Validation of a de-immunization strategy for monoclonal antibodies using cynomolgus macaque as a surrogate for human.

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human-cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336-672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.

Facilitating T Cell Infiltration in Tumor Microenvironment Overcomes Resistance to PD-L1 Blockade.

Immune checkpoint blockade therapies fail to induce responses in the majority of cancer patients, so how to increase the objective response rate becomes an urgent challenge. Here, we demonstrate that sufficient T cell infiltration in tumor tissues is a prerequisite for response to PD-L1 blockade. Targeting tumors with tumor necrosis factor superfamily member LIGHT activates lymphotoxin ß-receptor signaling, leading to the production of chemokines that recruit massive numbers of T cells. Furthermore, targeting non-T cell-inflamed tumor tissues by antibody-guided LIGHT creates a T cell-inflamed microenvironment and overcomes tumor resistance to checkpoint blockade. Our data indicate that targeting LIGHT might be a potent strategy to increase the responses to checkpoint blockades and other immunotherapies in non-T cell-inflamed tumors.

A model system for studying NK cell receptor signaling.

Study of NK cell receptor signaling in mouse NK cells has been difficult since there are no clones of murine NK cells. We describe here a model system that overcomes this problem. This system allows the study of many aspects of NK cell receptor function with complete control over the variables that may affect activity such as cis versus trans ligand engagement, homotypic interactions, multiple target types, receptor number, receptor-ligand affinity, and signaling adaptor molecule expression. Although we give examples only for 2B4, Ly49C, and CD48, any NK cell receptors could be studied using these methods. Since many NK cell receptors such as 2B4, CD48, and the Ly49 family can be expressed in T cells, this model system allows the study of not only NK cells but also T cells with NK cell receptors. A standardized system for determining the regulation of NK cell receptor signaling can be important for understanding the anti-tumor activities of NK cells.

Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

Structure of natural killer receptor 2B4 bound to CD48 reveals basis for heterophilic recognition in signaling lymphocyte activation molecule family.

Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3.

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.