Biotech Stack #2616357

January 23, 2018 Off By admin
Question Answer
Innate immunity Nonspecific immunity you're born with
Adaptive Immunity Specific immunity aquired over time provided by antibodies.
What are the two types of adaptive immunity? Antibody-mediated (hunoral) immunity

Cell-mediated immunity

Which cells produce antibodies? B cells
Where are antibodies found in the body? Lymphnodes, serum of blood
Serum Part of blood without RBC,wbc,platelets or clotting factors.
Plasma has clotting factors but no wbc rbc plate
Antiserum Serum with antibodies specific to 1 antogen
Antibody-mediated humoral immunity Antibody production by B cells
Cell-mediated immunity Activated T cells (helper, cyotoxic, regulatory); Directly attack cells with "non-self" antigens
B lymphocytes B cells in bone marrow. Active partcipants in humoral immunity.
Exposed to Ag in secondary lympiod organs.
Secondary lympoid organs Spleen, tonsils, lymph nodes, intestinal mucosa
Plasma B cells Actively secrete antibody Ab
Memory B cells Dormant (mount secondary reesponse, more potent, longer lasting)
Serology Interaction between antogens and antibodies
Antibody Natural component of immune system that attacks and neutralizes antigens
Antigen Foriegn object. Body recognizes as nonself.
Paratope Region of antibody that recognizes and binds to the antigen
Epitope Part of the antigen that the antibody can recognize.
Antibodies bind to either 1. Monoclonal antbody specofic epitope
2. Polycolonal: bind to multiple epitopes on antigen
IgA Heavy chain a, external secretions
IgD B Cell surface receptor; heavy chain delta
IgE Heacy chain Epsilon; Cells secrete histamines.
IgG Heacy chain gamma; main antibody in serum (humans) most stable. Promotes antibody-dependent cellular cytotoxicity (ADCC), compliment fixation (CDC: compliment dependent cytotoxicity)
Fab Fragment binding site binds to the antigen (2 total made of the VL, CL,VH,CH1; 2 variable and 2 constant units)
Fc Fragment crystalline. Determines functional properties of the antibody, effector recognition and binding, initiates immune response, determines clearance rate (binding to FcRn receptors) (1/2 life of antibody)
Fv Fragment variable. CdRs (complementary determining regions)=hypervariable regions=3=implies a paratope
Total variabke subunits in an antibody 4. 2 light 2 heavy
Disulfide linkages in antibody CH1-CH1; CH2-CH2; 2 linkages (CH1-CL)
Monoclonal antibodies source clones from a single parent B Cell
Monoclonal antibodies Number of epitope recognition/binding sites bind to a single epitope on a single specific antigen
Monoclonal antibodies sensitivity of Ag detection Low (specific: slight change in conformation-> no recognition)
Monoclonal antibodies Ease/cost of production expensive/ time consuming
Monoclonal antibodies Purity high (batch to batch homogenetiy)
Monoclonal antibodies Potential for cross-reactivity low
Polyclonal antibodies Source complex misture of clones from different B cells
Polyclonal antibodies Number of epitope recognition/binding sites bind to multiple epitopes on the same antigen
Polyclonal antibodies Sensitivity of Ag detection high
Polyclonal antibodies ease/cost of production easy/cheap
Polyclonal antibodies purity low (batch to batch variability)
Polyclonal antibodies potential for cross reactivity high
Mouse IgG (murinic) Aa Variable, contrast region, all of it is mouse; most immunogenic
Chimeric IgG Ab variable segment is mouse
Humanized IgG Ac CDR=Mouse
Human IgG Ad no mouse; least immunogenic
Murine source fully murine
Murine % similarity to humans 0%
Murine antibody nomenclature Momab (O) (mab=monoclonal antibody)
Murine antibodies produced in response HAMA (human anti-murine antibodies)
Murine Effectiveness of therapy Poor (poor bioavailability, poor tumor penetration, rapid clearance) Used against cancer.
Murine Probability of Hypersensitivity Highest/most
Murine Production Hybridoma technology
Chimeric source murine variable regions and human constant regions
Chimeric % similarity to humans 60-70%
Chimeric antibody nomenclature Ximab (Xi) Mab=monoclonal antibody
Chimeric antibodies produced in response HACA (human anti-chimeric antibodies)
Chimeric Effectiveness of therapy better bioavailablity than murinic. Increased 1/2 life.
