Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA

We explored the possibility of converting established antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximately 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximately 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.     

The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.     

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody‐dependent cellular cytotoxicity, we disrupted α‐1, 6 fucosyltransferase (FUT8 ) gene in CHO‐S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO‐S (FUT8 ^?/?) cells was comparable with wild type CHO‐S cells. FUT8 catalyzes the transfer of a fucose residue from GDP‐fucose to N‐glycans residue. Defucosylated IgG1 antibodies produced by FUT8 ^?/? cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody‐dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.     

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.     

Enhancement of DNA uptake in FUT8‐deleted CHO cells for transient production of afucosylated antibodies

Removal of the core α1,6 fucose from the glycans in the Fc region of IgG1 antibodies has been demonstrated to improve antibody‐dependent cellular cytotoxicity (ADCC) activity. In order to produce afucosylated antibodies using transient transfection, a FUT8 knockout (FUT8KO) cell line was generated in a CHO host cell line using the zinc finger nuclease technology. Transient transfection of DNA into mammalian cells using the cationic polymer, polyethylenimine (PEI), is commonly used for rapid generation of recombinant proteins. FUT8KO cells evaluated in PEI transfections yielded lower titers than parental CHO WT cells. FACS and HPLC analyses revealed that the FUT8KO cells had lower cell surface heparan sulfate (HS) levels than CHO WT. Removal of cell surface HS resulted in reduced uptake of PEI‐transfected DNA (PEI:DNA) and lower transfection titers suggesting that PEI:DNA relies on HS for binding and cellular entry. The absence of cell surface HS did not severely impact transfections performed with cationic liposomes. We undertook two approaches to improve transient production of afucosylated antibodies. First, we evaluated transfection of FUT8KO cells with cationic liposomes, which were observed to be less dependent on HS levels for uptake. Transfection of FUT8KO cells using the cationic liposome, DMRIE‐C, produced similar titers to CHO WT in both shake flask and large‐scale 10 L bioreactors. The second approach was to engineer a cell line overexpressing exostosin‐1 (EXT1), an enzyme responsible for HS chain elongation, to increase HS content. EXT1‐FUT8KO and CHO WT cells produced comparable levels of antibody from PEI transfections. Biotechnol. Bioeng. 2010;106: 751–763. © 2010 Wiley Periodicals, Inc.     

Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II

The effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein. Therefore several approaches have been developed to rationally manipulate these glycans and improve the biological functions of the antibody. Overexpression of recombinant beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in production cell lines leads to antibodies enriched in bisected oligosaccharides. Moreover, GnT-III overexpression leads to increases in non-fucosylated and hybrid oligosaccharides. Such antibody glycovariants have increased antibody-dependent cellular cytotoxicity (ADCC). To explore a further variable besides overexpression of GnT-III, we exchanged the localization domain of GnT-III with that of other Golgi-resident enzymes. Our results indicate that chimeric GnT-III can compete even more efficiently against the endogenous core alpha1,6-fucosyltransferase (alpha1,6-FucT) and Golgi alpha-mannosidase II (ManII) leading to higher proportions of bisected non-fucosylated hybrid glycans ("Glyco-1" antibody). The co-expression of GnT-III and ManII led to a similar degree of non-fucosylation as that obtained for Glyco-1, but the majority of the oligosaccharides linked to this antibody ("Glyco-2") are of the complex type. These glycovariants feature strongly increased ADCC activity compared to the unmodified antibody, while Glyco-1 (hybrid-rich) features reduced complement-dependent cytotoxicity (CDC) compared to Glyco-2 or unmodified antibody. We show that apart from GnT-III overexpression, engineering of GnT-III localization is a versatile tool to modulate the biological activities of antibodies relevant for their therapeutic application.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new strategy for generating completely non-fucosylated recombinant therapeutics

Currently, removal of core fucose from the Fc oligosaccharides of therapeutic antibodies is widely recognized as being of great importance for the effector function of antibody-dependent cellular cytotoxicity, and alpha-1,6-fucosyltransferase (FUT8) knockout cells have been generated as an ideal host cell line for manufacturing such therapeutics. Here, we attempted to identify genes other than FUT8 that could be targeted for the manufacture of non-fucosylated therapeutics. Loss-of-function analyses using siRNAs against three key genes involved in oligosaccharide fucosylation in Chinese hamster ovary (CHO) cells revealed that there was a positive correlation between the Fc oligosaccharide fucosylation and the mRNA expression through the origin in the cases of both GDP-fucose 4,6-dehydratase (GMD) and FUT8, but not for the GDP-fucose transporter, suggesting that there is no functional redundancy in GMD and FUT8. GMD knockout CHO/DG44 cells were successfully established, and were confirmed to be devoid of intracellular GDP-fucose and to produce completely non-fucosylated antibodies. GMD knockout cells recovered their fucosylation capability through the salvage pathway upon addition of l-fucose into the culture medium, and exhibited equable morphology, growth kinetics and recombinant protein productivity, demonstrating that loss of oligosaccharide fucosylation has no impact on these cellular phenotypes. Our results demonstrate that GMD knockout is a new strategy applicable to the manufacture of non-fucosylated therapeutic antibodies, and completely O-fucose-negative therapeutics as well.     

