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Antibodies and hybridomas. Presented by Nis giladi. Antibodies. Antibodies are the antigen binding protein present on the B-cell membrane and secreted by plasma cells. Secreted antibodies circulate in the blood, where they serve as the

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Antibodies and hybridomas

Antibodies and hybridomas

Presented by Nis giladi


Antibodies
Antibodies

  • Antibodies are the antigen binding protein present on the B-cell

    membrane and secreted by plasma cells.

  • Secreted antibodies circulate in the blood, where they serve as the

    effectors of humoral immunity by searching out and neutralizing

    antigens or marking them for elimination.

  • All antibodies share Structural features, bind to antigen, and

    participate in a limited number of effecter functions.


Antibodies are heterodimers
Antibodies Are Heterodimers

  • Antibody molecules have a common structure of four peptide chains.

  • This structure consists of two identical light (L) chains, polypeptides of about

    25,000 molecular weight, and two identical heavy (H) chains, larger polypeptides

    of molecular weight 50,000 or more.

  • Each light chain is bound to a heavy chain by a disulfide bond, and by such

    noncovalent interactions as salt linkages, hydrogen bonds, and hydrophobic

    bonds, to form a heterodimer (H-L).

  • Similar noncovalent interactions and

    disulfide bridges link the two identical heavy

    and light (H-L) chain combinations to each

    other to form the basic four-chain (H-L)2

    antibody structure, a dimer of dimers.


  • The first 110 or so amino acids of the amino-terminal region

    of a light or heavy chain varies greatly among antibodies of different

    specificity. These segments of highly variable sequence are called V

    regions:VL in light chains and VH in heavy.

  • All of the differences in specificity displayed by different antibodies

    can be traced to differences in the amino acid sequences of V regions. In fact,

    most of the differences among antibodies fall within areas of the V regions

    called CDR, and it is these CDRs, on both light

    and heavy chains, that constitute the antigen

    binding site of the antibody molecule.

  • By contrast, within the same antibody class,

    far fewer differences are seen when one

    compares sequences throughout the rest of

    the molecule. The regions of relatively

    constant sequence beyond the variable

    regions have been dubbed C regions.


Immunoglobulin g igg
Immunoglobulin G (IgG)

  • the most abundant class in serum, constitutes about 80% of the total serum

    immunoglobulin.

  • The IgG molecule consists of two heavy chains g and two k or two l light

    chains There are four human IgG subclasses, distinguished by differences in –

    chain sequence and numbered according to their decreasing average serum

    concentrations: IgG1, IgG2, IgG3, and IgG4.

    Functions:

    -IgG1, IgG3, and IgG4 readily cross the placenta

    and play an important role in protecting the

    developing fetus.

    -IgG3 is the most effective complement activator,

    -IgG1 and IgG3 bind with high affinity to Fc receptors

    on phagocytic cells and thus mediate opsonization.


Immunoglobulin m igm
Immunoglobulin M (IgM)

  • IgM accounts for 5%–10% of the total serum immunoglobulin,

    Monomeric IgM, with a molecular weight of 180,000, is expressed

    as membrane-bound antibody on B cells.

  • IgM is secreted by plasma cells as a pentamer in which five monomer

    units are held together by disulfide bonds that link their carboxyl-terminal heavy chain

    domains .

  • Each pentamer contains an additional Fc-linked polypeptide called the J (joining)

    chain, which is disulfide-bonded to the carboxyl-terminal cysteine residue of two of

    the ten chains.

  • IgM is the first immunoglobulin class produced in a primary

    response to an antigen, and it is also the first immunoglobulin to be synthesized by the

    neonate.

  • Because of its large size, IgM does not diffuse well and therefore is found in very low

    concentrations in the intercellular tissue fluids.


Immunoglobulin a iga
Immunoglobulin A (IgA)

  • Although IgA constitutes only 10%–15% of the total immunoglobulin

    in serum, it is the predominant immunoglobulin class in external secretions such

    as breast milk, saliva, tears, and mucus of the bronchial, genitourinary,and

    digestive tracts. In serum,IgA exists primarily as a monomer.


Immunoglobulin e ige
Immunoglobulin E (IgE)

  • extremely low average serum concentration (0.3 g/ml).

  • mediate the immediate hypersensitivity reactions that are responsible for the

    Symptoms of hay fever, asthma, hives, and anaphylactic shock.


Antibody mediated effector functions
Antibody-Mediated Effector Functions

  • The three major effector functions that enable antibodies to remove antigens

    and kill pathogens are:

    1.opsonization-which promotes antigen phagocytosis by macrophages

    and neutrophils.

    2. complement activation, which activates a pathway that leads to the

    generation of a collection of proteins that can perforate cell membranes.

    3. Antibody dependent cell-mediated cytotoxicity (ADCC), which can

    kill antibody-bound target cells.


In summary
In Summary

  • An antibody molecule consists of two identical light chains and two identical

    heavy chains, which are linked by disulfide bonds.

  • Each heavy chain has an amino-terminal variable region followed by a constant

    region..In any given antibody molecule, the constant region contains

    one of five basic heavy-chain sequences (m,d ,a ,e , or c) called isotypes and one

    of two basic light-chain sequences ( or ) called types.

  • The heavy-chain isotype determines the class of an antibody (, IgM; , IgG; , IgD; , IgA; and , IgE).


  • The five antibody classes have different effector functions,average serum

    concentrations, and half-lives.

  • Immunoglobulins are expressed in two forms: secreted antibody that is

    produced by plasma cells, and membrane-bound antibody.

