Monoclonal Antibodies

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Monoclonal Antibodies

• In Nuclear Medicine Understanding The Basics

2
Information on You Tube

• Suggested review prior to this lecture
• Role of B Lymphocytes
• http//www.youtube.com/watch?vZ36dUduOk1Y
• Production of Monoclonal Antibodies
• http//www.youtube.com/watch?vO-SrPqJuEVg

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Basic Concepts

• Antibodies Ab (Immunoglobulins Ig) are
  produced by plasma cells in response to foreign
  substances (Antigens Ag)
• Ag are usually 1,000 daltons or more in size
• Ig possess specific binding regions on the
  surface that recognize the shape of particular
  sites (determinants) on the surface of an Ag
• Ab binds to the Ag in an immunological response
  destroying the Ag
• Whats the size of a dalton?

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Ab Response

• Ag usually have several determinants or epitopes
• Each epitope stimulates one or more B lymphocytes
• B lymphocytes can differentiate into plasma cells
  that secrete a specific Ig response to a
  determinate on the Ag
• Hence B lymphocytes create plasma cells and the
  plasma cells produce a host of different Ab in
  response to the Ag

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How Antibodies are developed

• Using a mouse or rabbit it is immunized with Ag
  agent ex. Cancerous tissue is placed underneath
  the skin
• This creates an Ab to Ag response within the host
• Serum can then be extracted from the host that
  has the Abs which were created from the different
  epitopes on the Ag surface
• This is known as a polyclonal response because
  there are many types of Abs that were produced
  from the inoculation of a specific Ag
• Refer to the image on the next side to define the
  process

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Ab To Ag Response

• This side shows the
• mouse response
• to the Ag that creates
• polyclonal Ab. Important points
• Ag is injected into host
• Lymphocytes respond to Ag and produce Abs
• Taking lymphocytes from host you can fuse this
  with Myeloma Cells resulting in hybrid Ab

• These polyclonal Abs can be separated into MoAbs
• Refer to the
• side next to
• note MoAb
• Production

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Where Do MoAbs Come From?

• Lymphocytes or Plasma cells are extracted from
  the mouse and fused with myeloma cells
• This creates a hybrid myeloma cells
• They are cloned
• Specific MoAbs are then grown in culture

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A closer look at the process in which MoAbs are
produced. The process is discussed on the
following slides
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Creating the MoAb For Medical Use

• Extract the splenic lymphocytes from an immunized
  mouse along with the myeloma cell line
• Fusing these cells creates a hybridoma cells
• Fusing occurs in a polyethylene glycol solution
  where the cell will multiple
• Selected hybridoma cells are then grown in a
  hypoxanthene-aminopterin-thymidine (HAT) medium
  (only fused cells survive)
• These cells can then be separated for assay and
  are re-cultured (re-cloned) until the right MoAb
  is found

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The Immunoglobulins (Ig)

• There many types of Igs IgG, IgM, IgE, IgA, and
  IgD
• Usually hybridomas are developed from some form
  of IgG or its subclass
• The next slide demonstrates the structure of an
  IgG MoAb

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IgG Structure

• Variable region (light chain) respond to the
  different to the epitopes on the Ag surface
• Constant or heavy region remains the same
• These chains are held together by a disulfide
  bonds
• Note that the IgG structure can be fragmented via
  pepsin or papain

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Fragmented F(ab)2 ,Fab, and Fc

• Removal of most or all of the heavy change causes
• Reduce HAMA response
• Allows for faster clearance after injection
• MoAbs have been created from all of the above
  mentioned types
• Can you identify a whole IgG and fragmented IgG
  used in nuclear medicine?
• Discuss some of its imaging properties
• Whole IgG is (50,000 daltons or greater) that
  metabolize in the liver, while fragmented are
  much smaller and quickly excreted by the kidneys

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Some other points

• Total mol.wt. of an IgG is up to 150,000
  daltons
• Affinity refers to the strength of attraction
  between the Ab-Ag
• Avidity refers to the integrity of the Ab-Ag bond
• Both affinity and avidity are important in order
  for specific/strong tag to occur between the Ab-Ag

