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Antibodies - Development, Deficiency and Biologics - Coggle Diagram

- - - - Pro B
        
        DJ
        
        Iga Igb
      - CLP
      - Pre Pro B
        
        activates VDJ machinary
        
        none
  - - - Small Pre B
        
        VJ
        
        BCR (igM), Iga, Igb
        
        success signal when the BCR is complete and SLC replaced with real light chain
      - Large Pre B
        
        VDJ
        
        Pre BCR, lga, Igb
        
        success signal when heavy chain pairs with surrogate light
  - - - BCR (igM), Iga, Igb
        
        Checkpoint: test for tolerance by screening for autoreactivity
        
        If autoreactive: VDJ machinery turned on and given a second chance to rearrange alternate light chain, then tested again (K or L)
  - - - BCR (igM), IgD, Iga, igB
- - - - a. RAG1/RAG2 bind to RSS region, HMG1 enhances binding
      - b. RAG1/RAG2 bring together the D and J regions and form a nuclease that create double-stranded breaks at the RSS sites
      - c. cut ends are ligated to form coding joints (hairpins) on the end of the D and J regions, and
        
        coding joints (hairpins) on the end of the D and J regions,
        
        signal end joints which connects all intervening DNA
  - - - a. DNA-PKcs binds to coding joint hairpins and recruits Artemis, Ku70, Ku80
      - b. Autophosphorylation of DNA-PKcs activates artemis
      - c. CODING HAIRPIN JOINT Artemis opens coding hairpins, DNA-PKcs, Ku70/Ku80, Artemis initiate NHEJ repair
      - d. SIGNAL JOINT Ku70/Ku80 initiate NHEJ repir
  - - - d. SIGNAL JOINT Ligase IV and XRCC4 bind and ligate
      - b. CODING JOINT TdT enzyme recruited and adds random n neucleotides to coding end in 5' > 3' direction
      - a. CODING JOINT DNA-PKcs & XRCC4 (DNA ligase) align DNA ends
      - c. Exonucleases remove bases from coding ends and
        DNA polymerases fill in complementery nucleotides
      - d. Ligase IV and XRCC4 ligates ends
- - - - CSR
        
        AID enzyme (Activation Induced Cytosine Deaminase) introduces nicks into S DNA region
        
        AID is expressed only in activated B cells
        
        Enough nicks are made to create double stranded (ds) breaks in two S regions
        
        DNA Ends are joined, repaired by nonhomologous end joining (NHEJ), intervening DNA deleted
        
        Tanscription of H chain genes starts from promotor upstream of Vh segment,
        
        Thus exons encoding Ig C (constant) regions can’t be expressed until a V segment is placed upstream of them
      - SMH and AM
        
        AID introduces point mutations into V genes which may cause amino acid replacements
        
        changes improve of impairs binding of Ig/BCR to antigen
        
        Intense B cell proliferation induced by B cell
        
        tested by follicular DC's = low affinity B cells die by apoptosis, high affinity selected to survive
        
        SMH occurs in hypervariable complementarity determining regions (CDR1, 2, 3) in heavy and light chain regions
- - - - X-linked agammaglobulinaemia
        
        What is it
        
        inborn errors in Pre-BCR signalling (BTK) that prevents B-cell development
        
        incidence - 1: 58,000 wordwide
        
        clincal features
        
        Complete absence/very little serum Ig (all isotypes)
        
        Complete absence circulating B cells, normal T cells
        
        (may be some B cells if ‘milder’ mutation)
        
        Recurrent
        
        infections
        
        primarily Strep. pneumonia, Haemophilus, influenza b
        
        otitis in children and sinusitis in adults
        
        Average age of diagnosis 3 years old
        
        earlier (2.6 years) if family history
        
        maternal antibodies (igG) provide protection in the first year of life, reccurent infections manifest when these levels wane
        
        How
        
        genetic atiology
        
        inborn errors in Pre-BCR signalling that prevents B-cell development
        
        particularly in BTK which is responsible for sending sucess signal
        
        single inactivating mutation (95%) OR mutiple mutations none of which can be identified as the inactivating one
        
