Frederick C. Kull

Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (CH3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

Samarium-153 and Lutetium-177 Chelation Properties of Selected Macrocyclic and Acyclic Ligands

We describe a simple in vitro characterization of chelation that is useful when choosing an appropriate ligand-metal combination for clinical applications. These properties include the effect of concentration on chelation efficiency, time to maximum chelation, and stability in acidic and serum environments. The macrocyclic ligands nitro-DOTA and nitro-PADOTA, the acyclic ligands nitro-CHX-A-DTPA, nitro-MX-DTPA, DTPA, and a novel terpyridine ligand, TMT-amine, were evaluated as chelate complexes of both intermediate energy beta-emitting lanthanides lutetium-177 and samarium-153. The data were compared to results obtained in a previously published study with yttrium-90. Acid lability, time to achieve maximum chelation, and stability in human serum are properties unique to each ligand-metal combination and should be evaluated prior to choosing an appropriate combination for therapeutic applications. Concentration dependence and duration of chelation are general properties of lanthanide and yttrium chelation that can be applied to an appropriate ligand-metal combination to achieve optimum chelation efficiencies.

Estimation of cell number by neutral red content. Applications for proliferative and survival assays.

A simple photometric method for estimating viable cell number in culture is described. When cultured cells are allowed to internalize 0.005–0.01% neutral red for 1 h, the amount of accumulated dye is directly proportional to cell number. The linear relationship holds for adherent and suspended cell lines. Thus, dye content reflects cell number. Since dye content is easily measured by instruments that photometrically scan microtiter trays, proliferative and survival (cytotoxic) responses can be easily quantitated.

Tumoricidal Effector Mechanisms of Murine BCG‐Activated Macrophages: Role of TNF in Conjugation‐Dependent and Conjugation‐Independent Pathways

The roles of secreted and membrane‐associated TNFs were investigated in activated macrophage cytolysis of L929, EMT‐6, and P815 targets. While all three targets were susceptible to cytolysis in coculture, an anti‐TNF antiserum blocked lysis of L929 and EMT‐6 but not of the P815 targets. Of the three targets, recombinant human or mouse TNF could only lyse the L929 target; despite the fact that a role for TNF was invoked in lysis of EMT‐6 targets in coculture, the latter was strongly resistant to soluble rTNF, even at concentrations 30–40‐fold higher than the Ka for Its TNF‐receptor. Cytolysis of the L929 target occurred when it was cocultured with BCG‐activated macrophages even when these effector cells did not secrete TNF, either due to prior chemical crosslinking or to lack of exposure to a triggering level of lipopolysaccharide. Furthermore, by introduction of the anti‐TNF antiserum over a dose‐range, it was shown that macrophage cytolysis both of L929 and EMT‐6 targets occurred in the absence of bioavailable, fluid‐phase TNF. Thus, even for targets susceptible to fluid‐phase TNF, TNF‐dependent, direct macrophage‐mediated cytolysis appears to be a function independent of secreted TNF and one that utilizes effector‐target contact to express the action of a membrane form of the molecule.

Human Insulin Receptor Gene: Data Supporting Assignment to Chromosome 19

Somatic cell hybrid clones constructed by crossing human skin fibroblasts with mouse L cells have been examined for expression of human insulin receptors, using a monoclonal antibody directed against the human insulin receptor. Data obtained in this study support the assignment of the human gene for the insulin receptor to Chromosome 19.

Production and characterization of monoclonal antibodies against the vasoconstrictive peptide human urotensin-II.

We report the production and characterization of four monoclonal antibodies (MAbs) against human urotensin-II (hU-II). The antibodies were raised against human hU-II, which contains the C-terminus cyclic ring (CFWKYC) that is conserved across species. Multiple selection assays were applied to ensure antibody potency and reactivity against the ring structure. The MAbs reacted via ELISA with hU-II bound to plastic, immunoprecipitated [(125)I-Y(9)] hU-II, bound to biotinylated hU-II in BIAcore analysis and, by Western analysis, recognized the full-length human preprourotensin-II expressed in transfected HEK293 cells. All four MAbs cross-reacted with porcine A, porcine B, rat, mouse, and goby U-II in ELISA. By competitive RIA, hU-II(5-11) (identical to the C-terminus of goby U-II) reacted equivalently to hU-II and goby U-II. The IC(50)s were 0.8 nM for one MAb and 1.6 nM for the others. All four MAbs reacted 15-fold less potently with hU-II(5-10) and 50-fold less potently with hU-II(5-10) amide. Thus, the ring structure and terminal Val/Ile comprise the binding site for this group of MAbs. This panel of antibodies could be useful tools to help delineate the biology and pharmacology of U-II. They may also be of diagnostic value in monitoring hU-II in body fluids.

Monoclonal Antibodies as Probes for Insulin and Insulin-like Growth Factor-I Receptors

Receptors for polypeptide hormones are present in cell membranes in extremely small quantities and comprise only a small fraction of a percent of the total membrane protein. Therefore, any method that can be used for their detection and characterization must have exquisite sensitivity and specificity. Antibodies provide reagents with these essential features. In this chapter we described a series of monoclonal antibodies to receptors for insulin and insulin-like growth factor-I and their use in characterizing these receptors.