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Comment on HAMA

• A mouse contains murine Abs
• Human response to this could be a human anti-body
  (HAMA) reaction
• Human-human hybridomas should be considered to
  reduce the HAMA response
• HAMA is an allergic reaction
• Anaphylactic is the most server and if left
  unchecked could cause death
• Interferes with imaging can also occur creating
  a false negative image (the body has prevented
  Ab-Ag to occur because HAMA interferes)

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Finding the Right Radionuclide Tag

• Consider
• Therapy vs. diagnostic (beta vs. gamma)
• Type of radionuclide (Tc99m vs. In111)
• Tagging whole vs. fragmented IgG

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Therapy vs. Diagnostic

• Therapy
• If beta radiation is used particle radiation can
  destroy the disease
• Requires a strong/stable Ab-Ag reaction
• Diagnostic
• Gamma radiation is used
• Disease is identified
• Requires a strong/stable Ab-Ag reaction

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The Real Thing

• Is this whole or fragmented?
• Identify the parts of the MoAb
• What would you tag to it? Therapy vs.
  Diagnostic
• Where is the tag?
• What is this type of tag called?

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Type of Radionuclide

• If the radio-MoAb is the entire IgG
• Takes a long time to clear or get a good target
  to background (72 or more hours)
• Requires a radionuclide such as In111
• Fragmented IgG
• Clears quickly and gives a better target to
  background (usually within 24 hours)
• Tc99m can be used

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More on the Whole IgG MoAb

• When using In111
• DTPA is used so that the In111 tags to the heavy
  change of the MoAb
• In111 DTPA MoAb (on the heavy change)
• In111 is then introduced and tags to the DTPA
• Remember In111 must be used if the MoAb being
  used requires significant filtering by the body,
  over time
• What other gamma emitters could we use if a whole
  IgG MoAb is being used?

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MoAb Whole vs. Fragmented
Both agents are used to image Colon
cancer OncoScints MoAb is whole, its
tracer 111In, injection to scan is 3
days Arcitumomabs MoAb is fragmented, Tagged
to 99Tc, injection to scan is 24 hours What is
your assessment of background activity in both
procedures?
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Complications of the Radioactive Tag

• Radiolabeling may alter the biological activity
  of the MoAb, rendering it either less infective
• Immunoreactive fraction is a concern and results
  when free MoAb dissociates from the radioactive
  tag
• Immunospecificity is another issue where
  specificity of the agent can be lost. This can
  occur by any one of the following
• Blood flow
• Metabolism
• Capillary permeability

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Target to Background Ratios

• Minimal requirement 21, but 51 is preferred
• Digital subtraction and image contrast can help,
  however, a higher false-positive may occur
• Theoretically 1001 to 10001 ratios should be
  attained, however, this has never happened
• To make an idea MoAb consider the following
  MoAb clearance, reducing background, reducing
  dosimetry, and reducing HAMA favor the fragmented
  MoAb

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Clinical Status

• MoAbs have been created to identify
• Lung Cancer
• Prostate Cancer
• Colon Cancer
• Infection
• Lymphoma
• Still lacks sensitivity and specificity
• Is it the poor mans PET?
• When you look at the sensitivity and specificity
  of PET over MoAbs, you might conclude that MoAbs
  are the inferior scan (see PET lecture)

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Review of the Issues

• Excessive background (more so with whole IgG)
• Ionization of the radioactive tag (losses
  specificity)
• Cross-reactivity with non-specific Ag (goes where
  you dont want it to)
• Variation of expression Ab-Ag can result in a
  false negative study
• HAMA response to the IgG reduces image quality
• Alternate routes of administration should be
  considered

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ProstaScint and CEA Scan
This is an example of Prostate mets
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MDP vs MoAb
http//www.nature.com/ncpuro/journal/v3/n4/fig_tab
/ncpuro0452_F2.html
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For more information you can access the following
article Monoclonal Antibodies in Nuclear
Medicine, by AM. Keenan, Et al. Jour NM, May
1985 10/14
http//www.med.harvard.edu/JPNM/physics/pharms/rad
pharm/antibod/FDAnov96.html
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