Small pox Several classes of immune modulatory drugs
The main goal of these drugs is to prevent unwanted tissue damage, towards maintaining tissue function.
Conventional immunosuppresive drugs:
- corticosteroids (prednisone)
- cytotoxic (azathioprine)
- noncytotoxic but signal modulators (cyclosporin, rapamycin)
- cytokine receptor modulator
Precision proteins:
- mAbs
- fusion proteins (CTLA-4-Fc)
Part of glucocorticoid family ({mineralcorticoids, sex_steroids, glucocorticoid})
prednisone - a synthetic version of cortisol.
Activated intracellular receptors move into the nucleus where they control ~20% of genes leukocytes express.
Main mechanism:
Expression of AnxaI - inhibitor of phosophlipase A2 (Arachidonic Acid). Suppresses prostaglandin + leukotrienes.
Adverse effects:
- fluid retention (weak mineralocorticoid effects by upregulating sodium transporters in kidney)
- weight gain (promote fat creation and stimulate appetite)
- diabetes (increase glucose generation in liver + decrease insulin sensitivity in peripheral tissue)
- bone mineral loss (osteoporosis - bone cell proliferation, reduce calcium absorption)
- thinning of skin (reduce collagen + ECM protein production)
These guys mostly interfere with DNA synthesis and originally created to treat cancer. But found to suppress lymphocytes too.
High doses are only used to completely deplete lymphocyte populations eg. in HSC transplants.
azathioprine:
- converted to purine analog 6-thioguanine (6-TG) and competes with purine metabolism precursor to prevent A + G synthesis
- 6-TG can also directly incorporate itself into DNA where it increases risk of mutation when exposed to UV
- Competes with GTP for GTPase Rac1 and inhibits CD28 costim. Inhibiting this anti-apoptotic pathway leads to cell death.
mycophenolate mofetil:
- metabolized to mycophenolic acid: inhibits inosine monophosphate dehydrogenase blocking GMP (guanine monophosphate)
cyclophosphamide:
- becomes phosphoramide mustard and alkylates DNA with two "arms"
- part of nitrogen mustard family derived from mustard gas
- causes bladder hemorrhaging + inflammation
alkylating agents. Usually central nitrogen or sulfur with two alkylating "arms".
Side effects come from general tissue toxicity.
- cyclosporin A - Tolypocladium inflatum. Norway soil fungus. Binds to cyclophilins.
- tacrolimus - Streptomyces tsukabaensis. Japan filamentous bacteria. binds to FK binding proteins
Both bind to class of intracellular proteins called immunophilins. These are isomerases, but unrelated to mechanism. Rather form a complex and inhibit calcineurins.
Ca+ -> calmodulin -> calcneurin -> NFAT
Immunosuppresants of choice in clinic
- rapamycin - Streptomyces hygroscopicus. 'Rapa Nui' (polynesian name for Easter Island). Origin of name.
Downstream of PI 3-kinase + Ras/MAPK
mTOR1 (rapamycin specific)
- proliferation
- autophagy (clears out damaged cell components, like mitochondria)
mTOR2
- actin cytoskeleton remodeling
- adhesion
- migration
Rapamycin:FKBP disrupts mTOR1
Recognition of S1P by S1PR (actually a GCPR) Fingolimod (2010) is a competitive binder
Four members of JAK family: JAK1/2/3 + TYK2.
- Tofacitinib -> JAK3
- Ruxolitinib -> JAK1/2
Non mAB:
- late 1800s equine sera for diptheria and tetanus
- anti-lymphocyte globulin (pooled from exposure to human lymphocytes in mice)
- IVIG (pooled from many human donors to provide wide repertoire)
The use as targeted tx agents for specific immune components is recent development: 1986 - muromomab (OKT3)
Anti-CD52 mAB alemtuzumab now used. Still use anti-lymphocyte globulin - sometimes broader depletion favorable, cost and clinical familiarity
Broadly depleting and non-depleting.
