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Monoclonal antibodiesMonoclonal antibodies
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Wh at is an antibody?Wh at is an antibody?AnAn antibodyantibody is a protein used by the immune system to identify and neutralizeis a protein used by the immune system to identify and neutralize
foreign objects like bacteria and viruses. Each antibody recognizes a specificforeign objects like bacteria and viruses. Each antibody recognizes a specificantigen unique to its target.antigen unique to its target.
Polyclonal antibodiesPolyclonal antibodies are antibodies that are derived from different cell lines.are antibodies that are derived from different cell lines.
Monoclonal antibodiesMonoclonal antibodies ((mAbmAb ) are antibodies that are identical because they were) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. produced by one type of immune cell, all clones of a single parent cell.
IsotypesIsotypesAccording to differences in their heavy chain constant domains, immunoglobulinsAccording to differences in their heavy chain constant domains, immunoglobulinsare grouped into five classes, or isotypes:are grouped into five classes, or isotypes: IgG IgG ,, IgA IgA,, IgM IgM ,, IgD IgD , and, and IgE IgE ..
IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid.IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid.
IgA:IgA:IgA1 (90%) and IgA2 (10%), stomach and intestinesIgA1 (90%) and IgA2 (10%), stomach and intestinesIgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked withIgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked withdisulfide bondsdisulfide bonds
IgD:IgD:1% of proteins in the plasma membranes of B1% of proteins in the plasma membranes of B- -lymphocytes, function unknownlymphocytes, function unknownIgE: on the surface of plasma membrane of mast cells, play a role in immediateIgE: on the surface of plasma membrane of mast cells, play a role in immediate
hypersensitive and denfensive for parasitehypersensitive and denfensive for parasite
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T h e IgG Class of AntibodiesT h e IgG Class of Antibodies
All current therapeutic antibodies are of theAll current therapeutic antibodies are of theIgG class.IgG class.
When the objective of antibody therapy is toWhen the objective of antibody therapy is todirectly kill the target cell, the isotype of directly kill the target cell, the isotype of choice is IgG1, since this isotype is optimal for choice is IgG1, since this isotype is optimal for complement fixation.complement fixation.
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T h e structure of antibodiesT h e structure of antibodies
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F ab = F ragment, antigen bindingF ab = F ragment, antigen bindingF c = F ragment, crystallineF c = F ragment, crystalline
The F c fragment specifies biological propertiesThe F c fragment specifies biological propertiesof the molecule, such as complement fixation,of the molecule, such as complement fixation,
placental transfer, skin fixation and catabolic placental transfer, skin fixation and catabolicrate.rate.
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In a specific immune response, only those T and B cells that can bind to the antigensof the pathogen are selected to participate in the response.
Clonal selection of lymphocytes during the specific immune response
An antigen with 2 epitopes- red epitope, blue epitope
Mixture of T and B cellswith different antigenspecificities
Proliferation of cellswith receptors capableof binding epitopes of the antigen
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In 1975 , Georges Kohler and Cesar Milstein first fused lymphocytes to produce acell line which was both immortal and a
producer of specific antibodies. The two
scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this"hybridoma ." The value of hybridomas to thefield was not truly appreciated until about 1987,when MAbs were regularly produced in rodentsfor diagnostics.
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Polyclonal antibodies:
Polyclonal antibodies are a mixture of antibodies with different antigen binding sites that may bind todifferent epitopes or antigens of the immunizing agent with varying affinities. They may be of differentantibody classes.
The serum obtained from an immunized animal is referred to as a polyclonal antiserum.
A polyclonal antiserum contains antibody to different epitopes and different antigens that were presentin the immunizing inoculum.
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Epitopes
Immune Response Antibodies
A mixture of antibodies - all bind to epitopes of theoriginal antigen. Some bind with higher affinity thanothers.
Polyclonal antibodies
Protein
Immunize
B
B
B
B
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Monoclonal antibodies:
Antibodies produced from a single clone of B cells.
Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture.
Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same
epitope with the same affinity. They are all of the same antibody class (isotype).
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B
B
B
B
Polyclonal antibodies(Polyclonal antiserum)
B
B B B B B B B B
Harvest AbMonoclonal antibodies
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-- B lymphocytes can mutate into tumor B lymphocytes can mutate into tumor cells that result in a type of cancer termedcells that result in a type of cancer termedmyeloma.myeloma.
-- Myeloma cells become immortal andMyeloma cells become immortal andwill grow indefinitely in culture.will grow indefinitely in culture.
