuppressant that inhibits the clonal expansion of T cells.
It is metabolised to Teriflunomide, which inhibits dihydroorotate dehydrogenase and therefore pyrimidine synthesis.
Adverse effects include diarrhoea (20%), nausea, rash and alopecia (10%), abnormal liver function (5%), and teratogenicity.
Aminosalicylates - DMARDs
5-aminosalicylate (mesalazine) increases PPAR𝛾 receptor activation.
Activated PPARγ forms a heterodimer with retinoid X receptors (RXR). The heterodimer translocates to the nucleus, where it binds DNA response elements and regulates gene expression to reduce synthesis of inflammatory mediators.
Sulfasalazine is a prodrug metabolised to mesalazine. It is used in RA and Ulcerative colitis, as metabolic conversion is performed by bacteria in the colon.
- In UC, this limits drug release to the colon, where the therapeutic effect is exerted.
- Not as effective in Crohn’s disease - may be because in this condition inflammation can occur at any point of the digestive tract, so not all sites may be targeted by the active compound.
Cyclosporin
Cyclosporin is an immunosuppressant, acting as an inhibitor of calcineurin, a calcium-dependent phosphatase.
Calcineurin dephosphorylates the nuclear factor of activated T cell cytoplasmic (NFATc), leading to its activation.
NFATc is a transcription factor which upregulates gene transcription, including of IL-2, an inflammatory cytokine required for T cell activation.
Cyclosporin binds to the cyclophilin protein, and the cyclosporin-cyclophilin complex inhibits calcineurin, therefore inhibiting T cell activation and cytokine production.
It is used in autoimmune diseases like ulcerative colitis, RA and psoriasis, and for preventing organ transplant rejection.
Tacrolimus
Tacrolimus is another calcineurin inhibitor, like cyclosporin.
It binds to FKBP (FK506 binding proteins) to inhibit calcineurin.
It is used for organ transplant.