Chimeric probability of hyperesnsitivity less that murinic more than all else
Chimeric production genetic engineering using recombination technology
Humanized source Murine CDRs and human variable framework and constant region
Humanized % similarity to human mABs 90-95%
Humanized antibody nomenclature Zumab
Humanized antibodies produced in response HAHA (human anti-human antibody)
Humanized effectiveness of therapy improved bioavailability, increased 1/2 life over chimeric and murine.
Humanized probability of hypersensitivity less than chimeric more than human
Humanized production genetic engineering using recombination technoloy
Human source fully human
Human % similarity to human 100%
Human antibody nomenclature Mumab
Human antibody produced in response HAHA (human anti-human antibody)
Human Effectiveness of therapy more stable, high specificity, high therapeutic efficacy, long 1/2 life
Human probability of hypersensitivity low/ least
Human production phage display library or transgenic XenoMouse
Direct modulation of target antigen cause cell lysis (DNA damage) apotosis, phagocytosis. Bind and destroy directly
Fc binds to complement proteins C1q (complement-dependent cytotoxicity (CDC)
Fc binds to ___receptors Fcy receptors (CH2 domain) leads to antibody dependent cellular cytotoxicity (ADCC)
Delivery of cytotoxic agent drug, radioscope, toxin
Compliment system: classical: binding to antibodies produced against and attached to a particular foreign invader specifically activates the compliment cascade (an adaptive immune response) Fc part that binds to that effector=compliment protien.
Compliment system: Alternate pathway: binding directly to a foreign invader nonspecifically activates the complement cascade (innate immune response)
Compliment system: formation of membrane attack complex once activated (C5, C6, C7, C8, C9) aggregate to form a porelike channel in the plasma membrane of the target cell, resulting in leakage and destruction of the cell.
Compliment system: cascade sequence of events: proteins are designated (C1-C9); C1 serves as a recognition protein; C2-4: activators; C5-9: attack by attaching to a cell membrade and destroying it. Creates a large pore in membrane=water influx=cell lysis
Compliment Dependent Cytotoxicity (CDC) by Rituximab monocolonal antibody bind to receptor and initiate compliment system "complement cascade" end result is a membrane attack complex (MAC) which leads to cell lysis/death. CD 20: antigen expressed on surface of B cancer cell which will be bound to a C1q/r/a
Antibody-Dependent Cellular Cytotoxicity (ADCC) by Rituximab Fc gamma R1-3 receptors bind to antibodies Fc binding receptors, targets CD20 antigen on B cancer cells. Fc fragment of antibody bound to monocyte, macrophage, NK cell which engulf tumor cell and destroy it. NK cell secrete cytokines and recruit B cell
Enzymatic Degradation of Antibody with Papain leads to 2 intact Fab; 1 intact Fc (b/c disulfide bond still there)
Enzymatic Degradation of Antibody with Pepsin leads to intact Fab2 (a bound) fragment (with all the disulfide bonds) Fc fragment degraded
Enzymatic Degradation of Antibody with Pepsin + MEA Hcl leads to 2 intact Fab fragmetns with three sulfide molecules. No Fc.
MEA-HCl 2-Mercaptoethylamine-Hcl
Antibody Derivatives (fragments) Ig conjugate (cytokine, radioisotope, toxin, drug); F(ab)2 (2 antibody disulfide bond); Fab/scFv; Bispecific (2 antibodies for diff antigens disulfide bond); Fc fusion protein (cytokine, enzyme, receptor)
Prime determinant of 1/2 life in antibody Fc region
Antibody covalent linkage to: Radio- immunotherapeutic agents; antibody-drug conjugates; toxin-immunoconjucates RIT: trageted drug delievery system (radiation kill cancer cell) positive drug delivery system. Only link 1 region. ADC: site of action at antigen.
Monoclonal antibodies common adverse effects allergic: rash, itching, hives; flu-like symptoms: fever, chills, muscle ache, pain. N/V/Diarrhea/Headache
Monoclonal antibodies rare adverse effects Infusion: severe allergy/death; Low blood cell count: RBC, WBC, platelet low; cardiac complications: HF, MI; Excessive bleeding
Hybridoma technology Mouse/murine: fusion of a B cell (produces a spec antibody) with a myeloma cell (ability to grown in tissue) leads to hybrid cell line (Hybridoma)
Hybridoma fusion of B cell and myeloma cell to produce a monoclonal antibody (mAbs). Discovered in 1975, approved (OKT3) 1986.
