Monoclonal Antibodies and Cancer Therapy

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Monoclonal Antibodies and Cancer Therapy
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Definition

• Mono One
• Clone A strain of cells descended form a single
  cell
• Antibody A molecule of animal origin that has
  immunolgical activity only against the antigen to
  which it was made

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Whats an ANTIGEN?

• Antigens are soluble substances
• They may be toxins or foreign proteins
• They may be whole cells OR just parts of a cell,
  like a component of the cell wall
• Proteins usually make the best antigens
• Very small proteins (peptides) can be coupled
  (attached) to carriers so they make better
  antigens

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How to make a monoclonal antibody?

• Step 1 Immunize mice
• Step 2 Test the serum
• Step 3 Perform a fusion
• Step 4 Screen the fusion for the right cells
• Step 5 Grow the hybridomas
• Step 6 Harvest the antibody
• Step 7 Concentrate and purify the product

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Step 1 Immunize the mice - Housing

• Can uses Balb C mice to make monoclonals.
• The mice are housed in a laminar flow hood to
  minimize their exposure to other antigens.
• Each box contains mice for only one project.

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Step 1 Immunize the mice - Identification

• Each mouse has a microchip implanted to identify
  it.
• Each time the mouse is inoculated or bled, the
  chip is checked.

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Step 1 Immunize the mice - Inoculation

• The mice are aseptically inoculated with the
  antigen combined with an adjuvant.
• Inoculations are done either sub-cutaneously or
  intra-peritoneally.
• Normal dose per mouse is between 50 and 100
  micrograms of protein.
• Inoculations are performed every 21 to 28 days.

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Step 2 Test the serum - Bleeding the mice

• Blood is drawn into the tube.

• A capillary tube is applied to nicked vein.

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Step 2 Test the serum - In the Lab

• Whole blood from the immunized mice is
  transported to the lab.
• The whole blood is spun down in a centrifuge to
  separate the serum.
• The serum is diluted in a series from 1300 to
  1300,000.

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Step 2 Test the serum - ELISA

• Diluted serum is tested by an ELISA (Enzyme
  Linked ImmunoSorbent Assay)
• Color change in the ELISA substrate indicates the
  amount of antibody present in the diluted serum.

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Step 2 Test the serum - Decision time

• When the serum titer of the mice has reached a
  plateau, an additional ELISA test is performed to
  determine the predominant isotype present. The
  two isotypes that are most common in mouse serum
  are IgG and IgM.

• Sometimes additional testing is done (Western
  blots, immunoflurorescence) to determine whether
  the serum response is specific for the selected
  antigen.
• The mouse with the strongest, most specific
  response is chosen for the fusion.

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Step 3 Perform a fusion - Feeder Plates

• 1) Euthanize an ICR mouse.
• 2) Aseptically open the peritoneal cavity of the
  mouse.
• 3)Use sterile media and a pipet to flush
  macrophage cells from the peritoneal cavity.

• 4) Dilute the macrophage cells in media.
• 5) Distribute the diluted macrophage cells into
  96-well plates.
• 6) Allow the feeder cells to grow for 24 hours.

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Step 3 Perform a fusion - Prepare spleen cells

• 1) Anesthetize the best responder among the
  immunized mice.
• 2) Perform a cardiac puncture and withdraw whole
  blood from the mouse.
• 3) Euthanize the mouse.
• 4) Aseptically open the mouses abdomen.

• 5) Dissect away the peritoneal membrane to expose
  the spleen.
• 6) Carefully remove the spleen to a sterile petri
  dish.
• 7) Make a single cell suspension of the spleen.

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Step 3 Perform a fusion - Hybridomas

• 1) Dilute the spleen cells.
• 2) A special line of tumor cells (SP 2/0) has
  already been grown in a flask.
• 3) Mix the spleen cells with the tumor cells.

• 4) Add a chemical agent to the mixture to
  soften the cell membranes.
• 5) Place the now-fused spleen/tumor cells, called
  hybridomas, on top of the feeder cell layer in
  the 96-well plate.

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Step 3 Perform a fusion - Growing the cells
(hybridomas)

• Fusions are performed in a lab.

• Cells are grown in 96-well plates.

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Step 3 Perform a fusion - Growing the cells
(hybridomas)

• Cells are grown in a 37o C incubator.
• Cells are kept in an atmosphere of about 6 CO2.
• The cells are fed after 7 days of incubation.
• The cells are checked for growth after 10 days of
  incubation.

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Step 4 Screen the fusion for the right cells

• 1) When growth is detected in one of the wells of
  the 96-well plate, cell supernatant from that
  well is tested by ELISA.
• 2) If the well tests positive, cells from it are
  removed and diluted. The diluted cells are
  plated into new 96-well plates at a dilution that
  will contain only one cell per well.

• 3) The wells are tested again and positive wells
  are re-diluted. This process is called cloning.
• 4) The object of cloning is to obtain a
  population of identical cells all producing the
  same antibody - monoclonal cells.

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Step 5 Grow the hybridomas - 24-well plates

• Once a monoclonal population of cells is
  obtained, they are expanded to produce the
  desired amount of antibody.
• The antibody is produced by the cells and
  released into the cell supernatant.
• First, the monoclonal cells are expanded into
  24-well plates.

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Step 5 Grow the hybridomas - Flasks

• Then, when the cells in the 24-well plates are
  growing well and look healthy, they are
  transferred into flasks.
• Cells in the flasks are expanded, as needed.
• Some cells are preserved for future use by
  freezing.

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Step 6 Harvest the antibody

• 1) The antibody is produced by the cells and
  released into the cell supernatant.
• 2) Media is added to the flasks until the desired
  volume of antibody-containing supernatant is
  obtained.

• 3) When the desired volume is reached, the
  supernatant is refrigerated and tested by ELISA.
• 4) If the testing is successful, the supernatant
  can be used by the investigator, or concentrated
  and purified.

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Step 7 Concentrate and Purify the Product

• The antibody can be concentrated by precipitation
  with a solution of saturated ammonium sulfate.
• Concentrated antibody can then be purified by
  passing it through a column.

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So, why make monoclonal antibodies?

• Monoclonal antibodies can be used in three
  general ways
• to purify reagents for tests and research
• as labeling agents for detection assays
• for experimental therapy.

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More specifically, monoclonal antibodies can

• Be attached to a color agent of fluorescent
  chemical for immunological staining.
• Be attached to a medium in a column to purify
  other substances that will bind to them (affinity
  purification).
• Coat plates for ELISA testing, as for a
  diagnostic test.

• Be used as therapy by attaching to a particular
  target cell and marking it for lysis by natural
  killer cells or medicines
• Be radiolabeled (attached to an isotope) for
  diagnostic imaging such as PET (Posirton Emission
  Technology)

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Cancer Therapy
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Cancer therapy example 1
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Cancer therapy example 2
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