Stem from the use of animal models to generate the antibodies
- chimeric antibodies: splicing human constant region into mouse variable region
- generating antibodies for genetically engineered mice with human genes
- fully human monoclonals from human hybridomas
- plasma cells (antibody generating B cells) isolated from donors are terminally differentiated and short lived
- to induce long life, viral insertion of oncogenes OR fusion with cancer cells
directly is needed:
- EBV transformation
- hybridoma (fusion with myeloid)
omab - fully murine ximab - chimeric: CDR + framework fused with human constant zumab - humanized: only CDR spliced with human constant + framework umab - fully human
Rituximab CD20 "Eliminates B cells" "Non-Hodgkin's lymphoma" Alemtuzumab CD52 "Eliminates lymphocyte" "Chronic myeloid leukemia" Muromomab CD3 "Inhibits T-cell activation" "Kidney transplantation" Daclizumab IL-2R "Reduces T-cell activation" "Kidney transplantation" Basiliximab IL-2R "Reduces T-cell activation" "Kidney transplantation" Infliximab TNF-\alpha "Inhibit inflammation" "Chron's" Certolizumab TNF-\alpha "Inhibit inflammation" "RA" Adalimumab TNF-\alpha "Inhibit inflammation" "RA" Golimumab TNF-\alpha "Inhibit inflammation" "RA" Tocilizumab IL-6R "Block inflammation from IL-6" "RA" Canakinumab IL-1\beta "Block inflammation from IL-1" "Muckle Wells syndrome" Denosumab RANK-L "Inhibits activation of osteoclasts by RANK-L" "Bone loss" Ustekinumab IL-12/23 "Inhibits inflammation caused by IL-12 + IL-23" "Psoriasis" Efalizumab CD11a "Block lymphocyte trafficking" "Psoriasis(Withdrawn)" Natalizumab "\alpha_4 Integrin" ""Block lymphocyte trafficking" "MS" Omalizumab "IgE" "Removes IgE" "Chronic asthma" Belimumab "BLyS" "Reduces B-cell responses" "SLE" Ipilimumab "CTLA-4" "Increases CD4 T cell response" "Metastatic melanoma" Raxibacumab "Bacillus anthracis protective antigen" "Prevents actions of anthrax toxin" "Anthrax infection"
- anti-CD52 Alemtuzumab depletes T cells but still maintains some graft-vs-leukemia effect (for unknown reasons)
- anti-CD3: muromomab no longer used because of cytokine storm from reaction with constant chain. teplizumab has a two alanine substitution preventing this.
- anti-CD25: subunit of IL-2. daclizumab + basiliximab.
- CTLA-4-Ig fusion: abatacept prevents costim (also approved for RA)
TNF-\alpha:
mAB
- infliximab
- adalimumab
etanercept: TNF receptor subunit-Fc fusion
CD, ankylosing spondylitis, psoriatic arthropathy
IL-1
ankira: recombinant protein blocking IL-1 receptor Muckle-Wells, RA
IL-6 tocilizumab
INF-\beta Straight up cytokine: Avonex How does it work?
- inhibits components of inflammasome (cleave Il-1 pro-protein)
- reduces expression of IL-1 pro-protein
integrin \alpha_4:\beta_1 (VLA-4) (binds to VCAM-1)
integrin \alpha_4:\beta_7 (binds to MAdCAM-1)
natalizumab specific for \alpha_4, inhibits both CD, MS
But causes infection, including brain JC virus
CD28 (and B7 or CTLA-4) abatacept - CTLA-4-Ig RA, psoriasis
CD2 (CD58 or Lymphocyte Functioning Antigen, LFA-3) alefacept - CD58-IgG1
- Inhibits HMG-CoA reductase, reducing liver cholesterol biosynthesis -Somehow increases MHC-II expression on lymphocytes, likely due to lipid concentration changes in cell membrane
Bind to Vitamin D Receptors, which become TFs for a range of cytokines: IL-12 (DC), IL-2 + IFN-\gamma (CD4).
The doses needed cause hypercalcemia and bone resorption. Investaging alternatives.
