Subclasses of human IgG have a range of activity levels with different effector systems but each triggers at least one mechanism of cell destruction. We are aiming to engineer non‐destructive human IgG constant regions for therapeutic applications where depletion of cells bearing the target antigen is undesirable. The attributes required are a lack of killing via Fcγ receptors (R) and complement but retention of neonatal FcR binding to maintain placental transport and the prolonged half‐life of IgG. Eight variants of human IgG constant regions were made with anti‐RhD and CD52 specificities. The mutations, in one or two key regions of the CH2 domain, were restricted to incorporation of motifs from other subclasses to minimize potential immunogenicity. IgG2 residues at positions 233 – 236, substituted into IgG1 and IgG4, reduced binding to FcγRI by 104‐fold and eliminated the human monocyte response to antibody‐sensitized red blood cells, resulting in antibodies which blocked the functions of active antibodies. If glycine 236, which is deleted in IgG2, was restored to the IgG1 and IgG4 mutants, low levels of activity were observed. Introduction of the IgG4 residues at positions 327, 330 and 331 of IgG1 and IgG2 had no effect on FcγRI binding but caused a small decrease in monocyte triggering.

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Le(y)-reactive monoclonal antibodies (mAbs) were generated in mice by immunization with synthetic Le(y) neoglycoproteins or with Le(y)-expressing cells. Serological analysis indicated that mAbs raised against synthetic Le(y) (i) reacted strongly with synthetic Le(y) but poorly with natural Le(y), (ii) cross-reacted with Le(x) or H-type 2 structures, and (iii) were IgG1, IgG2a, or IgG2b. mAbs raised against Le(y)-expressing cells (i) reacted with both synthetic Le(y) and natural Le(y), (ii) were of two types: cross-reactive with Le(x) or H-type 2 structures or specific for Le(y), and (iii) were IgM or IgG3. One of the mAbs raised against natural Le(y), mAb 3S193 (IgG3), showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y) containing glycoproteins and glycolipids; it also reacted strongly in rosetting assays and cytotoxic tests with Le(y)-expressing cells. mAb 3S193 did not lyse O, A, AB, and B human erythrocytes in the presence of human complement. In flow cytometry, there was weak reactivity with granulocytes, a reactivity also observed with two previously described highly specific Le(y) mouse mAbs--BR55-2 (IgG3) and B3 (IgG1). A humanized version of mAb 3S193 has been constructed, and the specificity pattern and reactivity for Le(y) remain very similar to mouse mAb 3S193.

Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation.

Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogenous ADCs, disulfide modification is considered an effective, low-cost and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallisable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibodydependant cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g. CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of inter-chain covalent linkages. It was found that by natively re-bridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g. thermal stability/aggregation), the natively re-bridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to sub-standard non Fc-specific attributes (thermal stability). These results advocate native disulfide re-bridging as an excellent synthetic strategy for forming homogenous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.