  • Unlike polyclonal antibodies that arise from many B cell clones and have a

    heterogeneous collection of binding sites, a monoclonal antibody is derived

    from a single B cell clone and is a homogeneous collection of binding sites.


Monoclonal antibodies
Monoclonal antibodies. functions,average serum


Monoclonal antibodies as therapy
Monoclonal antibodies as therapy functions,average serum

  • mAbs are biomolecules that are generally indistinguishable from endogenous

    antibodies.

  • The targets of clinically useful mAbs are usually secreted molecules such as

    cytokines or the extracellular portions of transmembrane proteins such as

    growth factor receptors (GFRs) and adhesion molecules.

  • Such extracellular targets are representative of relevant targets in a wide

    variety of diseases. Examples include ErbB family members and single-pass

    receptor tyrosine kinases (cancer);tumor necrosis factor (TNF)-a and other

    cytokines (inflammatory diseases such as ulcerative colitis, rheumatoid

    arthritis, juvenile rheumatoid arthritis, ankylosis spondylitis,psoriatic arthritis,

    and psoriasis.



  • Several chimeric mAbs (eg, rituximab, cetuximab ,Erbitux ) have been

    associated with markedly fewer AARs compared with murine mAbs.

    Nevertheless, the development of the human anti-chimeric antibody (HACA)

    response remains a potentially significant problem that requires close

    monitoring and. HACA response rates of up to 61% have been reported with

    infliximab and have been associated with shorter duration of effect and

    increased risk for infusion reactions.

  • Reducing the immunogenicity associated with mAbs is a critical goal. Because

    of immunogenicity, the successful use of many murine mAbs has been

    restricted to acute indications (eg, acute graft rejection, imaging applications).



Human monoclonal technologies
Human Monoclonal Technologies ‘‘humanization’’


1 hybridoma
1.Hybridoma ‘‘humanization’’

1.Mice are immunized with antigen A and given an intravenous booster immunization three days before they are killed, in order to produce a large population of spleen cells secreting specific antibody. Spleen cells die after a few days in culture. In order to produce a continuous source of antibody.

2. Spleen cells are fused with immortal myeloma cells by using polyethylene glycol (PEG) to produce a hybrid cell line called a hybridoma.


3.The myeloma cells are selected by HAT medium because they lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT). The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture because of the malignant potential contributed by the myeloma cells. Therefore, unfused myeloma cells and unfused spleen cells die in the HAT medium.

4.Individual hybridomas are then screened for antibody production, and cells that make antibody of the desired specificity are cloned by growing them up from a single antibody-producing cell.

5. The cloned hybridoma cells are grown in bulk culture to produce large amounts of antibody. As each hybridoma is descended from a single cell, all the cells of a hybridoma cell line make the same antibody molecule, which is thus called a monoclonal antibody.


2 xenografting transplantation in scid
2. lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT). The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture because of the malignant potential contributed by the myeloma cells. Therefore, unfused myeloma cells and unfused spleen cells die in the HAT medium.Xenografting transplantation in SCID

  • One way to circumvent murine sequence-related complications is to reconstitute a human immune system in severe combined immune deficiency (SCID) mice.

  • By xenografting fetal human tissues in these mice, it is possible to recapitulate

    the diversity of the human immune system within an animal that can be

    immunized and killed for retrieval of splenocytes for subsequent fusion.

  • The major disadvantagein that the human xenograft is not passed on through

    the germ line, necessitating regeneration of xenograft mice for

    development of each specific mAb.


  • Two alternative strategies now at the forefront of human lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT). The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture because of the malignant potential contributed by the myeloma cells. Therefore, unfused myeloma cells and unfused spleen cells die in the HAT medium.

    mAb production largely circumvent the need for human

    donors and are contributing numerous therapeutic

    molecules and candidates.


3 phage display based generation of human antibodies
3.Phage Display-Based Generation of lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT). The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture because of the malignant potential contributed by the myeloma cells. Therefore, unfused myeloma cells and unfused spleen cells die in the HAT medium.Human Antibodies

  • The technique takes advantage of filamentous phage (bacterial viruses) to

    isolate genes based on their protein products Phage display synthesis of

    human mAbs. Variable (VH and VL) domains from hybridomas or pools of B

    cells are cloned by reverse transcriptase polymerase chain reaction (PCR). The

    PCR fragments are inserted into gene III offilamentous phage. The domains

    are joined by a short peptide linker and associate via framework region-

    mediated interactions,generating a scFv.


Display of the pIII-scFv fusion on the phage coat allows it to interact with

antigen. High-affinity scFvs bind phage to a immobilized antigen during

multiple highstringency washes, whereas low-affinity phage are removed

(‘‘panning’’). Adherent phage are then eluted and used to

infect Escherichia coli. The scFv can then be altered through

directed or random mutagenesis, creating subpools of phage

with novel binding characteristics.

When optimized, the VH and VL domains can then be cloned into heavy and

light chain expression vectors

and transfected into hybridoma

cells for expression of

conventional mAbs.


4 fully human antibodies from transgenic mice
4.Fully Human Antibodies From to interact with Transgenic Mice

  • Generation of transgenic mice in which the murine immunoglobulin genes

    have been disrupted and replaced with human immunoglobulin gene clusters.

  • Conventional transgenic strategies had to be adapted, because the

    immunoglobulin genes are arranged in large clusters, rendering their cloning

    quite arduous:the human heavy, l-light, and k-light chain loci,located on

    chromosomes 14, 22, and 2, respectively, span more than 1 megabase each.


D end to interact with


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