Provided herein are activatable anti-human CTLA-4 antibodies comprising a heavy chain comprising a VH domain and a light chain comprising a masking moiety (MM), a cleavable moiety (CM), and a VL domain. Such activatable anti-human CTLA-4 antibodies have CTLA-4 binding activity in the tumor microenvironment, where the masking moiety is removed by proteolytic cleavage of the cleavable moiety by tumor-specific proteases, but exhibit greatly reduced binding to CTLA-4 outside the tumor. In this way, the activatable anti-human CTLA-4 antibodies of the present invention retain anti-tumor activity while reducing the side effects associated with anti-CTLA-4 activity outside the tumor.

Systems and methods for characterizing low molecular weight (LMW) protein drug product impurities are provided. One embodiment uses hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry analysis. After removal of the N-linked glycans from the protein drug product, for example an antibody drug product, the elution of LMW impurities from the HILIC column was determined by the size of the molecular weight species. In some embodiments, the HILIC separation is performed under denaturing conditions, making the detection of LMW forms using this method highly comparable to both SDS-PAGE and CE-SDS methods. LMW drug product impurities include, but are not limited to light chain, half antibody, H2L, H2, HL, HC, peptide backbone-truncated species, and combinations thereof.

Herein is reported a method for the generation of multispecific antibodies by a half-antibody exchange reaction between two 2/3-IgGs destabilized in one half by asymmetric perturbing mutations fostering the generation of correctly assemble full length bispecific antibodies. The method can be performed in the absence of reducing agents and does not require hinge region disulfide bonds in the starting 2/3-IgGs.

Herein is reported a method for the generation of multispecific antibodies by a half-antibody exchange reaction between two 2/3-IgGs destabilized in one half by asymmetric perturbing mutations fostering the generation of correctly assemble full length bispecific antibodies. The method can be performed in the absence of reducing agents and does not require hinge region disulfide bonds in the starting 2/3-IgGs.

The present invention provides heterodimeric antibodies and fragments thereof and methods for their preparation, wherein the pairing of heavy and light chains has been improved. Interface residues were mutated such that each light chain strongly favoured its cognate heavy chain when two different heavy chains and two different light chains were co-transfected and co-expressed in the same cell to assemble a functional, heterodimeric antibody or fragment thereof.

The present disclosure provides methods of degrading an EGFR protein on a target cell. The present disclosure further discloses antigen binding molecules that bind to an EGFR protein and a membrane-associated internalizing protein, such as ITGB6.

Genetically modified non-human animals and methods and compositions for making and using them are provided, wherein the genetic modification comprises a deletion in an immunoglobulin constant region CH1 gene (optionally a deletion in a hinge region) of an IgG, IgA, IgD, and/or IgE, and wherein the mouse is capable of expressing a functional IgM. Genetically modified mice are described, including mice having a functional IgM gene and modified to have a deletion of a CH1 domain and a hinge region in a heavy chain constant domain that is not an IgM, e.g., in an IgG heavy chain constant domain. Genetically modified mice that make human variable/mouse constant chimeric heavy chain antibodies (antibodies that lack a light chain), fully mouse heavy chain antibodies, or fully human heavy chain antibodies are provided.

The present invention provides heterodimeric antibodies and fragments thereof and methods for their preparation, wherein the pairing of heavy and light chains has been improved. Interface residues were mutated such that each light chain strongly favoured its cognate heavy chain when two different heavy chains and two different light chains were co-transfected and co-expressed in the same cell to assemble a functional, heterodimeric antibody or fragment thereof.

Heterodimerizing domains with orthogonal mutations that drive heterodimerization, in particular heterodimerizing antibody CH3 domains, heterodimeric polypeptides comprising such heterodimerizing domains, and antibody constructs comprising such heterodimeric polypeptides, are provided.