Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.Monoclonal antibodies are a highly established technology in drug development and the majority of currently approved therapeutic antibodies are derived from immunized rodents (1). The advent of display libraries and engineered animals that can produce “fully human” antibody v-gene sequences has had a significant positive impact on antibody drug discovery success (1), but these technologies are mostly the domain of biopharmaceutical companies. Antibodies from wild-type animals that are already extant, or can be freely developed, will therefore continue to be a rich source of therapeutic candidates. In addition, phylogenetically distant hosts such as rabbits and chickens may become a valuable source of monoclonals with clinical potential against challenging targets (2, 3).Chimerization of murine antibodies can reduce anti-IgG responses in man (4), but murine v-domains may still have provocative T-cell epitope content, necessitating “humanization” of their framework regions (5, 6). Classical humanization “grafts” murine CDRs into human v-gene sequences (7), but this typically leads to significant reduction in affinity for target, so murine residues are introduced at key positions in the frameworks (a.k.a. “back-mutations”), to restore function (8). Importantly, humanized antibodies do elicit lower immunogenicity rates in patients in comparison with chimerics (9).Alternative humanization methods have also been developed based on rational design or empirical selection (10–17), but current methods still all suffer from flaws, such as: high non-germ-line amino acid content retention (5, 6); grafting into poorly understood frameworks (13); resource-intensive, iterative methods (15, 18); requirement for homology modeling of the v-domains, which is often inaccurate (19, 20), or a cocrystal structure with the target antigen (14). Methods that allow humanization into preferred frameworks can add numerous framework mutations (18, 21), which may destabilize the v-domains (22), encode new T-cell epitopes, or introduce random amino acid mutations in CDRs (12, 13) that can drive polyspecificity and/or poor PK properties (23).Critically, testing of protein therapeutics in monkeys has been shown to be nonpredictive of immune responses in man (24) and animal immunogenicity testing has been suggested to be of little value in biosimilar development (25). Current evidence suggests that the main risk factors for antibody immunogenicity in man are human T-cell epitope content and, to a lesser extent, T-cell independent B-cell responses (6). B-cell epitopes are challenging to predict and B-cell-only responses to biotherapeutics appear to be driven by protein aggregates (26). The key attributes to reduce antibody immunogenicity risk in the clinic appear to be: low T-cell epitope content, minimized non-germ-line amino acid content and low aggregation potential (27).In recent years, several reports have strongly suggested that CDRs might be malleable in ways that could not be predicted a priori. Random mutagenesis and reselection of a classically humanized rat antibody found that individual framework back mutations and CDR residues could revert to human germ-line sequence, while maintaining or even improving the function of the antibody (28). A number of humanization studies have now also shown that a small number of positions in the CDRs could be substituted for human germ-line residues, through a rational design cycle of reversion mutations (5, 29). In addition to these observations, a number of structural analyses have illustrated the common redundancy of sequence space in antibody binding interfaces. Despite typically large buried interfaces between antibodies and protein targets, only a subset of residues in the CDRs of antibodies usually makes contact with antigen (30–32). Alanine scanning of CDR loops has also shown that only a limited number of residues directly affect antigen binding affinity (33). Indeed, it has even been shown that redundant paratope space in a single antibody may be exploited to engineer binding specificity to two separate targets (34). Additionally, CDR loop structures are known to be restricted to a limited number of canonical classes, despite amino acid variation within those classes at specific positions (35–38). These observations led us to hypothesize that, under the right experimental conditions, a large proportion of residues in grafted animal CDRs could be concurrently replaced by the residues found at the corresponding positions in a given destination human germ-line v-gene.In this study, we generated combinatorial libraries on the basis of a design principle we have named “Augmented Binary Substitution” (ABS). Each library was based on a single starting antibody: rat anti-RAGE (28), rabbit anti-A33 (2), and chicken anti-pTau (3). These libraries were built into human germ-line frameworks of high predicted stability and solubility, then interrogated via phage display and screened to identify lead clones with epitope specificity and affinity equivalent to the parental clone. ABS proved to be a facile, rapid method that retains only the functionally required CDR content of the parental animal antibody, without the need for prior crystal-structure insight. Notably, this CDR germ-lining approach generated highly stable and soluble human IgGs, from multiple key antibody discovery species, that have minimized predicted human T-cell epitope content. The reproducibility of these findings across three antibodies from three disparate species demonstrates a fundamental plasticity in antibody paratopes that can be broadly exploited in therapeutic antibody optimization.     

