Immunotherapy • Treatment that uses the immune system to fight disease • mAb's – Ab’s made from cloned B cells – Same specificity – Benefits
mAB Production • There are currently two widely adopted methods used to produce monoclonal Ab. • In vivo & in vitro • There are advantages and disadvantages for both methods. • The generation of mAb producing cells are typically done in mice. • This method involves fusing a B cell with a myeloma cell (tumor cell)
Preparations of mAb • Step 1: Immunization of mice • Mice are immunized with an antigen (attached to adjuvant). The antigen can be whole cells, membrane fragment, or complex molecules. • Mice serum’s are screened using various techniques such as ELISA. • When sufficient titer is reached the mice are euthanize and spleen is removed as a source of cells for cell fusion. • Step 2: Preparation of Myeloma Cells • Myeloma cells are immortalized cells that are capable of dividing indefinitely. • These cells are treated with 8-azaguanine to ensure sensitivity to HAT
Preparations of mAb • Step 3: Fusion of myeloma cells with Spleen cells • Spleen cells harvested from mice are fused with myeloma cells. • Fission is done through co-centrifusing in polyethylene glycol. • Cells are plated in selection medium • hypoxanthine-aminopterin-thymidine (HAT 1) selection medium – inhibitor of aminoterin which blocks nucleotide synthesis. • Only fused cells with grow on HAT! • Cells are distributed on feeder cells (murine bone-marrow 2) to promote growth of the hybridomal cells.
Preparations of mAb • Step 4: Cloning of Hybridoma cells. • A mouse is inoculated with the cell and thereby becomes a factory for producing the mAB. • Ascites are collected from the mouse. • • • • • •
Step 5: Ab are screened and Purified Ab are screened using specific Ag binding. Advantage of in vivo process Relatively inexpensive and easy Disadvantage: Ethical concerns with using animals.
Preparations of mAb â€˘ Step 6: Desired Ab are cloned â€˘ This is done in vitro on culture bottles
Problems with using mouse mAb • The therapeutic use of rodent monoclonal antibodies in humans is limited by their immunogenic, short circulating half-life, and inability to efficiently trigger human effectors mechanisms. • This is due to differences between the mouse and humans. • Also severe allergic response in human when mouse mAb are introduced to a patients. • Also constant region of marine mAb are not effective in interacting with human effectors molecules.
Chimeric mAb • mAb are genetically engineered using a molecular approach • Chimeric Ab are obtained by genetically fusing the mouse variable domains to human constant domains [Boulianne et al.,1984; Morrison et al., 1984;Wright et al., 1992] • Variable regions are Isolated using polymerase chain reaction (PCR).
Issues with Chimeric mAb â€˘ There are problems that can arise from using mousehuman Ab. sometimes the body may elicit an antichimeric Ab in the present of these genetically engineered Ab.
Humanized mAb • To address this problem, the complementary regions (CDRs), which are the responsible for antigen binding within the variable regions, have been transferred to human frameworks creating ‘‘CDRgrafted’’ or ‘‘humanized’’ antibodies. This is, in essence a human Ab with small segments containing mouse Ab genes.
Examples of FDA approved treatments of certain cancers: MAb Name
Used to Treat:
Acute myelogenous leukemia (AML)
Chronic lymphocytic leukemia (CLL)
Colorectal cancer Head & neck cancers
Monoclonal antibodies ď Ž
MAbs produced from a single clone of B cells
Monoclonal antibodies all have identical antigenbinding sites. Thus they all bind to the same epitope with the same affinity
Mostly produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture
HYBRIDOMA TECHNOLOGY •
1) Immunize animal (mouse or rabbit)
2) Isolate spleen cells (containing antibodyproducing B cells)
3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol)
4) Allow unfused B cells to die
5) Add HAT culture to kill unfused myeloma cells
6) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells)
7) Screen supernatant of each clone for presence of the desired antibody (ELISA)
8) Grow the chosen clone of cells in tissue culture indefinitely.
9) Harvest antibody from the culture supernatant.
Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells PEG (polyethylene glycol) FUSION (by SPLEEN CELLS
making the cell membranes more MYELOMA CELLS permeable) HGPRT-
Plating of Cells in HAT selective Medium
Scanning of Viable Hybridomas
HYBRIDOMA CELLS HAT Medium
Hypoxanthine-guanine phosphoribosyltransferase - is an enzyme in purine metabolism.
Hybridoma Selection The “HAT Trick” •
Myeloma cells have been genetically engineered (HGPRT-) 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
The hybridomas now are ready to be diluted and grown, thus obtaining a number of different colonies, each producing only one type of antibody. The desired antibodies from the different colonies are then should be tested for their ability to bind to the antigen ( ELISA), and the most effective one is picked out.
Reclone and cultivate positive clones â€˘Monoclonal antibodies can be produced in cell culture or in animals. â€˘Unethical to inject hybridoma cells in mice!!!
There is a problem with mAbs derived from â€Śmice
Main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies")
This problem derives from the fact that although antibodies show some conservation there are many sequence differences between rodent antibodies and human antibodies
Human antibodies directly from !humans
It has proven technically much more difficult to immortalize and clone human B-cells and human hybridomas
Also it raises ethical problems of immunizing human donors
Other types of mAb designed
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)
Humanized mAbs: contained only the CDRs of the rodent variable region grafted onto human variable region framework
Recombinant monoclonal antibodies. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. Phage display library: construction of VH and VL gene libraries and expression of them on a filamentous bacteriophage. The phage expressing an antigen-bonding domain specific for a particular antigen to screen the mAbs.
mAbs development â€˘
Transgenic animals: transgenic (genetically engineered) mouse to produce more human-like antibodies (Abgenix,CA)
Such mice have human antibody gene loci inserted into their bodies (using the embryonic stem cell method) Their own genes for making antibodies are "knocked out"
The remarkable specificity of antibodies makes them promising agents for human therapy
Imagine, for example, being able to make an antibody that will bind only to the cancer cells in a patient delivering a cytotoxic agent (e.g. a strong radioactive isotope or a toxin) to that antibody, and then giving the complex to the patient so it can seek out and destroy the cancer cells (and no normal cells) OR MAbs may 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
Affinity chromatography: 1.
Bind antibody to a support matrix (e.g. sepharose gel)
Add protein mixture - antigen binds to antibody on support
Wash to remove unbound material
Lower pH - antibody releases the antigen - which is now free of contaminants
Polyclonal antibodies: If an animal is immunized with a protein, a wide array of B cells will be stimulated to produce anti-protein antibodies. Antibodies may be made to a number of different epitopes of the protein. Even antibodies that bind to the same epitope may have different antigen-binding sites and bind the epitope with different affinity. The mixture of antibodies produced in response to an antigen are referred to as polyclonal antibodies (they are produced by many different clones of B cells).
Protein Immunize Antibodies Immune Response Epitopes
A mixture of antibodies - all bind to epitopes of the original antigen. Some bind with higher affinity than others.
Polyclonal antibodies B B
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.
Affinity chromatography - antibody purification. Antigen can be bound to the support matrix in order to purify antigen-specific antibody from a polyclonal antiserum.
Polyclonal antibodies (Polyclonal antiserum)
Hybridomas Technique B lymphocytes can mutate into tumorcells that result in a type of cancer termed .myeloma Myeloma cells become â€œimmortalâ€? and.will grow indefinitely in culture Fusion of a single activated B cell and amyeloma cell will create a hybridoma .that can grow indefinitely in culture
Secrete antibody but don't grow in tissue culture
Grow indefinitely in cell culture but don't secrete the desired antibody
Grow indefinitely in cell culture AND secrete antibody
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).
Hybridoma Selection The â€œHAT Trickâ€? 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
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 (e.g. using PEG - polyethylene glycol) 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 youâ€™re a biotech company) charge about $1,000-$2,000 per mg.
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
A mixture of different Ab classes (isotypes)
All of a single Ab class
A mixture of Abs with different antigen-binding sites
All Abs have the same antigen binding site
Potential for cross-reactivity: High
Uses 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