Hybridoma technology step 1-3 1. immunization of a mouse with specific antigen. 2. isolate antibody-producing B cells from spleen (mouse). 3. cultivation of myeloma cells (different mouse) from bone marrow (infinite growth capacity)
Hybridoma technology step 4-5 4. Fusion of B cell and myeloma cell using polyethylene glycol PEG (fuse tissue cells)->hybridoma cell. a. antibody producing capacity (b cell). b. continous growth capability (myeloma cell). 5. HAT selection: seperation of cell lines in selective medium
HAT selective hypoxanthine aminopterin thymidine medium (only pure hypridoma cells survive)
Hybridoma technology step 6-8 6. screen suitable cells. 7. in viro (tube) or in vivo (animal) multiplicaiton. 8. Harvest antibodies
Pure Synthesis Pathways: HAT medium Salvage pathway: helped/stimulated
De novo pathway: inhibited
Pure synthesis Pathways (2) Salvage: use old/recycled DNA HGPRT; De novo synthesis: fresh DNA, make diff purines and pyrimidines
Pure Synthesis Pathways HAT selection myeloma cells: de novo (inhibit) not fused or fused w/ each other. B cells: both pathways stim salvage, inhibit de novo
Salvage pathway requires Hypoxathine and thymidine (HaT): intermediates in DNA syn; Hypoxanthine guanine phosphoribosyl transferase (HGPRT) enzyme. HGPRT gene mutated in myeloma cells (cancer cells can't use it so dies in HAT)
De novo pathway requries blocked by aminopterin (folate metabolism inhibitor) (hAt)
HAT selection principle fused hybridoma: suvive: contains immortality from myeloma cells, rescued ability to synthesize DNA due to restored HGPRT function (b cell)
Phage Display Library Human IgG: Fab genes inserted into bacteriophage. Fabs displaying phage presented to immovilized/antigen of our choice. Nonspecific washed away/ only bacteriophage molecule you need sticks "screneing". Infects host bactera and reproduces
Transgenic XenoMouse Model human IgG: make one mouse incapable of producing mous eantibodies. Make one mouse producing humand and mouse antibodies. Mate the mice. mouse strain producing himan IgG and not expressing mouse antibodies made.
Monoclonal antibodies advantages high specificty (for a single epitope); high homogeneity (from same B cells); unlimited quantity
Monoclonal antibody disadvantages affinity lower than polyclonal, conformation changes in epitopes leads to poor reactivity to mAbs, Immune reactions, Time and effort
Small molecule drugs PKPD binding non specific
Small molecule drugs PKPD PK linear PK (most)
Small molecule drugs PKPD Route of Admin oral and others
Small molecule drugs PKPD Metabolism CYP450 (and other enzymes in liver)
Small molecule drugs PKPD MDR transporter involve mutlidrug resistance transporter utilization (MDR) make cell resistant to drug/ push drug out of cell
Small molecule drugs PKPD protein binding binding to tissues High Vd
Small molecule drugs PKPD half life relatively short
Small molecule drugs PKPD clearance renal clearance
Monoclonal antibodies PKPD binding specific binding to target antigens/target protiens
Monoclonal antibodies PKPD PK linear/ non linear
Monoclonal antibodies PKPD route of admin parenteral/subcut/intramusc
Monoclonal antibodies PKPD metabolism No involvement of CYP 450 enzymes. catalyzed to peptide fragments/ recycled from protein synthesis
Monoclonal antibodies PKPD involvement of MDR no involvement of MDR transporter
Monoclonal antibodies PKPD protein binding distributed to blood of extraceeelular space low Vd
Monoclonal antibodies PKPD half life long 1/2 life. role of FcRn receptors
Monoclonal antibodies PKPD Clearance no renal clearance of intact antibodies
Antibody disposition antibody Fc binds to FCR receptor, taken into lysozome, any antibody boudn to FcRn won't get degreaded/ only recycled back onto the surface
antibody nomenclature variable-target substem- source substem-stem-additional words (in some cases)
-anibi- angiogenesis inhibitor (target)
-ba- bacterium (target)
-ci circulatory system (target)
-fu- fungus (target)
-ki- interleukin (target)
-les- inflammatory lesions (target)
-li- immune system (target)
-mul- musculoskeletal system (target)
-n(e)- nervous system (target)
-s(o)- bone (target)
-tox(a)- toxin (target)
-tu- tumor (target)
-vi- virus (target)
-o-/-omab mouse (source)
-xi-/-ximab chimeric (source)
-zu-/-zumab humanized (source)
-u-/-umab human (source)
-a- Rat (source)
-e- hamster (source)
-i- primate (source)
-axo- rat/murine hybrid
pegol PEGlyated: linked to polyethylene glycol, slow enzymatic degradation, lower immunogenicity. EX: alacizumab pegol
vedotin Cyotoxic agent: linked, "magic bullet" to make the antibody more targeted, carried to site of action, EX: glembatumamb vedotin
chelator used to link a drug/raioisotope (name precedes antibody names)
ex: indium (11ln) cpromab pendetide
Mechanism of action of mAbs 1. CDCC; 2. ADCC; 3. Delivery of cytotoxic compound to site of action
mAb therapeutic appliction in oncology, transplantation/immunosuppression, autoimmune disease, inflammatory disease, infection disease
ADEPT Ab-directed enzyme prodrug therapy: attaching a specific enzyme ot the monoclonal. monoclonal attaches to the antigen and enzyme attached prodrug given/converted ot active form using the enzyme and active drug used on intended site.
