No abstract

An engineered variant of subtilisin BPN', termed subtiligase, which efficiently ligates esterified peptides in aqueous solution, was used for the complete synthesis of ribonuclease (RNase) A that contains unnatural catalytic residues. Fully active RNase A (124 residues long) was produced in milligram quantities by stepwise ligation of six esterified peptide fragments (each 12 to 30 residues long) at yields averaging 70 percent per ligation. Variants of RNase A were produced in which the catalytic histidines at positions 12 and 119 were substituted with the unnatural amino acid 4-fluorohistidine, which has a pKa of 3.5 compared to 6.8 for histidine. Large changes in the profile of the pH as it affects rate occurred for the single and double mutants with surprisingly little change in the kcat for either the RNA cleavage or hydrolysis steps. The data indicate that these imidazoles function as general acids and bases, but that the proton transfer steps are not rate-limiting when the imidazoles are present in their correct protonation states. These studies indicate the potential of subtiligase for the blockwise synthesis of large proteins.

A variant of subtilisin BPN', which we call subtiligase, has been used to ligate esterified peptides sitespecifically onto the N termini of proteins or peptides in aqueous solution and in high yield. We have produced biotinylated or heavy-atom derivatives of methionyl-extended human growth hormone (Met-hGH) by ligating it onto synthetic peptides containing biotin or mercury. Polyethylene glycol (PEG)-modified atrial natriuretic peptide (ANP) was produced by ligating ANP onto peptides containing sites for PEG modifi'cation. We have established the N-terminal sequence requirements for efficient ligation onto proteins, using either synthetic substrates or pools of ifiamentous phage containing Met-hGH with random N-terminal sequences (substrate phage). To facilitate ligations involving proteins with highly structured or buried N termini, a more stable subtiligase was designed that effectively ligates peptides onto Met-hGH even in 4 M guanidine hydrochloride. The use of subtiligase should expand the possibilities for protein semisynthesis and rational protein design.Coupling of synthetic peptides onto a protein or protein fragment is a potentially powerful means of introducing natural or nonnatural constituents to probe and design protein function (for reviews, see refs. 1-3). For example, enzymatic and chemical peptide ligation methods have been used to produce a variety of semisynthetic proteins (4-14). However, in virtually all cases, coupling relied on noncovalent or covalent preassociation or some other special secondary structural feature ofthe fragments to be ligated. Thus, a more general method for the semisynthesis of any protein would be useful.Recently, a variant of the serine protease subtilisin BPN', in which the catalytic Ser-221 was converted to cysteine and Pro-225 was converted to alanine, was shown to ligate dipeptides onto tetrapeptide esters with high kcat values (20 s'1) and with little hydrolysis of the ester substrate or proteolysis of the amide product (15). Here, we evaluate the sequence requirements for efficient peptide ligation using this double mutant of subtilisin BPN', termed "subtiligase," and design more stable variants of it. These studies suggest that subtiligase may be generally useful for site-specific ligation of peptides containing affinity handles, isotopic labels, heavy atoms, or other nonnatural constituents onto the N terminus of proteins or protein fragments.MATERIALS AND METHODS Materials. Enzymes for DNA manipulations were from New England Biolabs or BRL. Streptavidin-horseradish peroxidase (SAHRP) was from GIBCO/BRL, column resins were from Pharmacia, and atrial natriuretic peptide (ANP) was from Bachem. Oligonucleotides were synthesized by the Oligonucleotide Synthesis Group at Genentech.Peptide Synthesis. All peptides were synthesized by standard methods (16). Peptides esterified with glycol-conjugated phenylalanylamide (glc-F-amide) were synthesized as described (15). For ligations onto immobilized supports, peptides were synthesized on 96-well (---0.17 nmol p...

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.

The accumulation of leukocytes in various organs contributes to the pathogenesis of a number of human autoimmune diseases such as asthma, rheumatoid arthritis, Crohn s disease, ulcerative colitis, hepatitis C, and multiple sclerosis. The inflammatory processes leading to tissue damage and disease are mediated in part by the alpha4 integrins, alpha4beta1 and alpha4beta7, expressed on the leukocyte cell surface. These glycoprotein receptors modulate cell adhesion via interaction with their primary ligands, vascular cell adhesion molecule (VCAM) and mucosal addressin cell adhesion molecule (MAdCAM), expressed in the affected tissue. Upon binding, the combined integrin/CAM interactions at the cell surface result in firm adhesion of the leukocyte to the vessel wall followed by entry into the affected tissue. Elevated cell adhesion molecule (CAM) expression in various organs has been linked with several autoimmune diseases. Monoclonal antibodies specific for alpha4 integrins or their CAM ligands can moderate inflammation in animal models suggesting such inhibitors may be useful for treating human inflammatory diseases. The alpha4 integrins have become well validated drug targets for pharmaceutical companies and numerous publications describing alpha4 integrin antagonists have recently appeared. This article discusses the rationale for targeting alpha4 integrins for the treatment of autoimmune disorders and reviews some currently known antagonists. The methods used to identify lead molecules and the progress of selected antagonists toward becoming new drugs will is also discussed. (131 references).