Cover Article: Control Conjugates – The Perfect Companion for Targeted Toxins

Dr. Douglas Lappi, Chief Scientific Officer, Advanced Targeting Systems

The field of targeted toxins has made enormous strides in the years since Advanced Targeting Systems introduced the first research targeted toxin in 1994. We now offer 16 different targeted toxins, with more on the way. The number of important papers in high impact journals continues to rise, and the sophistication of the studies is very impressive.

Much of the research using targeted toxins has been dedicated to characterizing the lesions caused by these molecules. We studied this important work to understand where ATS could be most helpful. The result of our analysis is the offering this year of new control molecules that chemically resemble the targeted toxins, but are not targeted to any cell type.

Saporin alone (not conjugated) has been used by some researchers as a control. But without a stand-in for the targeting agent, there are fundamental differences in physiochemical structure. The new controls solve this problem. Some researchers have included the targeting agent mixing the antibody or peptide with saporin. While this has the components of the targeted toxin, it does not include the structural features of the conjugation chemistry. The new controls solve this difficulty.

ATS targeted toxins fall into three categories: immunotoxins, ligand toxins and second immunotoxins. To provide researchers with more verifiable targeting tools, each of these now has a dedicated control.

Immunotoxins: This category can be further divided into those with antibodies that are based on mouse monoclonals (192-Saporin, OX7- SAP,anti-DBH-SAP,ME20.4-SAP), and those with antibodies based on rat monoclonals (Mac-1-SAP, mu p75-SAP).

For the mouse monoclonals, we offer Mouse-IgG-SAP (Cat. #IT-18), produced by conjugation of mouse IgG to saporin. Mouse IgG replaces the murine monoclonal IgG that performs the specific targeting, and saporin is used in both. The molecular structure of the two is similar, even to the chemistry of conjugation. The only difference is the replacement of the targeting agent with mouse IgG that has no target. Figure 1 shows the same lack of targeted cytotoxicity as saporin alone and approximately 1000-fold less than targeted material.

The rat immunotoxins also have a negative control. Rat IgG-SAP, Cat. #IT-17, is made with rat IgG coupled to saporin with the same chemistry. Figure 2 shows that rat IgG-SAP has no more cytotoxicity to cells than saporin alone, while the targeted toxin is more than four orders of magnitude more toxic.

For the second immunotoxins (Mab-ZAP, Rab-ZAP), use Goat IgG- SAP, Cat. #IT-19. It works on the same easy principle as Mouse IgG- SAP and Rat IgG-SAP.

Ligand-toxins: Blank-SAP (Cat. #IT-21) is constructed from a nonsense peptide, a re-arranged alpha melanocyte- stimulating hormone. This sequence contains amino acids common to the ligands of G protein-coupled receptors, but has no known homology. Like all of our peptide- toxins, it has a 1:1 molar ratio of saporin to peptide, and is void of any free peptide or non-conjugated saporin. It’s a perfect match to the peptide ligand-toxins SP-SAP, SSP- SAP, orexin-SAP, dermorphin-SAP, and CRF-SAP. Figure 3 is an illustration of Blank-SAP “shooting blanks” relative to a targeted toxin. The targeted toxin is more than two orders of magnitude more potent than Blank-SAP or saporin alone.

The great thing about these new controls is they are ready to use. You don’t have to make strange calculations to figure how much of each component to add; you just use the same amount of control as you do the targeted toxin, and that’s it! Couldn’t be much simpler.