1. Monoclonal Antibodies
In Nuclear Medicine – Understanding The Basics

2. Information on You Tube
Suggested review prior to this lecture
Role of B Lymphocytes
Production of Monoclonal Antibodies

3. 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
What’s the size of a dalton?

4. 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

5. 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

6. 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

7. 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

8. A closer look at the process in which MoAbs are produced.
The process is discussed
on the following slides

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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)

15. Finding the Right Radionuclide Tag
Consider
Therapy vs. diagnostic (beta vs. gamma)
Type of radionuclide (Tc99m vs. In111)
Tagging whole vs. fragmented IgG

16. Therapy vs. Diagnostic Therapy Diagnostic
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

17. 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?

18. 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

19. 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?

20. MoAb – Whole vs. Fragmented
Both agents are used to image
Colon cancer
OncoScint’s MoAb is whole, it’s tracer
111In, injection to scan is 3+ days
Arcitumomab’s MoAb is fragmented,
Tagged to 99Tc, injection to scan is
24 hours
What is your assessment of
background activity in both
procedures?

21. 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

22. Target to Background Ratios
Minimal requirement 2:1, but 5:1 is preferred
Digital subtraction and image contrast can help, however, a higher false-positive may occur
Theoretically 100:1 to 1000:1 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

23. 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 man’s 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)

24. 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 don’t 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

25. ProstaScint and CEA Scan
This is an example of
Prostate mets

26. MDP vs MoAb

27. For more information you can access the following article:
“Monoclonal Antibodies in Nuclear Medicine”, by AM. Keenan,
Et al. Jour NM, May 1985
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