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Small amounts of allergens shift response from IgE to IgG/IgA
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Oral ingestion of antigens in mice has shifted CD4 autoimmmune response to T_regs
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Limited success in people
Rather than oral ingestion:
- Copaxone contains 4 amino acids in ratios that mimic MBP (myelin basic protein). It induces T_H2 response + reduces relapse by 30%
- "altered peptide ligands"
Congenic mice: Identical except for single genetic region Historically, MHC-congenic strains developed after induction of tumor.
Syngeneic tumor (genetically identical donor) always grow differently. Introduction of irradiated tumor cells confer some immune response, specific to tumor. Tumor rejection antigens
- Paul Ehrlich, 1908 Noble Prize, first to suggest immune system kills cancer
- Frank Burnet + Lewis Thomas, 1960 NP, formulated 'immune surveillance':
Elimination -> Equilibrium -> Escape
Evidence:
- Mice: lacking perforin (lymphomas), lacking RAG + STAT1 (gut epithelial + breast), lacking gamma/delta (skin cancer)
- Transplant: melonoma develop 1/2 years after kidney transplant, post-transplant lymphoproliferative disorder from unsuppressed EBV expansion
1/ Loss of antigen: mutations prevent expression of antigens in the first place 2/ Treated as self-antigen: antigens presented with lack of co-stims tolerize T cells 3/ Low immunogenicity: lose MHC expression, adhesion molecules, co0stims 4/ Immunosuppresive molecules: TGF-\beta (discovered in this context, hence name), IL-10 (suppressive, eg. DC in gut), IDO (degrades tryptophan), PD-L1 5/ Physical barrier
MDSCs (Myeloid Derived Suppressor Cells): pathologically activated immature myeloid cells. Suppress different lymphocytes. Accumulate in TME.
- Neoantigens: Point mutations to existing binders/allow new proteins to bind to MHC
- cancer-testis: Usually expressed in male germ cells without MHC. Cancer can start expressing them, eg. MAGE (melanoma-associated antigens). ex: NY-ESO-1 (New York esophageal squamous cell carcinoma-1)
- tissue differentiation antigen: eg. CD19 B cell
- overexpression antigen: HER-2/neu
- abnormal PTM: underglycosylated mucin
- skipped introns:
- viral oncogenes: HPV in cervical carcinoma
- tyrosinase (melanin production pathway) glycoproteins:
- gp100
- gp75
- MART1
Melanin is synthesized in special organelles called melanosomes and distributed across keratinocytes.
Piece of chrom 9 (ABL) breaks of and attaches to chrom 22 (BCR).
Bcr allows Abl (non receptor kinase) to bind to itself. Constitutively active molecule leads to growth / proliferation and Chronic Myeloid Leukemia.
You can then identify reactive T-cells to Bcr-Abl using tetramers
Expose donor lymphocyte infusion to leukemia specific peptides in-vivo for an increased leukemia killing + potentially less graft-vs-host response.
ALL (acute lymphoid leukemia)
1/ CD19 2/ 3 ITAMs from zeta chain of CD3 3/ 4-1BB (TNF)
Can achieve complete clinical remissions with ALL!
- Competing with receptor ligand, preventing growth effect
- Recruiting immune response to large molecule bound to cell
Herceptin binding to HER-2/neu is a flagship example
Problems?
- Inefficient killing of cells after bound
- Hard to penetrate solid tumor
- Soluble antigens mopping up antibody
Different types of conjugation, either to mAB or just Fv fragment
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immunotoxins (usually a protein toxin): ricin A chain + Pseudomonas
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small molecule
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radioisotope
-
ADEPT (antibody directed enzyme / pro drug therapy)
- 2005 clinical trial: HPV vacccines 100% effective in preventing cervical cancer
- Similar mechanism with liver cancer and hepatitis
- Trying to get tumor to express costims like B7 doesn't seem to work that well
- CTLA-4. Expressed on activated conventional T cell + T_regs. Competes with CD28 for B7 ligands. Ipilimumab
- PDL1. pembrolizumab.