-- F usion of a single activated B cell and aF usion of a single activated B cell and amyeloma cell will create a hybridomamyeloma cell will create a hybridomathat can grow indefinitely in culture.that can grow indefinitely in culture.
H ybridomas T ech nique
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Myeloma cells have been geneticallyMyeloma cells have been geneticallyengineered such that they can not useengineered such that they can not usehypoxanthine, aminopterin, and thymidinehypoxanthine, aminopterin, and thymidine(HAT medium) as a source for nucleic acid(HAT medium) as a source for nucleic acid
biosynthesis and will die in culture. biosynthesis and will die in culture.Only B cells that have fused with theOnly B cells that have fused with theengineered myeloma cells will survive inengineered myeloma cells will survive inculture when grown in HAT medium.culture when grown in HAT medium.
H ybridoma SelectionT h e H AT T rick
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B
Harvest AbMonoclonal antibodies
M
M
M
M
M
M
M
M
MMyeloma cells
Grow indefinitely in cellculture but don't secretethe desired antibody
FUSE Hybridoma cells
Secrete antibody but don't growin tissue culture
Grow indefinitely in cellculture AND secreteantibody
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Polyclonal antibodies Monoclonal Antibodies
Produced by: Many B cell clones A single B cell clone
Bind to: Multiple epitopes of all A single epitope of a singleantigens used in the antigenimmunization
Antibody class: A mixture of different All of a single Ab class Ab classes (isotypes)
Ag-binding sites: A mixture of Abs with All Abs have the same antigendifferent antigen-binding binding sitesites
Potential for cross-reactivity: High Low
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Purified antigenmolecule
Inject antigen into mouse
spleen
Kill t h e mouse and removeth e spleen (containing antibody
generating cells)
Monoclonal Antibodies
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E ac h cell may h ave produced a different antibody
spleen
dissociate+
immortalizedcell
fuse
A H ybrid cell t h at can be cultured
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Select one of t h ese " h ybrid " cells to subculture .
and produce amono clonal population
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All the cells secrete onlyAll the cells secrete only oneone type of antibody directedtype of antibody directedagainstagainst oneone epitopeepitope
antigen
H igh degree of specificity ...
but Low degree of sensitivity--ability to detect small amounts of antigen
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Practical steps in monoclonal antibody production:
1) Immunize animal
2) Isolate spleen cells (containing antibody-producing B cells)
3) F use spleen cells with myeloma cells (polyethylene glycol, virus, by electroporation)
4) Allow unfused B cells to die
5) Add aminopterin to culture to kill unfused myeloma cells
6) Clone remaining cells (place 1 cell/well and allow each cell to grow into a clone of cells)
7) Screen supernatant of each clone for presence of the desired antibody.
8) Grow the chosen clone of cells in tissue culture indefinitely.
9) Harvest antibody from the culture supernatant.
10) (If youre a biotech company) charge about $1,000-$2,000 per mg.
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Kuby Figure 4-22
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Rasing ascitic fluid
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T h e types of mAbT h e types of mAb
A. Murine source mAbs: rodent mAbs with excellent affinities and
specificities, generated using conventional hybridoma technology.Clinical efficacy compromised by HAMA(human anti murineantibody) response, which lead to allergic or immune complexhypersensitivities.
B. Chimeric mAbs: chimers combine the human constant regions withthe intact rodent variable regions. Affinity and specificity unchanged.
Also cause human antichimeric antibody response (30% murineresource)
C. Humanized mAbs: contained only the CDRs of the rodent variableregion grafted onto human variable region framework
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Mouse monoclonal antibodies have beenMouse monoclonal antibodies have beengenetically engineered to replace all of thegenetically engineered to replace all of theantibody molecule with human counterpartsantibody molecule with human counterparts
except the hyper variable regions directlyexcept the hyper variable regions directlyinvolved with antigen binding.involved with antigen binding.Humanized monoclonal antibodies areHumanized monoclonal antibodies are
currently be tested in human clinical trials.currently be tested in human clinical trials.