Global cost of correcting vision impairment from uncorrected refractive error

Objective

To estimate the global cost of establishing and operating the educational and refractive care facilities required to provide care to all individuals who currently have vision impairment resulting from uncorrected refractive error (URE).

Methods

The global cost of correcting URE was estimated using data on the population, the prevalence of URE and the number of existing refractive care practitioners in individual countries, the cost of establishing and operating educational programmes for practitioners and the cost of establishing and operating refractive care facilities. The assumptions made ensured that costs were not underestimated and an upper limit to the costs was derived using the most expensive extreme for each assumption.

Findings

There were an estimated 158 million cases of distance vision impairment and 544 million cases of near vision impairment caused by URE worldwide in 2007. Approximately 47 000 additional full-time functional clinical refractionists and 18 000 ophthalmic dispensers would be required to provide refractive care services for these individuals. The global cost of educating the additional personnel and of establishing, maintaining and operating the refractive care facilities needed was estimated to be around 20 000 million United States dollars (US$) and the upper-limit cost was US$ 28 000 million. The estimated loss in global gross domestic product due to distance vision impairment caused by URE was US$ 202 000 million annually.

Conclusion

The cost of establishing and operating the educational and refractive care facilities required to deal with vision impairment resulting from URE was a small proportion of the global loss in productivity associated with that vision impairment.     

High-dose cytosine arabinoside and daunorubicin as consolidation therapy for acute nonlymphocytic leukemia in first remission: a pilot study

High-dose (HD) cytosine arabinoside (ARA-C) is more effective treatment than conventional-dose ARA-C regimens for patients with relapsed acute nonlymphocytic leukemia (ANLL). We report here that HD ARA-C given during the first remission of ANLL has resulted in long remission durations and a high proportion of patients who survive more than three years free of disease. From August 1979 to September 1983, 36 adult patients with ANLL in first remission received one to three courses of HD ARA-C (3 g/m2 by one-hour infusion every 12 hours for 12 doses on days 1 through 6) alone or with daunorubicin (30 mg/m2 for two or three doses on days 7 through 9). Three patients died of sepsis or hemorrhage during consolidation, and 14 patients have relapsed from five to 48 months after diagnosis. The remaining 19 patients are in continued complete remission (CCR) from 11 to 62 months. Denoting all deaths in remission as relapse, the actuarial probability of CCR is 42% at 62 months, with an apparent plateau in the survival curve. Of the first 22 patients treated, ten remain in CCR from 37 to 62 months with no therapy for at least three years. Due to its heightened anti-leukemic activity, HD ARA-C allows brief but effective consolidation of ANLL in first remission, with long-term disease-free survival comparable to other approaches.     

Allogeneic marrow transplantation for refractory anemia: a comparison of two preparative regimens and analysis of prognostic factors

