Monoclonal Antibodies

Monoclonal Antibodies In Nuclear Medicine – Understanding The Basics

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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?

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

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

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)

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

ProstaScint and CEA Scan This is an example of Prostate mets

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

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

Monoclonal Antibodies