Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.

Keywords: antibody(s) antibody drug(s) antibody drug conjugate(s) (ADC) physicochemical properties pharmacokinetics monoclonal antibody(s) immunotherapy IgG antibody(s) drug design developability a b s t r a c t Antibody-based proteins have become an important class of biologic therapeutics, due in large part to the stability, specificity, and adaptability of the antibody framework. Indeed, antibodies not only have the inherent ability to bind both antigens and endogenous immune receptors but also have proven extremely amenable to protein engineering. Thus, several derivatives of the monoclonal antibody format, including bispecific antibodies, antibody-drug conjugates, and antibody fragments, have demonstrated efficacy for treating human disease, particularly in the fields of immunology and oncology. Reviewed here are considerations for the design of antibody-based therapeutics, including immunological context, therapeutic mechanisms, and engineering strategies. First, characteristics of antibodies are introduced, with emphasis on structural domains, functionally important receptors, isotypic and allotypic differences, and modifications such as glycosylation. Then, aspects of therapeutic antibody design are discussed, including identification of antigen-specific variable regions, choice of expression system, use of multispecific formats, and design of antibody derivatives based on fragmentation, oligomerization, or conjugation to other functional moieties. Finally, strategies to enhance antibody function through protein engineering are reviewed while highlighting the impact of fundamental biophysical properties on protein developability.

The rate of salivary glucose clearance was determined in 6 subjects after rinsing with solutions ranging in concentration from 0.3 to 50%. 6 repeated rinses of 10% glucose by 1 subject gave clearance times, i.e. time to reach 10 mg%, ranging from 18.7 to 24.6 min, while the clearance time of single rinses by each of the subjects ranged from 13.6 to 23.3 min. The coefficients of variation were 9.8 and 21.7%. Plotting the logarithm of the saliva concentrations of glucose against time gave linear curves (r close to ––1), with slopes which decreased with increase in rinse concentration. The mean half-time, i.e. the time required for a given concentration to reach half its value, was 0.8 min for the 0.3% rinse, increasing to 2.8 min for the 50% rinse. The time of clearance from the initial concentration to a baseline value of 10 mg% increased linearly with the logarithm of the rinse concentrations, and ranged from a mean of 3.2 min for the 0.3% rinse to a mean of 27.5 min for the 50% rinse. There was considerable between-subject variation in the rate of clearance, the rate for a given subject being consistently slow or consistently fast for all rinse concentrations.

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The ability of cooked starch (CS) and raw starch (RS)to cause intraoral enamel demineralization was determined by the iodide permeability (Ip) test which registers demineralization of enamel by an increase in Ip (+ΔIp) and mineralization by a decrease in Ip (––ΔIp). 5 subjects, who wore a palatal prosthesis holding 8 bovine enamel blocks covered by a layer of Streptococcus mutans swished 15 g of a 10% jelly of CS or 15 ml of a suspension of RS in the mouth for 1 min, and the change in Ip was determined after a period of 45 min. The salivary clearance of maltose during the experiment was also determined. CS gave + ΔIps which were statistically significant in 2 of the 5 subjects. The mean ± SE was +3.6 ± 1.47·10^––10 M of I, indicating mild demineralization. RS gave ΔIps in 4 of the 5 subjects (mean ––6.7 ± 1.87). The rate of salivary clearance of maltose varied considerably among the subjects but CS consistently showed higher initial concentrations than RS. Maximal salivary maltose concentrations ranged from 1,920 to 3,052 mg% for CS and from 523 to 1,041 mg% for RS. The range of the clearance time was from 19.3 to 44.9 min for CS and from 14.7 to 48.2 min for RS. Plotting maltose concentrations against time gave curves which followed an exponential equation. The areas under the curves were calculated, and correlation with the respective Δlps gave r = 0.78. It was concluded that RS had no demineralization potential, and that the demineralizing ability of CS was related to rapid hydrolysis and high early concentrations of maltose in saliva, and unrelated to clearance time.

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.

A newly-developed intra-oral enamel demineralization test was used to evaluate the effect of supplementation of a 10% sucrose solution with various components on enamel demineralization induced by the sucrose. Five human subjects wore a palatal prosthesis holding eight blocks of subsurface bovine enamel covered with a layer of S. mutans cells. The test involved rinsing with sucrose solution or with sucrose solution supplemented with 0.162 mol/l of different calcium salts or equivalent concentrations of Na-, K-, and Sr salts; rinsing was for one min at times zero and 45 min of the 90-minute test period. Ca-propionate, Ca-acetate, and Ca-levulinate completely inhibited sucrose-induced enamel demineralization; Ca-chloride, Ca-lactate, and Ca-ascorbate gave from 65-75%, and K-acetate, Na-lactate, and Sr-lactate 39, 25, and 18% inhibition, respectively. Consideration of the anion dissociation constants and the Ca-anion association constants of the salts suggests that the observed inhibition is caused mainly by common ion effects and, to a lesser extent, by buffer effects.

Six subjects wore intraoral devices carrying bovine enamel blocks covered with a layer of Streptococcus mutans. They swished solutions of 5% glucose or maltose, or sols or gels of 3, 5, 10, 15, or 20% gelatinized wheat starch in the mouth for 3 × 1 min. Demineralization was measured after 45 min by determining the change in iodide permeability (delta Ip) of the enamel. Spittings of the administered materials and samples of saliva, taken at intervals during the test, were analyzed for maltose, and the time of clearance was calculated. Demineralization was greatest for glucose followed by maltose, the starch gels, and the sols. The latter gave close to zero scores. The salivary clearance time increased with increase in concentration of the starch. Although the starch was hydrolyzed rapidly in the mouth, its oral retentiveness was greater than that of the maltose rinse. Demineralization was closely correlated with the final pH of the S. mutans cell layer. The data indicate that the starch in baked or cooked foods may have a significant demineralization potential and that it enhances oral retentiveness.