Jeffry D. Watkins

Monoclonal Antibody Clearance IMPACT OF MODULATING THE INTERACTION OF IgG WITH THE NEONATAL Fc RECEPTOR

The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-α (TNFα)). The T250Q/M428L anti-TNFα variant displayed an ∼40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFα variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased ∼500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFα T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFα variant displayed improved pharmacokinetics, characterized by an ∼2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.

Optimization of protein therapeutics by directed evolution.

Directed evolution is a broadly applicable technology platform that is ideally suited to address the need for protein optimization and to fully exploit the therapeutic potential of biologics. The approach takes advantage of the remarkable structural and functional plasticity of proteins and permits the rapid remodeling of biologics into new entities with improved functions. The ability to ameliorate virtually any characteristic of a protein can translate into significant clinical benefits, including decreased immunogenicity, higher potency, greater efficacy and improved safety profile, and can considerably increase the probability of successfully developing and commercializing biotherapeutics.

Cloning, isolation and characterization of human tumor in situ monoclonal antibodies.

Abstract. A bacterially expressed human antibody (Ab) library (diversity ≈105) was generated from tumor-infiltrating B lymphocytes present in tissue isolated from a colon tumor. Immunoglobulin (IgG) heavy and light chain variable regions were amplified without isolating or enriching B cells, cloned into a phage-expression vector, and soluble antigen-binding fragment (Fabs) from >105 members of the library were screened rapidly by two distinct and complementary methodologies. In the first approach, soluble Fabs were screened by enzyme-linked immunosorbent assay (ELISA) on tumor cell monolayers. Alternatively, tumor cell surface antigens were selectively biotinylated with a plasma membrane-impermeable reagent, solubilized with non-ionic detergent, and were used to screen >105 members of the Ab library by capture lift. Reactive Fabs were partially characterized for tumor cell specificity and cross-reactivity, resulting in the identification of multiple Abs that bind cultured tumor cells but not normal human fibroblasts. The Fabs clustered into at least three distinct epitope specificity groups based on multiple criteria, including differential reactivity on two tumor cell lines and distinct antigen recognition patterns on western blot and immunoprecipitation. Moreover, DNA sequencing of the Ab variable regions demonstrated that the majority of the tumor-reactive Fabs were distinct and substantially different from the corresponding most homologous Ab germline gene. The relatively small size of the tumor-derived library allowed direct screening of soluble Fab of every member of the library, permitting the characterization of multiple human monoclonal antibodies (mAbs) that might not be discovered using alternative approaches, such as hybridoma technology or phage-display.

Use of a Peptide Mimotope to Guide the Humanization of MRK-16, an Anti-P-glycoprotein Monoclonal Antibody

A mimotope-guided strategy for engineering antibodies directed against orphan targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb). MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprotein (P-gp). Elevated expression of P-gp on tumor cells is associated with resistance to cytotoxic drugs, a major obstacle in chemotherapy. Murine MRK-16 was used to enrich and screen a phage-displayed peptide library to identify reactive mimotopes. One peptide, termed ALR1, was enriched to a greater extent than others and subsequently was expressed as a fusion protein with glutathione S-transferase. ALR1 fusion protein bound MRK-16 specifically and inhibited binding of MRK-16 to cells expressing elevated levels of P-gp. To humanize MRK-16, the murine complementarity determining regions were grafted onto homologous human heavy and light chain variable region frameworks. Framework residues that differed between the murine MRK-16 and the homologous human templates were analyzed and subsequently, five framework positions potentially important for maintaining the specificity and affinity of MRK-16 were identified. A combinatorial library consisting of 32 variants encoding all possible combinations of murine and human residues at the five differing framework positions was expressed in a phage system. In the absence of purified P-gp, ALR1 fusion protein was used as surrogate antigen to screen the antibody library to identify the framework combination that most preserved the binding activity of the mAb. On the basis of the initial screening against the mimotope four antibody variants were selected for further characterization. The binding affinity of these variants for the ALR1 fusion protein correlated with their binding to cells expressing elevated levels of P-gp. Thus, peptide mimotopes which can be identified for virtually any antibody including those that recognize conformational or carbohydrate epitopes, can serve as antigen templates for antibody engineering.

In Vitro Affinity Maturation of Human IgM Antibodies Reactive with Tumor-Associated Antigens

Human lymphocytes secreting tumor cell-specific IgM antibodies were enriched in vitro following the stimulation of allogeneic human splenocytes from nontumor-bearing donors with cytostatic tumor cells or tumor cell plasma membrane fractions. The antibodies were generally of the IgM class and displayed low intrinsic affinity (K(d) > 100 nM). Nonetheless, the avidity arising from multivalent binding sites permitted the identification of multiple monoclonal antibodies (MAbs) displaying specificity for cultured tumor cells. Five antibodies were cloned from the B cells and two of these were expressed as human Fabs with IgG(1) constant regions. Although the avidity of the human IgM antibodies was sufficient to permit detection in the original screening, the monovalent Fabs displayed low binding activities, consistent with their low intrinsic affinity. Thus, in vitro affinity maturation was used to rapidly generate multiple variants of both antibodies displaying greater than 100-fold higher affinity. Two of the antibodies were characterized further and shown to have distinct specificities. One of the targets, LH11238, is associated both with the plasma membrane and with lysosomes and is rapidly internalized following incubation of the antibody with intact live cell monolayers. The second antigen, designated LH13, is a secreted antigen that has been enriched 200-fold from conditioned media and consists of two reactive bands at 42 and 45 kDa on denaturing Western blots. The stimulation and enrichment of human lymphocytes in culture coupled with rapid in vitro affinity maturation of low affinity antibodies potentially enables the discovery of human antibodies to a broader range of epitopes, including those that might be of greater therapeutic relevance.