        Residual BTK signalling and function = mild, later onset OR complete loss of funtion = severe, early onset
        
        only effects XY pairs as BTK is encoded on the X chromosome
        
        diagnosis
        
        Clinical presentation ± family history
        
        measure serum Ig levels (if >1 year)
        
        enumerate CD19+ B cells
        
        ± BTK sequencing
        
        Gene-based Dx
        
        Prenatal (chorionic villus sampling, CVS; amniocentesis)
        
        BTK Sanger sequencing for infants
        
        Gene panel
        
        KRECs newborn screening identify infants (TRECs normal)
        
        treatment
        
        Ig replacement therapy (IRT)
        
        IVIg
        
        SCIg
  - - - SCID
        
        how
        
        diagnosis
        
        Universal Newborn Screening (PCR)
        
        Using KRECS: may or may not be present = b cells present or not
        
        Using TRECS: None = no T cell = SCID
        
        USA, Europe, Australia
        
        Lymphocyte count
        
        -T cells, +/- B cells & NK
        
        Serum Ig level
        
        very little of any isotype
        
        Genetic test
        
        20 known genes for 90% of cases
        
        treatments
        
        depend on the genetic cause
        
        Include
        
        HSCT
        
        timley HSCT is curative
        
        Gene Therapy
        
        US, UK, Europe only
        
        Enzyme therapy
        
        IVRT for ALL
        
        What is it
        
        T cells always absent, +/- B and NK cells
        
        Prevelance: 1:58,000 births worldwide
        
        clinical features
        
        Complete absence/very little serum Ig (all isotypes)
        
        Complete absence of circulating T cells, May be circulating B cells and NK cells
        
        Recurrent
        
        infections
        
        viral, fungal and bacterial infections
        
        Usually diagnosed in first year of life
        
        if untreated, lethal in early childhood
        
        Defects along the pathway of early T cell development (and +/- B and NK cells)
        
        genetic atieology
        
        20 genes discovered so far
        
        T-B-NK+
        
        RAG1/2
        
        Treatments
        
        2 more items...
        
        Artemis
        
        T-B+NK-
        
        g(gamma)c chain (IL2RG)
        
        Treatments
        
        2 more items...
        
        JAK3
        
        T-B+NK+
        
        IL7Ra (IL7R)
        
        Treatment
        
        1 more item...
        
        T-B-NK-
        
        Adenosine
        deaminase (ADA)
        
        Treatments
        
        3 more items...
    - - Hyper-IgM (HIGM) Syndromes
        
        how
        
        defects in help signalling pathways impact...
        
        SHM & AM
        
        CSR
        
        B cell proliferation and GC formation
        
        what is it
        
        clinical features
        
        Usually present in first or second year of life
        
        Decreased IgG, normal/abnormally high levels IgM
        
        Variable symptoms and severity of disease
        
        reccurent
        
        infections
        
        pus-producing (pyogenic) bacterial infections of upper & lower respiratory tract
        
        1 more item...
        
        suseptibilites
        
        Opportunistic infections
        
        pneumonias, intestinal tract infections, fungal infections
        
        Chronic diarrhea > malabsorption > failure to thrive
        
        Autoimmunity
        
        neutropaenia, haemolytic anemia, and/or thrombocytopenia)
        
        Organ hypertrophy
        
        tonsils, hepatosplenomegaly (liver), lymphadenopathy (lymph nodes)
        
        Increased risk of lymphatic cancers
        
        Hodgkin’s/non-Hodgkin’s lymphoma (GI tract & liver)
        
        prevelance
        
        CD40L deficiency; 2:1,000,000 males
        
        AID deficiency ~1:1 000,000 newborns
        
        CD40 deficiency; ?? Very few reported cases
        
        molecular atieology
        
        XL CD40L deficiency (CD40LG; HIGM1)
        
        X-linked
        
        defective B cell proliferation > no GCs > no CSR + no SHM & AM
        
        AR CD40 deficiency (CD40, HIGM3)
        
        Autosomal Recessive
        
        no GCs > no CSR + no SHM & AM
        
        no immune memory, no increased affinity
        
        AR/AD-AID deficiency (AICDA, HIGM2, 4)
        
