ABSTRACT
We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antineoplastic Agents, Immunological/administration & dosage , Fibronectins/chemistry , Melanoma, Experimental/drug therapy , Protein Engineering/methods , Receptors, IgG/immunology , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacology , HEK293 Cells , Humans , Melanoma, Experimental/immunology , Mice , Models, Molecular , Phagocytosis , Receptors, IgG/chemistry , Structural Homology, Protein , Xenograft Model Antitumor AssaysABSTRACT
The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.
Subject(s)
Glycosylation , Immunoglobulin G/metabolism , Immunologic Factors/metabolism , Protein Engineering , Receptors, IgG/metabolism , Recombinant Proteins/metabolism , Animals , Biophysical Phenomena , Cell Line, Tumor , Disease Models, Animal , Half-Life , Humans , Immunoglobulin G/genetics , Immunologic Factors/genetics , Immunologic Factors/pharmacokinetics , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Mice , Models, Theoretical , Neoplasm Metastasis/prevention & control , Phagocytosis , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/pharmacokineticsABSTRACT
The number of total knee arthroplasty (TKA) procedures performed continues to rise. This is the first 1:1 matched cohort analysis of functional outcomes from a large prospective total joint registry. Our hypothesis is that a previous ipsilateral knee surgery is not associated with worse outcome scores following TKA. A total of 1,473 patients who underwent TKA were reviewed: 469 with a history of previous ipsilateral knee surgery and 1,004 without. After 1:1 matching, 469 patients were included in each cohort. Patients in both cohorts had statistically similar postoperative functional outcome scores, although patients without previous ipsilateral knee surgery had an increased Western Ontario and McMaster Universities Arthritis Index stiffness score. This study supports the hypothesis that previous ipsilateral knee surgery is not associated with worse functional outcomes following primary TKA.
