Monoclonal Antibodies

Monoclonal antibodies

What is an antibody?

An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Polyclonal antibodies are antibodies that are derived from different cell lines. Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Isotypes According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE. IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid. IgA:IgA1 (90%) and IgA2 (10%), stomach and intestines IgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked with disulfide bonds IgD:1% of proteins in the plasma membranes of B-lymphocytes, function unknown IgE: on the surface of plasma membrane of mast cells, play a role in immediate hypersensitive and denfensive for parasite

The IgG Class of Antibodies 



All current therapeutic antibodies are of the IgG class. When the objective of antibody therapy is to directly kill the target cell, the isotype of choice is IgG1, since this isotype is optimal for complement fixation.

The structure of antibodies

  

Fab = Fragment, antigen binding Fc = Fragment, crystalline The Fc fragment specifies biological properties of the molecule, such as complement fixation, placental transfer, skin fixation and catabolic rate.

In a specific immune response, only those T and B cells that can bind to the antigens of the pathogen are selected to participate in the response. Mixture of T and B cells with different antigen specificities

An antigen with 2 epitopes  ­ red epitope, blue epitope

Proliferation of cells  with receptors capable of binding epitopes of the antigen

Clonal selection of lymphocytes during the specific immune response

In 1975, Georges Kohler and Cesar Milstein first fused lymphocytes to produce a cell 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 the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.

Polyclonal antibodies: Polyclonal antibodies are a mixture of antibodies with different antigen binding sites  that may bind to different epitopes or antigens of the immunizing agent with varying  affinities.  They may be of different antibody 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 present in the immunizing inoculum.

Protein Immunize Immune Response

Antibodies

Epitopes

B B B B

A mixture of antibodies ­ all bind to epitopes  of the original antigen.  Some bind with higher  affinity than others. 

Polyclonal antibodies

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 antigen­binding sites.  Thus they all  bind to the same epitope with the same affinity.  They are all of the same  antibody class (isotype).

B B

Polyclonal antibodies    (Polyclonal antiserum)

B B

B

Harvest Ab

B

B

B

B

B

B

B

B

Monoclonal antibodies

Hybridomas Technique - B lymphocytes can mutate into tumor cells that result in a type of cancer termed myeloma. - Myeloma cells become “immortal” and will grow indefinitely in culture. - Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture.

Hybridoma Selection The “HAT Trick” ✦

✦

Myeloma cells have been genetically engineered such that they can not use hypoxanthine, aminopterin, and thymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture. Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium.

Secrete antibody but don't grow  in tissue culture

B

Myeloma cells

M M M

FUSE

M

M

M M

M

M

Grow indefinitely in  cell culture but don't  secrete the desired  antibody Grow indefinitely in  cell culture AND  secrete antibody

Hybridoma cells

Harvest Ab

Monoclonal antibodies

Polyclonal antibodies            Monoclonal Antibodies Many B cell clones

A single B cell clone

Multiple epitopes of all antigens used in the  immunization

A single epitope of a single antigen

Antibody class:

A mixture of different Ab classes (isotypes)

All of a single Ab class

Ag­binding sites:

A mixture of Abs with different antigen­binding sites

All Abs have the same antigen binding site

Produced by: Bind to:

 

Potential for cross­reactivity: High

Low

Monoclonal Antibodies Purified antigen  molecule

Inject antigen into mouse

Kill the mouse and remove  the spleen (containing antibody  generating cells) spleen

Each cell may have produced a  different antibody

spleen

fuse dissociate

+

A Hybrid cell that can be cultured

“immortalized”   cell

Select one of these "hybrid" cells to subculture….

… and produce a  mono clonal population

All the cells secrete only one type of antibody directed against one epitope

High degree of specificity ... …but Low degree of “sensitivity”­­ ability to detect small amounts of  antigen

antigen

Practical steps in monoclonal antibody production: 1) Immunize animal 2) Isolate spleen cells (containing antibody-producing B cells) 3) Fuse 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 you’re a biotech company) charge about $1,000-$2,000 per mg.

Kuby Figure 4­22

Ras ing ascit ic f luid

The 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 murine antibody) response, which lead to allergic or immune complex hypersensitivities. B. Chimeric mAbs: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity unchanged. Also cause human antichimeric antibody response (30% murine resource) C. Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework

Humanized Monoclonal Antibodies ✦

✦

Mouse monoclonal antibodies have been genetically engineered to replace all of the antibody molecule with human counterparts except the hyper variable regions directly involved with antigen binding. Humanized monoclonal antibodies are currently be tested in human clinical trials.

USES Measuring protein and drug levels in serum Typing tissue and blood Identifying infectious agents Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas Identifying tumor metastasis Identifying and quantifying hormones Immunoaffinity Purification









Used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e.g. monoclonal antibodies to HCG used in pregnancy test kits or diagnosis of AIDS by the ELISA test. monoclonal antibody can be coupled to another molecule like a fluorescent molecule to aid in imaging the target Or with a strongly-radioactive atom, such as Iodine-131 to aid in killing the target. Used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only cancerous cells.









Monoclonal antibodies can be used to treat viral diseases, traditionally considered "untreatable". In fact, there is some evidence to suggest that antibodies may lead to a cure for AIDS. Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria gonorrhoeae can be typed using mAB. To identify and to trace specific cells or molecules in an organism. OKT3, an antibody to the T3 antigen of T cells, is used to alleviate the problem of organ rejection in patients who have had organ transplants.

Affinity chromatography: 3.

Bind antibody to a support matrix (e.g. sepharose gel)

5.

Add protein mixture ­ antigen binds to antibody on support

7.

Wash to remove unbound material

9.

Lower pH ­ antibody releases the antigen ­ which is now free of contaminants

Figure 6.24

Affinity chromatography ­ antibody purification. Antigen can be bound to the support matrix in  order to purify antigen­specific antibody from a  polyclonal antiserum.

From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html

Tumor diagnosis in vitro

in vivo

99m

Tc­mAb

Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. 

mAbs act directly when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function.



mAbs can be modified for delivery of a toxin, radioisotope, cytokine or other active conjugates.



it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell



Herceptin (Trastuzumab) is an anti-cancer antibody that acts on HER2/neu (erbB2) receptor, which is overexpressed in breast cancer. Only cells overexpressing this receptor are susceptible.



Gemtuzumab ozogamicin (Mylotarg) monoclonal antibody attacks the CD33 receptor, which is found in most leukemic blast cells, but not in normal hematopoietic stem cells

Until Feb 28, 2005, 18 mAbs were approved by FDA, which were applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic malignancies and psoriasis.

The first approved mAbs was OKT-3, which is a murine IgGa2 protein to deplete T cells in patients with acute rejection of renal allotransplant