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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.

slide4
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).
slide14
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 as therapy
Monoclonal antibodies as therapy
  • 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.

slide17
Because the patient’s immune response is limiting the therapeutic utility of

mAbs there are major effort to reduce inherent mAb immunogenicity.

by production of chimeric and Humanized mAbs .

slide18
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).

slide19
The next step in mAb evolution referred to as ‘‘humanization’’

intended to replace all rodent sequences except the CDRs regions with

human sequences.

1 hybridoma
1.Hybridoma

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.

slide22
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.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.

slide24
Two alternative strategies now at the forefront of human

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 ofHuman 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.

slide26
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 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.

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