131 Tositumomab (Bexxar) Target: CD-20 on Lymphomic B cells.
90 Y Ibritumomab (Zevalin) Target: CD-20 on Lymphomic B cells.
Rituximab (Rituxan) Target: CD-20 on Lymphomic B cells.
Brentuximab vedotin (Adcetris) Target: CD-30 on lymphomic cells (Hodgkin's)
Gemtuzumab calicheamicin (Mylotarg) Target: CD-33 on myeloid (acute myeloid leukemia AML)
Alemtuzumab (Campath) Target: CD-52 on B-cell for chronic lymphocytic leukemia (CLL)
Cetuximab (Erbitux) Target: EGFR1 (stop growth) +ve colorectal cancer
Panitumumab (Vectibix) Target: EGFR1 (stop growth) +ve colorectal cancer
Taraztuzumab (Herceptin) Target: HER2 (EGFR2) +ve breast cancer
Bevacizumab (Avastin) Target: VEGF (growth factor) non-small cell lung cancer NSCLC colorectal cancer
Nivolumab (Opdivo) Target: PD1 (metastatic melanoma) NSCLC
Phosphate and Ca levels always ___ each other oppose. Ca up PO down. vice versa.
131 Tositumomab (Bexxar) mechanism binds to CD20 antigen on normal and malignant B lymphocytes. 2. Cross fire effect of iodine-131 will cause damage to tumor and adjacent normal tissue
Alemtuzumab (Campath) mechanism 1. binds to CD52 surface marker on chronic lymphocytic leukemia cell. 2. trigger complement-dependent cytotoxicity and NK cell action (ADCC/CDCC)
Cetuximab (Erbitux) mechanism 1. target EGFR and bind with higher affinity than natural ligand. 2. internalization of antibody receptor complex without activation of intrinsic tyrosine kinase=> signal transduction blocked=> tumor growht inhibited=> apotosis.
Taraztuzumab (Herceptin) mechanism 1. antibodies latch on to HER2 receptors blocking sites and shutting down excess growth signals. (cancer cells have more HER2 than normal cells on cell surface 25-30% in breast cancer)
Bevacizumab (Avastin) mechanism tumor releasing VEGF (more BV formed to feed tumor)=> drug inhibits this by binding
Nivolumab (Opdivo) mechanism T cell attacking tumor cell. PD1 receptor= programmed death receptor on T cell. Tumor cells use this to kill PD1 receptor. PDL1 receptor: pgrogrammed cell death ligand in cancer which'll bind to PD1. (NIV blocks this ligand from killing T cells).
Monoclonal antibodies in organ translplant target antigens in b and t cell to immunosuppress. Use: desensitize potential transplant recipients before transplant; reduce immunity during; maintain immunosup after; treat trans rejection
monoclonal antibodies in organ transplant adverse effects infections, infusion-related reactions, post-transplant maliganncies
Muromonab (OKT3) Target: CD3 (blocks kidney allograft rejection, steroid-resistant heart transplant)
Daclizumab (Zibryta) Target: CD-25 (IL2 receptor) for acute rejection of kidney transplants
Basiliximab (Simulect) Target: CD-25 (IL2 receptor) for acute rejection of kidney transplants
Monoclonal antibodies in hypercholestroama target: PCSK9; degrade the receptor that kills LDL lipids and the receptor at once and keeps LDL receptors from breaking down (recycling them back onto surface to catch more LDL)
Alirocumab (Praluent) target PCSK9 (enzyme in blood proprotein convertase subtillisin kexin type 9) to lower cholestrol
Evolocumab (Repatha) target PCSK9 (enzyme in blood proprotein convertase subtillisin kexin type 9) to lower cholestrol
Ethical consideration animal testing, XenoMouse modle, adverse effects with death.