This disclosure is based, at least in part, on an unexpected discovery that the novel nanobodies and variants thereof are able to specifically bind afucosylated or sialylated IgG Fc glycoforms. Glycosylation of the IgG Fc domain is a major determinant of the strength and specificity of antibody effector functions, modulating the binding interactions of the Fc with the diverse family of Fc? receptors. These Fc glycan modifications, such as removal of the core fucose residue, are newfound clinical markers for predicting severity of diseases, such as diseases caused by dengue virus (DENV) or SARS-CoV-2. However, it remains challenging to accurately distinguish specific IgG glycoforms without costly and time-intensive methods. The novel glycol-specific nanobodies and variants thereof, as disclosed herein, can be used as rapid clinical diagnostics or prognostics to risk stratify patients with viral and inflammatory diseases.

Provided herein are compositions with enhanced protein content, proteins with high solubility, protein combinations and methods for the preparation thereof.

This invention describes a new analytical method to determine the quantity and distribution of fucose per Fc within an antibody preparation.

The present invention provides for recombinant Endo-S mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sialylated oxazoline or synthetic oligosaccharide oxazoline is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor. Such recombinant Endo-S mutants are useful for efficient glycosylation remodeling of IgG1-Fc domain to provide different antibody glycoforms carrying structurally well-defined Fc N-glycans.

The present invention provides for recombinant Endo-S2 mutants (named Endo-S2 glycosynthases) that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sugar chain is added to a fucosylated or nonfucosylated GlcNAc-IgG acceptor. As such, the present invention allows for the synthesis and remodeling of therapeutic antibodies thereby providing for certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and/or ability to deliver a therapeutic agent.

Methods are provided for making bispecific antibodies and antibody conjugates comprising site-specifically cross-linking two or more antibodies, antibody fragments or Fc-fusion proteins. Also provided are compositions and uses for the bispecific antibodies and antibody conjugates. The bispecific antibodies may be used to treat a disease or condition. Also provided are methods for site-specifically conjugating a liposome, an mRNA or an siRNA to an antibody, and uses of the antibody-conjugated liposome, mRNA or siRNA.

A simpler ADC production method is provided. A method for producing an antibody-drug conjugate including the following step: the step of conjugating a glycan-homogenized antibody and a glycosylated drug. The glycosylated drug consists of a drug bound to a glycan having an oxazolinated reducing end via a linker. The glycan-homogenized antibody and the glycosylated drug can be conjugated with an endo-P-N-acetylglucosaminidase mutant enzyme.

Methods for making a site-specific modification of a glycoprotein are provided which preserve the charge and glycoform of the glycoprotein. One or more glycans are trimmed from the glycoprotein, while keeping a core glycan attached to the glycoprotein. The removal of the one or more glycans provides access to a targeted amino acid for a site-specific reaction. The attached core glycan can optionally be utilized for modulation of the glycoprotein, or one or more glycans or reagents can be attached to the core.

The present disclosure provides compounds useful for enzymatic glycan remodeling of a glycoprotein. Also provided is a method for remodeling a glycoprotein using M6P-glycan oxazolines in a one-pot deglycosylation/transglycosylation process, which may enable site selective M6P-glycan remodeling of glycoproteins to obtain homogeneous products. The remodeled glycoprotein (such as a recombinant human acid ?-glucosidase) may have enhanced affinity for the CI-MPR, increased uptake by a cell, and improved therapeutic efficacy compared to the original glycoprotein. A method of treating Pompe disease using a glycan remodeled lysosomal enzyme is also provided.

The present application relates to the field of glyco-engineering, more specifically to glycosylation-engineered fungal cells, more specifically glycosylation-engineered yeast cells, optimized to produce highly homogenous forms of complex N-glycans on recombinant proteins. The invention specifically relates to methods to obtain pharmaceutical compositions comprising recombinant glycoproteins which have homogenous forms of complex N-glycans. In addition, the invention relates to novel pharmaceutical compositions which result from the methods of the invention.