β-Lactam-based approach for the chemical programming of aldolase antibody 38C2

Abstract

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped targeting modules. A model study was first performed with β-lactam conjugated to biotin. This conjugate efficiently and selectively modified the catalytic site lysine (LysH93) of mAb 38C2. We then conjugated a β-lactam to a cyclic-RGD peptide to chemically program mAb 38C2 to target integrin receptors α v β 3 and α v β 5 . The chemically programmed antibody bound specifically to the isolated integrin receptor proteins as well as the integrins expressed on human melanoma cells. This approach provides an efficient and versatile solution to irreversible chemical programming of aldolase antibodies.Monoclonal antibody (mAb) 38C2 was generated by reactive immunization with a 1,3-diketone hapten. 1 This antibody possesses a lysine residue at position 93 of the heavy chain, LysH93, that has an unusually low pK a of ~6.0 and is essential for the catalytic mechanism of the antibody. 2 mAb 38C2 can be chemically programmed to selectively bind to cells or other targets by selective reaction of LysH93 with β-diketone (Scheme 1A) or an acetone aldol adduct of a vinylketone (Scheme 1B) linker equipped with a targeting ligand or module. 3-8 Many of our studies have used antagonists of α v β 3 and α v β 5 integrins as targeting modules for chemical programming of aldolase antibody specificity. 3-7 In the first case, a reversible covalent attachment of a diketone compound is achieved through the formation of an imine, which isomerizes to the more stable enaminone derivative. In the latter case, an acetone aldol adduct of the vinylketone is used as a prolinker, because the vinylketone linker is highly reactive. mAb 38C2 catalyzes the retro-aldol reaction of the prolinker to give free vinylketone linker, which then undergoes Michael addition, in situ, with the catalytic Lys residue in the antibody binding site. This approach provides irreversible attachment of the targeting modules; however, preparation of the prolinker equipped targeting modules requires complicated multistep synthesis and is not compatible with a wide range of substrates.In earlier studies we had explored a β-lactam based modification strategy for covalent attachment of thiazolium cofactor into the active site of aldolase antibodies in order to prepare the first thiazolium-dependent catalytic antibodies. 9 Given the success of this study and the chemical stability of the resulting covalent modification, we were encouraged to examine this approach as a route to chemically programmed antibodies. We now report the selective reaction of the catalytic site LysH93 with β-lactam-equipped targeting modules (Scheme 1C); the synthesis of the targeting module was straightforward and this strategy provides for irreversible Correspondence to: Carlos F. Barbas, III. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manu...

Chemically programmed antibodies

“…Prompted by this finding, 1,3-diketone derivatives were also shown to serve as ARMs for endogenous antibodies triggered by immunization [63]. With the preassembled cpAb being a preferred IND entity, however, subsequent studies with mAbs 38C2 and h38C2 switched the electrophilic group of the synthetic component from 1,3-diketone to 2-azetidinone (β-lactam), which affords irreversible covalent conjugation to K99 [64]. Irreversible covalent conjugation to mAb 38C2 and other aldolase mAbs was also achieved with a vinylketone released from its stable acetone aldol adduct by the catalytic activity of the reactive lysine residue [65].…”

Instant immunity through chemically programmable vaccination and covalent self-assembly

“…JW-KLH and JW-BSA were prepared as described (4). Targeting agents SCS-873, SCS-397, and cRGD-dk were prepared in accord with published methodologies (10,13,36). cRGD peptide [cyclo(ArgGly-Asp-D-Phe-Lys)] was obtained from Peptides International, Inc.…”

N -Sulfonyl-β-lactam hapten as an effective labeling reagent for aldolase mAb

“…14-16 We reported an effective labeling system of the aldolase monoclonal antibody (mAb) 38C2 17-21 , which was generated by conjugating 1,3-diketone 1 (see Figure 2 for the structure of 1 ) and carrier protein KLH via N -acyl β-lactam (NABL)-mediated amidation (Scheme 1). 13,22-26 In this case, the NABLs react selectively and irreversibly with the two amino groups of the lysine residues to form stable covalent bonds with mAb 38C2.…”

“…13,22-24 Therefore, we synthesized N -sulfonyl-β-lactam 3a (Scheme 2). At first, the commercially available 4-(bromomethyl)benzene-1-sulfonyl chloride ( 4 ) was converted to the corresponding methylsulfonate 5 29 by methanolysis in the presence of Et 3 N. Methylsulfonate 5 was etherified with alcohol 6 , 30 which is derived from tetraethyleneglycol, to produce 7 .…”