1. Monoclonal Antibodies In Nuclear Medicine – Understanding The Basics

2. Information on You Tube Suggested review prior to this lecture Role of B Lymphocytes Role of B Lymphocytes http://www.youtube.com/watch?v=Z36 dUduOk1Yhttp://www.youtube.com/watch?v=Z36 dUduOk1Yhttp://www.youtube.com/watch?v=Z36 dUduOk1Yhttp://www.youtube.com/watch?v=Z36 dUduOk1Y Production of Monoclonal Antibodies Production of Monoclonal Antibodies http://www.youtube.com/watch?v=O- SrPqJuEVghttp://www.youtube.com/watch?v=O- SrPqJuEVghttp://www.youtube.com/watch?v=O- SrPqJuEVghttp://www.youtube.com/watch?v=O- SrPqJuEVg

3. Basic Concepts Antibodies [Ab] (Immunoglobulins [Ig]) are produced by plasma cells in response to foreign substances (Antigens [Ag]) 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 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 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 Ab binds to the Ag in an immunological response destroying the Ag

4. Ab Response Ag usually have several determinants or epitopes Ag usually have several determinants or epitopes Each epitope stimulates one or more B lymphocytes 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 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 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 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 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 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 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 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 Lymphocytes or Plasma cells are extracted from the mouse and fused with myeloma cells This creates a hybrid myeloma cells This creates a hybrid myeloma cells They are cloned They are cloned Specific MoAbs are then grown in culture 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 Extract the splenic lymphocytes from an immunized mouse along with the myeloma cell line Fusing these cells creates a hybridoma cells Fusing these cells creates a hybridoma cells Fusing occurs in a polyethylene glycol solution where the cell will multiple 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) 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 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 There many types of Igs: IgG, IgM, IgE, IgA, and IgD Usually hybridomas are developed from some form of IgG or its subclass Usually hybridomas are developed from some form of IgG or its subclass The next slide demonstrates the structure of an IgG MoAb 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 Variable region (light chain) respond to the different to the epitopes on the Ag surface Constant or heavy region remains the same Constant or heavy region remains the same These chains are held together by a disulfide bonds These chains are held together by a disulfide bonds Note that the IgG structure can be fragmented via pepsin or papain 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 Removal of most or all of the heavy change causes Reduce HAMA responseReduce HAMA response Allows for faster clearance after injectionAllows for faster clearance after injection MoAbs have been created from all of the above mentioned types MoAbs have been created from all of the above mentioned types Can you identify a whole IgG and fragmented IgG used in nuclear medicine? Can you identify a whole IgG and fragmented IgG used in nuclear medicine? Discuss some of its imaging properties 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 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 Total mol.wt. of an IgG is up to ~150,000 daltons Affinity refers to the strength of attraction between the Ab-Ag Affinity refers to the strength of attraction between the Ab-Ag Avidity refers to the integrity of the Ab-Ag bond 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 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 A mouse contains murine Abs Human response to this could be a human anti-body (HAMA) reaction Human response to this could be a human anti-body (HAMA) reaction Human-human hybridomas should be considered to reduce the HAMA response Human-human hybridomas should be considered to reduce the HAMA response HAMA is an allergic reaction HAMA is an allergic reaction Anaphylactic is the most server and if left unchecked could cause deathAnaphylactic 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) 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 Consider Therapy vs. diagnostic (beta vs. gamma)Therapy vs. diagnostic (beta vs. gamma) Type of radionuclide (Tc99m vs. In111)Type of radionuclide (Tc99m vs. In111) Tagging whole vs. fragmented IgGTagging whole vs. fragmented IgG

16. Therapy vs. Diagnostic Therapy Therapy If beta radiation is used particle radiation can destroy the diseaseIf beta radiation is used particle radiation can destroy the disease Requires a strong/stable Ab-Ag reactionRequires a strong/stable Ab-Ag reaction Diagnostic Diagnostic Gamma radiation is usedGamma radiation is used Disease is identifiedDisease is identified Requires a strong/stable Ab-Ag reactionRequires a strong/stable Ab-Ag reaction

17. Type of Radionuclide If the radio-MoAb is the entire IgG 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)Takes a long time to clear or get a good target to background (72 or more hours) Requires a radionuclide such as In111Requires a radionuclide such as In111 Fragmented IgG Fragmented IgG Clears quickly and gives a better target to background (usually within 24 hours)Clears quickly and gives a better target to background (usually within 24 hours) Tc99m can be usedTc99m can be used

18. More on the Whole IgG MoAb When using In111 When using In111 DTPA is used so that the In111 tags to the heavy change of the MoAbDTPA is used so that the In111 tags to the heavy change of the MoAb In111 – DTPA – MoAb (on the heavy change)In111 – DTPA – MoAb (on the heavy change) In111 is then introduced and tags to the DTPAIn111 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 timeRemember 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?What other gamma emitters could we use if a whole IgG MoAb is being used?

19. Complications of the Radioactive Tag Radiolabeling may alter the biological activity of the MoAb, rendering it either less infective 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 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 Immunospecificity is another issue where specificity of the agent can be lost. This can occur by any one of the following Blood flowBlood flow MetabolismMetabolism Capillary permeabilityCapillary permeability

20. Target to Background Ratios Minimal requirement 2:1, but 5:1 is preferred Minimal requirement 2:1, but 5:1 is preferred Digital subtraction and image contrast can help, however, a higher false-positive may occur 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 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 To make an idea MoAb consider the following: MoAb clearance, reducing background, reducing dosimetry, and reducing HAMA favor the fragmented MoAb

21. Clinical Status MoAbs have been created to identify MoAbs have been created to identify Lung CancerLung Cancer Prostate CancerProstate Cancer Colon CancerColon Cancer InfectionInfection LymphomaLymphoma Still lacks sensitivity and specificity Still lacks sensitivity and specificity Is it the poor man’s PET? 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) When you look at the sensitivity and specificity of PET over MoAbs, you might conclude that MoAbs are the inferior scan (see PET lecture)

22. Review of the Issues Excessive background (more so with whole IgG) Excessive background (more so with whole IgG) Ionization of the radioactive tag (losses specificity) Ionization of the radioactive tag (losses specificity) Cross-reactivity with non-specific Ag (goes where you don’t want it to) 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 Variation of expression Ab-Ag can result in a false –negative study HAMA response to the IgG reduces image quality HAMA response to the IgG reduces image quality Alternate routes of administration should be considered Alternate routes of administration should be considered

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

24. MDP vs MoAb http://www.nature.com/ncpuro/journal/v3/n4/fig_tab/ncpuro0452_F2.html

25. For more information you can access the following article: “Monoclonal Antibodies in Nuclear Medicine”, by AM. Keenan, Et al. Jour NM, May 1985 http://www.med.harvard.edu/JPNM/physics/pharms/radpharm/antibod/FDAnov96.html Return to the Table of Content