Production of therapeutic antibodies with controlled fucosylation

ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; Fc, antibody constant region; FcγRIIIa, human Fcγ-receptor IIIa; IgG, immunoglobulin G; NK cell, natural killer cell; CHO, Chinese hamster ovary; EPO, erythropoietin; Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine; Gal, galactose; NANA; N-acetylneuraminic acid; FUT8, α-1,6 fucosyltransferase; GMD, GDP-mannose 4,6-dehydratase; FX, GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase; GFT, GDP-fucose transporter; siRNA, short interfering RNA; GnTIII, β-1,4-N-acetylglucosaminyltransferase; ManII, α-mannosidase II     

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal (“afucosylation”). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.     

Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity

Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies.^1,2 The most advanced uses of this approach, however, have been limited to E. coli as an expression host to produce non-glycosylated antibodies. Here, we applied the technology to mammalian host expression systems to produce glycosylated, effector-function competent heterodimeric antibodies. In our mammalian host system, each arm is secreted as a heavy chain-light chain (H-L) fragment with either the knob or hole mutations to allow for preferential heterodimer formation in vitro with low levels of homodimer contaminants. Like full antibodies, the secreted H-L fragments undergo Fc glycosylation in the endoplasmic reticulum. Using a monospecific anti-CD20 antibody, we show that full antibody-dependent cell-mediated cytotoxicity (ADCC) activity can be retained in the context of a knobs-into-holes heterodimer. Because the knobs-into-holes mutations convert the Fc into an asymmetric heterodimer, this technology was further used to systematically explore asymmetric recognition of the Fc. Our results indicate that afucosylation of half the heterodimer is sufficient to produce ADCC-enhancement similar to that observed for a fully afucosylated antibody with wild-type Fc. However, the most dramatic effect on ADCC activity is observed when two carbohydrate chains are present rather than one, regardless of afucosylation state.     

Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells.

In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells.

Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab.

The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Functional polymorphisms of Fc receptors in human monocyte-mediated cytotoxicity towards erythrocytes induced by murine isotype switch variants

Human monocytes can be triggered to antibody-dependent cell-mediated cytotoxicity (ADCC) by murine antibodies. In this study, a series of H chain isotype switch variant antibodies against glycophorin A on human RBC was used to study the influence of isotype on the induction of ADCC. Furthermore, it was studied whether the functional heterogeneity in responsiveness to IgG1 and IgG2b anti-CD3 antibodies, as found among different donors in T cell proliferation induction experiments, was reflected in ADCC. Whereas IgG2a induced ADCC to the same extent in monocytes from all donors, IgG1 showed a heterogeneous pattern, which corresponded to the heterogeneity in T cell proliferation studies. IgG1 anti-CD3 nonresponder monocytes could, however, be induced to ADCC by IgG1 antiglycophorin, although they needed a much higher antibody density on the target cell than did responder monocytes. IgG2b antiglycophorin at a high density induced ADCC in monocytes from all donors irrespective of responsiveness to IgG2b anti-CD3, whereas IgE and IgA antiglycophorin were barely effective in monocytes from all donors. By specific blocking with mAb, the FcR that were involved in ADCC directed by the various isotypes were characterized. ADCC by IgG2a was predominantly mediated by FcRI and could be specifically enhanced by culturing the monocytes with rIFN-gamma. ADCC by IgG1 was predominantly mediated through FcRII in both anti-CD3 responder and nonresponder monocytes. FcRII was also involved in ADCC by IgG2b, although other receptors seemed to contribute significantly to ADCC. When FcRII or FcRI were blocked, IgG1 and IgG2a could also functionally interact with FcRI and FcRII, respectively, provided that the target cells were sensitized to a high degree. These findings indicate that FcRI and both forms of FcRII can mediate cytotoxicity and that the specificity of human FcR for murine isotypes is relative.     

Ocaratuzumab,an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells. In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.     

Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII.

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.     

Antitumour activity of a chimeric antibody against the leucocyte antigen CD48

Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3 and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma. Received: 5 May 1999 / Accepted: 12 August 1999     

Analysis of effector cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies

We examined purified human large granular lymphocytes, peripheral monocytes, and T cells for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) with murine monoclonal antibodies. We also evaluated the effects of pretreatment of cells with interleukin 2 and interferon to augment ADCC activity. MB3.6, a murine monoclonal antibody directed against the GD3 ganglioside, induced high levels of ADCC. This ADCC was mediated predominantly, if not completely, by human killer cells (large granular lymphocytes) whereas other effector cell populations demonstrated no significant cytotoxic activity in 6- or 18-hr assays. The IgG2a an anti-melanoma antibody 9.2.27 generated low or no ADCC with most normal donors or melanoma patients. IL 2 was a very potent booster of ADCC activity. Interferon alpha also was effective, whereas interferon gamma did not augment but rather inhibited reactivity. We tested a large panel of antibodies of various isotype against colon carcinoma cells and found that gamma-3 isotype antibodies more frequently generated ADCC and produced higher levels of cytotoxic activity than did IgG1 or IgG2 antibodies. It appears that a variety of parameters can affect ADCC reactions, including the type of effector cell and its level of activation, the isotype of the antibody, and properties of the target cell line such as its susceptibility to lysis.