H umanized Monoclonal Antibodies
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M easuring protein and drug levels in serumT yping tissue and bloodIdentifying infectious agentsIdentifying clusters of differentiation for theclassification and follow-up therapy of leukemias and lymphomasIdentifying tumor metastasisIdentifying and quantifying hormonesImmunoaffinity Purification
USES
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Used in several diagnostic tests to detect small amountsUsed in several diagnostic tests to detect small amountsof drugs, toxins or hormones, e.g. monoclonal antibodiesof drugs, toxins or hormones, e.g. monoclonal antibodies
to HCG used in pregnancy test kits or diagnosis of AIDSto HCG used in pregnancy test kits or diagnosis of AIDS by the ELISA test. by the ELISA test.monoclonal antibody can be coupled to another moleculemonoclonal antibody can be coupled to another moleculelike a fluorescent molecule to aid in imaging the targetlike a fluorescent molecule to aid in imaging the target
Or with a stronglyOr with a strongly- -radioactive atom, such as Iodineradioactive atom, such as Iodine- -131131to aid in killing the target.to aid in killing the target.Used in the radioimmunodetection andUsed in the radioimmunodetection andradioimmunotherapy of cancer, and some new methodsradioimmunotherapy of cancer, and some new methodscan even target only cancerous cells.can even target only cancerous cells.
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Monoclonal antibodies can be used to treat viral diseases,Monoclonal antibodies can be used to treat viral diseases,traditionally considered "untreatable". In fact, there istraditionally considered "untreatable". In fact, there issome evidence to suggest that antibodies may lead to asome evidence to suggest that antibodies may lead to acure for AIDS.cure for AIDS.Monoclonal antibodies can be used to classify strains of aMonoclonal antibodies can be used to classify strains of a
single pathogen, e.g.single pathogen, e.g. Neisseria
gonorrh
oeae Neisseria
gonorrh
oeae
can be typedcan be typedusing mAB.using mAB.To identify and to trace specific cells or molecules in anTo identify and to trace specific cells or molecules in anorganism.organism.OKT3, an antibody to the T3 antigen of T cells, is used toOKT3, an antibody to the T3 antigen of T cells, is used toalleviate the problem of organ rejection in patients whoalleviate the problem of organ rejection in patients whohave had organ transplants.have had organ transplants.
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Figure 6.24
Affinity chromatography:
1. Bind antibody to a support matrix (e.g. sepharose gel)
2. Add protein mixture - antigen binds to antibody on support
3. Wash to remove unbound material
4. Lower pH - antibody releases the antigen - which is now free of contaminants
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F rom http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html
Affinity chromatography - antibody purification.
Antigen can be bound to the support matrix in order to
purify antigen-specific antibody from a polyclonalantiserum.
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99 mTc-mAb
in vitro in vivo
T umor diagnosis
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Monoclonal antibodies for cancer treatmentMonoclonal antibodies for cancer treatment
Three mechanisms that could be responsible for the cancer treatment.Three mechanisms that could be responsible for the cancer treatment.
mAbs act directly when binding to a cancer specific antigens andmAbs act directly when binding to a cancer specific antigens andinduce immunological response to cancer cells. Such as inducinginduce immunological response to cancer cells. Such as inducingcancer cell apoptosis, inhibiting growth, or interfering with a keycancer cell apoptosis, inhibiting growth, or interfering with a keyfunction.function.
mAbs can bemAbs can be modified for delivery of amodified for delivery of a toxintoxin ,, radioisotoperadioisotope ,,cytokinecytokine or other active conjugates.or other active conjugates.
it is also possible to designit is also possible to design bispecific bispecific antibodies that can bindantibodies that can bindwith their F ab regions both to target antigen and to a conjugate or with their F ab regions both to target antigen and to a conjugate or effector celleffector cell
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Herceptin (Trastuzumab) is an antiHerceptin (Trastuzumab) is an anti- -cancer antibodycancer antibodythat acts on HER2/neu (erbB2) receptor, which isthat acts on HER2/neu (erbB2) receptor, which isoverexpressed in breast cancer. Only cellsoverexpressed in breast cancer. Only cellsoverexpressing this receptor are susceptible.overexpressing this receptor are susceptible.
Gemtuzumab ozogamicin (Mylotarg) monoclonalGemtuzumab ozogamicin (Mylotarg) monoclonalantibody attacks the CD33 receptor, which is found inantibody attacks the CD33 receptor, which is found inmost leukemic blast cells, but not in normalmost leukemic blast cells, but not in normal
hematopoietichematopoietic stem cellsstem cells
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U ntil Feb 28, 2005, 18 mAbs wereapproved by FDA, which were appliedin the treatment of organ transplant,Cancer, Asthma, Hematopoieticmalignancies and psoriasis.
The first approved mAbs was OKT-3,which is a murine IgGa2 protein todeplete T cells in patients with acuterejection of renal allotransplant