        Autosomal Recessive
        
        GC can form but no CSR, no SHM and AM
        
        Autosomal Dominant
        
        GC can form but no CSR, but SHM and AM normal
        
        UNG deficiency (UNG, HIGM5)
        
        acts downstream of AID
        
        Autosomal Recessive
        
        GC can form but no CSR, no SHM
        
        treatment and managment
        
        Management
        
        Prophylactic antibiotics, corticosteroids for neutropaenia
        
        Avoiding exposure to infections
        
        coordinated specialist care
        
        immunologists, haematologists, pulmonary clinicians, gastroenterologists
        
        Treatment
        
        Immunoglobulin replacement therapy (IVIG or SCIg)
        
        For XL-HIGM
        
        bone marrow transplantation & gene therapy clinical trials may be considered
      - Common Variable Immunodeficiency
        
        what is it
        
        most prevalent PID
        
        1/25,000
        
        clinical features
        
        susceptibilities
        
        co-morbidites (CM along with infections 70%)
        
        +/- failure to thrive/poor growth
        
        autoimmunity
        
        GI disease
        
        malignancy
        
        lymphoproliferation
        
        infection related (infection only 30%)
        
        recurrent bacterial infections
        
        immune dysfunction
        
        poor vaccine responses
        
        Hypogammaglobulinaemia
        
        reduced IgG; 50% patients also have also reduced IgM, IgA)
        
        diagnosis
        
        exclude all other PIDS
        
        Impaired antibody in response to vaccine challenge
        
        pneumococcal & tetanus
        
        No/reduced isotype switched memory B cells (could be normal)
        
        T cell numbers/function usually normal
        
        Difficult to diagnose young children <4 yr
        
        diagnosis often 9+ years after first symptoms
        
        Delay in diagnosis of 1 year: 4% increased risk of mortality
        
        serum Ig
        
        reduced IgG in all; also reduced IgA/M for 50%
        
        treatment and managment
        
        treatment
        
        lifelong Ig replacement therapy
        (IVIG /SCIg infusions)
        
        management
        
        of complications
        
        prophylactic antibiotics,
        corticosteroids
- - - - From Mice
        
        b. Transgenic Mouse
        
        a. Mouse Hybridoma
        
        Mouse makes plasma cells (from polyclonal resposne)
        
        Plasma cells fused with myeloma to make hybridoma
        
        Fused cells selected for in HAT medium, sorted into single cells, screening for cells that diaplay desired Ab specificty (ELISA)
        
        Expand slected hybridoma to produce mAbs
        
        Mouse immunised with specific antigen
      - From humans
        
        d. Single B cell
        
        Identify new epitopes; sequence and rank important epitopes
        
        Define the structure of Ab necessary for neutralisation
        
        Make vaccine constructs that elicit only certain Abs
        
        eg. mAbs for HIV
        
        c. Phage Display
        
        Step 1: Make a phage library
        
        Step 2: Screen and selection
    - - Mouse -o
      - Chimeric -xi
      - Humanized -zu
        
        via CDR grafting
      - Human -u
      - nomenclature
        
        eg. Rituximab (anti CD20)
    - - Naked Whole mAb
        
        unmodified
        
        antibody-drug-conjugates
      - Fragment mAb
        
        Fab mAb
        
        F(ab) 2 mAb
        
        Fc mAb
        
        scFv mAb
      - Fc fragment fusion mAb
        
        Fc fusion mAb
        
        scFv Fusion mAb
      - Bispecific mAb
      - Fragment, multispecifc mAb
        
        Fab and scFv Multispecific mAb formats
        
        F(ab) 2 bispecific/ F(ab) 2 trispecific
        
        BITE (Bi-specific T cell engagers)
        
        DART (Dual-affinity Re-Targeting proteins)
  - - - CSR, activate effector functions, half life, dissue distribution
    - - 7 amino acid regions = 4 Framework (FW) + 3 hypervariable CDR
        
        CDR1, CDR2 in germline, CDR3 through recombination
  - - - Anti-thymocyte globulin (ATG) in acute renal transplantation rejection
      - Anti-snake venom
      - Pregnancy testing
      - COVID-19 Rapid Ag test
    - - inject with antigen, B cells differentiate into memory B and plasma B, isolate antibodies in serum