Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T_M) of the C_H2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T_M of the C_H2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.     

Control of locomotion in vitro: II. Chemical stimulation.

Previous studies have described the presence of alternating activity induced in left and right ventral roots of the neonate rat in vitro brainstem-spinal cord preparation, following application of certain neuroactive substances to the bathing solution. The present findings show the presence of chemically induced, adult-like coordinated airstepping demonstrated by electromyographic recordings in the hindlimb-attached in vitro brainstem-spinal cord preparation. Analysis of muscular activity demonstrated alternation between antagonists of one limb and between agonists of different limbs, as well as a proximodistal delay in agonists active at different joints of the same limb. Neuroactive agents were applied independently to either the brainstem or spinal cord bath. The substances surveyed in the present studies included some of those used previously, as well as additional compounds: bicuculline and picrotoxin (gamma-aminobutyric acid-ergic antagonists), N-methyl-D-aspartic acid (excitatory amino acid agonist), substance P, acetylcholine, carbachol (cholinergic agonist), and serotonin. Application of these substances to the brainstem bath produced rhythmic airstepping. Application of dopamine, aspartate, glutamate, and N-methyl-D-aspartic acid to the spinal cord bath also produced rhythmic airstepping, while application of acetylcholine produced tonic, long-lasting co-contractions. These findings reveal the presence of several neurochemical systems in the central nervous system that can be activated at birth to induce coordinated airstepping in the neonate rat in vitro brainstem-spinal cord preparation.     

Experimental simulation of an estrous cycle — gonadotropin surges in estradiol‐infused ovariectomized rats

Abstract

Dynamic relations between the circulating estrogen and the hypophyseal gonadotropin secretion in the estrous cycle were investigated by replacing the ovaries by an infusion pump in freely moving rats. Female rats were ovariectomized in the morning at certain stages of the 4‐day estrous cycle, and simultaneously infused with estradiol (E₂) at a constant rate of 0.35 ng/min up to 120 h through a cannula chronically inserted into the jugular vein. They were killed at 6 h‐intervals. Rats ovariectomized at the second day of diestrus and at estrus showed a sharp rise in LH 36 h and 84 h, respectively, after the initiation of E₂ infusion, when the proestrous surge would occur in normal rats. During the other periods, blood levels of LH were very low, exhibiting a small daily rise in the evening. Similarly ovariectomized rats infused with vehicle only showed a gradual rise of gonadotropin secretion, never reaching the surge level. Rats ovariectomized at proestrus and infused with E₂ showed a LH surge 12 h later as expected. However, surge‐like LH secretions followed every evening thereafter. Thus, the constant supply of E₂ alone could simulate at least one 4‐day cyclic LH surge in ovariectomized rats. E₂ infusion caused a daily peak of FSH synchronized with the LH rises, but could not suppress the post‐operative hypersecretion. It is discussed that if the suppressing effect of progesterone endogenously secreted from the ovaries is cleared, a circadian pattern of the LH/FSH surge may appear under the signal from the cerebral clock mechanism and the effect of circulating estrogen. The failure to suppress the FSH hypersecretion by E₂ might indicate the involvement of inhibin in the regulatory mechanism. Time‐course changes in uterine and vaginal weights are also dealt with and discussed in relation to the constant E₂ exposure.     

Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, froman early stage of cultured tobacco mesophyll protoplasts bya differential display method. The expression of this gene wasspecifically induced by natural and synthetic cytokinins includingN-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-typecytokinin, although the simultaneous presence of auxin was alsorequired. It seems that the preceding treatment of the tobaccomesophyll protoplasts by auxin is necessary for the gene torespond to cytokinin. The addition of a cytokinin antagonist,compound 182, which suppressed the induction of cell divisionin tobacco mesophyll protoplasts, completely abolished the expressionof this gene. Though the predicted gene product of H13 did notsuggest us any sequences of defined functions, two domains ofthe predicted sequence had significant homology to several reportedsequences in the data base. The gene product of H13 is proposedto have a role in regenerating cell wall in cultured protoplasts,since a cDNA clone E6, from cotton fiber cells, which has themost closely related structure to H13, has been isolated fromcells which showed active cellulose synthesis. This suppositionis supported by the evidence that in the absence of cytokinin,cell wall regeneration was significantly suppressed, resultingin failure of the induction of cell division. Thus, the geneproduct of H13 is supposed to have a role in regenerating cellwalls and facilitating the progression of the cell cycle, resultingin the sustained cell division of tobacco mesophyll protoplasts. ¹These authors are equally contributed to this work.     

Biosynthesis of Biotin from Dethiobiotin by Intact Cells of Biotin-producing Bacterium

Intact cells of a biotin-producing bacterium, KY–21–1–25, were found to synthesize biotin from dethiobiotin. Optimal conditions for the biosynthesis of biotin from dethiobiotin by intact cells were investigated. Intact cells harvested from adenine-supplemented medium showed intensive biosynthesis. However, the biosynthesis of biotin by intact cells was strongly inhibited by the addition of adenine or adenosine. The inhibitory activity of adenine was about 10-fold greater than that of adenosine. Formation of several unidentified biotin-vitamers was observed in both reaction mixtures incubated with and/or without addition of adenine.