- variolation from corpse pustule in medical literature for 1000s of years
- Jenner - small pox vaccine. First to intentionally vaccinate with cowpox (-vacca)?
- Pasteur, extended name in his honor with chicken cholera vaccine
Most vaccines happen to be good because they stimulate neutralizing antibodies. But pathogens that require more complex response need better vaccines.
Specific examples:
1/ Malaria. Cycles between the liver and blood cells. Express different surface proteins. Hide inside cells. 2/ Tuberculosis. Hides inside macrophages. Can be sequestered with granulomas but create latent, living disease. 3/ HIV. Direct infection (and hiding) of T cell. High mutation. Latency.
- attenuated: reduced pathogenicity, eg. genetically modified that continues into present for malaria (no vaccines currently)
- killed: vaccines can cause lethal responses, especially in immunosupressed
"Reverse immunogenetics" to identify candidate peptide antigens that activate innate sensors (eg. TLRs) as adjuvants
1/ Infections are insufficient to generate protective immunity:
- malaria
- tuberculosis
- HIV
2/ Cost, storage, deploying vaccines in developing countries difficult
1/ Neutralize exotoxin produced by pathogen. Diphtheria + tetanus. 2/ Neutralize pathogen itself to prevent re-infection.
- Because of linked recognition, pathogens must have epitopes recognized by both T and B cells.
- Safe. Some can die from toxin or catch disease.
- Work in large proportion of population.
- Long lived. Must prime B + T cells.
- Cheap.
Herd immunity is possible but with large percentages of vaccinations. For mumps, estimated to be @ 80%.
2004-2005 in UK saw uprise in mumps because MMR (mumps / measles / rubella) was swapped with just MR because of supply.
Both paramyxovirus species Enter from respiratory droplets into resp. tract.
Measles: 1/ alveolar macrophages + dendritic cells -> lymph nodes -> T / B cells. 2/ primary viremia enters blood stream with T / B cells 3/ secondary viremia distributes to many organs
Mumps: More localized to parotid glands.
Live viruses are more effective at recruiting active arms of immune system. Attenuation can be achieved by selection for growth in non-human cells. Pose risks to immunodeficient patients Recombinant viruses offer path forward
Passaged BCG. Over 13 years + 200 passages to lose infectivity.
Malaria. Knockouts in sporozoites prevent transformation from liver to blood infection.
Injection is not only expensive and difficult at scale, it does not mimic natural route of entry.
Example: live attenuated polio developed by Sabin. Vaccine can also spread like normal virus in eg. fecal swimming pools.
Also, Proteins bind to epithelial cells and are resistant to proteases make good adjuvants:
- E. coli derived heat-labile toxin (HBT). Ex: introduce with oral tetanus toxin as adjuvant to build tolerance against lethal challenge.
Counter example: influenza. Antibodies developed in mucus and systemically for upper and lower respiratory respectively. Lower respiratory is what causes death. Oral ingestion would only prevent mild illness from upper resp.
Attenuated bacteria used to treat Whooping cough in early 1900s.
In 1972, Japan, whooping cough cases increased because of fear of encephalitis (brain swelling) pushed vaccination age from 3 months to 2 years.
Lead to development of acellular vaccine: single protein component isolated + purified. More effective and without side effects.
T cell independent (TI) antigens stimulate B cells without T cell help.
Capsular polysaccharides (eg. meningococcus, pneumococcus) are such TI antigens and can be used on their own with adults.
However young children (<2) cannot mount TI response: immature marginal zone B cells (the cells that handle TI), signaling pathways maturing.
Cross link protein to carrier protein recognized by T cell.
- Adjuvant approved in specific vaccine use
- Alum (aluminum salt) is the only adjuvant approved.
- MF59 (oil-in-water emulsion)
Trigger TLRs, NOD-like receptors, NLRP3
Stable and safe, but weak response. Encoding cytokines helps.
Trasnfecting directly in dendritic cells allows efficient presentation. Trials underway.
1/ Obvious immune response but fails to eliminate
- Helminth
- Tuberculosis 2/ Pathogen is "hiding"
- HIV
- herpes