From 1990 to 1993 we performed a prospective study of busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) in 30 patients with refractory anemia (RA) undergoing related (n = 17) or unrelated (n = 13) donor marrow transplantation. Nineteen patients survive disease free (63% 3- year actuarial disease-free survival [DFS]) and no patient relapsed. These results were compared to those of 38 historical controls with RA treated with cyclophosphamide and total body irradiation, of whom 22 are disease-free survivors and 1 relapsed. After correcting for significant variables between the two treatment groups, we found no statistically significant difference in outcome based on preparative regimen. Combining data from these 68 patients plus 2 additional patients with RA treated before 1993 with busulfan and cyclophosphamide, we identified four variables independently associated with improved survival: younger age, shorter disease duration, lower neutrophil count pretransplant, and lower hematocrit pretransplant. We also found that 15 patients 40 to 55 years of age had a 46% 3-year actuarial DFS and 26 patients receiving unrelated or mismatched related donor marrow had a 50% 3-year actuarial DFS. We conclude that there does not appear to be any significant difference in outcome based on preparative regimen in this patient population. In addition, allogeneic bone marrow transplantation may be a reasonable approach to therapy of RA early after diagnosis. However, whether early intervention with transplantation prolongs survival over that expected without transplantation cannot be ascertained with certainty from available data.     

Urinary metabolites of [1,2,3-13C]acrylonitrile in rats and mice detected by 13C nuclear magnetic resonance spectroscopy.

Acrylonitrile, a carcinogen in rats, undergoes extensive metabolism via two routes: direct glutathione conjugation or epoxidation. Metabolism to cyanoethylene oxide may mediate the carcinogenic and toxic activity of acrylonitrile. To characterize comprehensively the metabolism in vivo of acrylonitrile, the detection and identification of metabolites in urine of rodents dosed with acrylonitrile have been carried out using NMR spectroscopy. Following administration of [1,2,3-13C]acrylonitrile to male Fisher 344 rats (10 or 30 mg/kg, po) or B6C3F1 mice (10 mg/kg, po), urine samples were collected for 24 h. Carbon-13 NMR spectra were acquired directly on the urine samples after centrifugation and addition of 10-25% D2O. Resonances were assigned to carbons of acrylonitrile metabolites on the basis of chemical shift, proton multiplicity, carbon-carbon coupling, and calculated values of shift, and by comparison with standards. The proton multiplicity of each carbon was determined by heteronuclear 2D J-resolved spectroscopy (HET2DJ), and the carbon-carbon connectivities of resonances were determined using incredible natural abundance double quantum transfer spectroscopy (INADEQUATE). The metabolites identified in rat urine were thiocyanate, N-acetyl-S-(2-cyanoethyl)cysteine, N-acetyl-S-(2-hydroxyethyl)cysteine, N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine, thiodiglycolic acid, thionyldiacetic acid, and S-(carboxymethyl)cysteine or its N-acetyl derivative. These metabolites were also identified in mouse urine. Metabolites were quantitated by integrating metabolite carbon resonances with respect to that of dioxane added at a known concentration. Thiodiglycolic acid and (carboxymethyl)cysteine (or its N-acetyl derivative) were the major metabolites in the mouse, while N-acetyl-S-(2-cyanoethyl)cysteine and N-acetyl-S-(2-hydroxyethyl)cysteine were the major metabolites in the rat. Metabolites derived from cyanoethylene oxide (CEO) accounted for approximately 60% of the products excreted in rat urine, compared with 80% in the urine from mice. Differences between rat and mouse in the further metabolism of CEO were also observed. The proportion of the dose metabolized via CEO may be an important determinant of the toxicity and carcinogenicity of acrylonitrile.     

Pars triangularis asymmetry and language dominance.

The pars triangular is a portion of Broca's area. The convolutions that form the inferior and caudal extent of the pars triangularis include the anterior horizontal and anterior ascending rami of the sylvian fissure, respectively. To learn if there are anatomic asymmetries of the pars triangularis, these convolutions were measured on volumetric magnetic resonance imaging scans of 11 patients who had undergone selective hemispheric anesthesia (Wada testing) to determine hemispheric speech and language lateralization. Of the 10 patients with language lateralized to the left hemisphere, 9 had a leftward asymmetry of the pars triangularis. The 1 patient with language lateralized to the right hemisphere had a significant rightward asymmetry of the pars triangularis. Our data suggest that asymmetries of the pars triangularis may be related to speech-language lateralization.