Improved cancer therapy and molecular imaging with multivalent, multispecific antibodies

Abstract

Antibodies are highly versatile proteins with the ability to be used to target diverse compounds, such as radionuclides for imaging and therapy, or drugs and toxins for therapy, but also can be used unconjugated to elicit therapeutically beneficial responses, usually with minimal toxicity. This update describes a new procedure for forming multivalent and/or multispecific proteins, known as the dock-and-lock (DNL) technique. Developed as a procedure for preparing bispecific antibodies capable of binding divalently to a tumor antigen and monovalently to a radiolabeled hapten-peptide for pretargeted imaging and therapy, this methodology has the flexibility to create a number of other biologic agents of therapeutic interest. A variety of constructs, based on anti-CD20 and CD22 antibodies, have been made, with results showing that multispecific antibodies have very different properties from the respective parental monospecific antibodies. The technique is not restricted to antibody combination, but other biologics, such as interferon-alpha2b, have been prepared. These types of constructs not only allow small biologics to be sustained in the blood longer, but also to be selectively targeted. Thus, DNL technology is a highly flexible platform that can be used to prepare many different types of agents that could further improve cancer detection and therapy.

FIG. 1.

Example of a tri-Fab bispecific antibody prepared by the dock-and-lock procedure. The C_H1 of an antitumor Fab is modified with a short linker that attaches a DDD2 unit to the Fab (A). This structure will spontaneously form a homodimer consisting of two Fab units (A′). Another cell line expressing the antihapten, Fab-AD2 (B), is prepared, and when mixed with the antitumor, Fab-DDD2, a tri-Fab structure is formed (C). The strategically placed cysteine (S) residues in both the DDD2 and AD2 peptides allowing the structure to be covalently linked for greater stability.

FIG. 2.

Pretargeting using a tri-Fab bispecific antibody and a radiolabeled di-HSG-peptide. The tri-Fab bispecific antibody is given intravenously, and over a few days, it has localized in the tumor and cleared sufficiently from the blood so that the radiolabeled di-HSG-peptide can be given. This peptide can be prepared in a manner for radiolabeling with a number of different radionuclides. The divalent HSG residues enhance avidity and permit crosslinking of adjacent bsMAb on the tumor surface. The small peptide very quickly distributes throughout the fluidic volume of the body, being rapidly eliminated in the urine. The mouse image illustrates the targeting of a subcutaneous human colon tumor by an anti-CEA bsMAb used to pretarget a ^99mTc-labeled di-HSG-peptide. Within 1 hour, the tumor is clearly seen, with the kidneys (K) being the only major normal tissue seen in the background, and the urinary bladder (UB) filled with the eliminated ^99mTc-peptide.

FIG. 3.

Multivalent, multispecific antibodies or multispecific antibody-interferon constructs formed by the dock-and-lock method. (A) The C_H3 terminus of an anti-CD22 IgG heavy chain is modified with the AD2 peptide sequence, forming an IgG with two AD2 moieties. Fab-DDD2 of an anti-CD20 antibody form homodimers that will bind and lock on the IgG-AD2 to form an IgG structure with four anti-CD20 Fab-binding arms. (B) The same IgG-AD2 structure can also be used to bind to different cytokines, in this example alpha-interferon-2b, with the interferon modified with the AD2 sequence so that the final structure is an IgG capable of targeting four interferons.