Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen^TM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling^® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.     

Extending human IgG half-life using structure-guided design

Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.     

An engineered human IgG1 antibody with longer serum half-life

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was approximately 2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.     

Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.     

Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life

ABSTRACT

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.     

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')₂ (t_1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')₂, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t_1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t_1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.     

A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties.     

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies.

In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI).

Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities.

Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.     

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.     

pH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.     

Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody

Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases.     

Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)

We describe here the functional implications of an increase in IgG binding to the neonatal Fc receptor. We have defined in a systematic fashion the relationship between enhanced FcRn binding of a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody (MEDI-524) and the corresponding biological consequences in cynomolgus monkeys. The triple mutation M252Y/S254T/T256E (YTE) was introduced into the Fc portion of MEDI-524. Whereas these substitutions did not affect the ability of MEDI-524 to bind to its cognate antigen and inhibit RSV replication, they resulted in a 10-fold increase in its binding to both cynomolgus monkey and human FcRn at pH 6.0. MEDI-524-YTE was efficiently released from FcRn at pH 7.4 in both cases. We show that MEDI-524-YTE consistently exhibited a nearly 4-fold increase in serum half-life in cynomolgus monkeys when compared with MEDI-524. This constituted the largest half-life improvement described to date for an IgG in a primate. For the first time, we demonstrate that these sustained serum levels resulted in an up to 4-fold increase in lung bioavailability. Importantly, we also establish that our non-human primate model is relevant to human. Finally, we report that the YTE triple substitution provided a means to modulate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of a humanized IgG1 directed against the human integrin alpha(v)beta3. Therefore, the YTE substitutions allow the simultaneous modulation of serum half-life, tissue distribution and activity of a given human IgG1.     

Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life

Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood clearance in both strains of mice correlated with the hierarchy of binding obtained using soluble FcRn. Consequently, in vitro interaction analysis of engineered IgGs regarding their cross-species FcRn binding ability provides information for prediction of in vivo pharmacokinetics.     

Monoclonal antibodies directed against human FcRn and their applications

The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics. The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs) directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn and for the evaluation of therapeutic FcRn blockade strategies.Key words: FcRn, IgG, monoclonal antibody, albumin, therapy     

Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T_M) of the C_H2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T_M of the C_H2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.     

Interaction with Both Domain I and III of Albumin Is Required for Optimal pH-dependent Binding to the Neonatal Fc Receptor (FcRn)

Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.     

Influence of improved FcRn binding on the subcutaneous bioavailability of monoclonal antibodies in cynomolgus monkeys

Engineering monoclonal antibodies (mAbs) with improved binding to the neonatal Fc receptor (FcRn) is a strategy that can extend their in vivo half-life and slow their systemic clearance. Published reports have predominantly characterized the pharmacokinetics of mAbs after intravenous administration. Recently, studies in mice suggest FcRn may also play a role in affecting the subcutaneous bioavailability of mAbs. Herein, we examined whether five mAbs engineered with the T250Q/M428L Fc mutations that improved their FcRn interactions, and subsequently their in vivo pharmacokinetics after intravenous administration, had improved subcutaneous bioavailability compared with their wild-type counterparts in cynomolgus monkeys. Similar to the intravenous administration findings, the pharmacokinetic profiles of our variant mAbs after subcutaneous injection showed improved half-life or clearance. In contrast, a clear effect was not observed on the subcutaneous bioavailability. We expect that while FcRn may play a role in determining mAb subcutaneous bioavailability, multiple biopharmaceutical and physiological factors are likely to influence the success of engineering strategies aimed at targeting this pathway for improving bioavailability.     

Influence of improved FcRn binding on the subcutaneous bioavailability of monoclonal antibodies in cynomolgus monkeys

Engineering monoclonal antibodies (mAbs) with improved binding to the neonatal Fc receptor (FcRn) is a strategy that can extend their in vivo half-life and slow their systemic clearance. Published reports have predominantly characterized the pharmacokinetics of mAbs after intravenous administration. Recently, studies in mice suggest FcRn may also play a role in affecting the subcutaneous bioavailability of mAbs. Herein, we examined whether five mAbs engineered with the T250Q/M428L Fc mutations that improved their FcRn interactions, and subsequently their in vivo pharmacokinetics after intravenous administration, had improved subcutaneous bioavailability compared with their wild-type counterparts in cynomolgus monkeys. Similar to the intravenous administration findings, the pharmacokinetic profiles of our variant mAbs after subcutaneous injection showed improved half-life or clearance. In contrast, a clear effect was not observed on the subcutaneous bioavailability. We expect that while FcRn may play a role in determining mAb subcutaneous bioavailability, multiple biopharmaceutical and physiological factors are likely to influence the success of engineering strategies aimed at targeting this pathway for improving bioavailability.     

The human natural killer cytotoxic cell line NK-92, once armed with a murine CD16 receptor,represents a convenient cellular tool for the screening of mouse mAbs according to their ADCC potential

To take advantage of the large number of well-characterized mouse immunoglobulins (IgGs) for the study of antibody-dependent cell-mediated cytotoxicity (ADCC) in human cells, we armed human cytotoxic lymphocytes with a mouse receptor for the Fc portion of IgG antibodies. The human ΝΚ−92 natural killer cell line was transduced with a mouse receptor gene (mCD16), which was stably expressed on the cell surface (referred to as NK-92^mCD16). When tested against a B-lymphoblastoid cell line (BLCL) coated with mouse anti-CD20 IgG1, IgG2a or IgG2b monoclonal antibodies (mAbs), the newly expressed mouse Fc receptor enabled the NK-92^mCD16 cells to kill the BLCL by ADCC. Next, using the NK-92^mCD16 we compared mouse mAbs directed at B lineage specific CD antigens for their ability to induce ADCC against human Epstein-Barr virus- infected B lymphoblastoid (for anti-CD19, -CD20 and -CD21) or against myeloma (for anti-CD38 and –CD138) target cells. Our results demonstrated that the “NK-92^mCD16 assay” allows convenient and sensitive discrimination of mouse mAbs for their ability to mediate ADCC in a human cellular system. In addition, our results provide examples of dissociation between opsonization and target cell killing through ADCC. These “murinized” human effector cells thus represent a convenient cellular tool for the study of ADCC.     

Engineering therapeutic antibodies for improved function

The binding sites on human IgG1 for human Fc gamma receptor (Fc gamma R) I, Fc gamma RIIa, Fc gamma RIIb, Fc gamma RIIIa and neonatal FcR have been mapped. A common set of IgG1 residues is involved in binding to all Fc gamma Rs, while Fc gamma RII and Fc gamma RIII utilize distinct sites outside this common set. In addition to residues which abrogated binding to the Fc gamma R, several positions were found which improved binding only to specific Fc gamma Rs or simultaneously improved binding to one type of Fc gamma R and reduced binding to another type. Selected IgG1 variants with improved binding to Fc gamma RIIIa were then tested in an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay and showed an enhancement in ADCC when either peripheral blood mononuclear cells or natural killer cells were used.