Characterization of a Mutated IgA2 Antibody of the m(1) Allotype against the Epidermal Growth Factor Receptor for the Recruitment of Monocytes and Macrophages*

Background: IgA constitutes a promising antibody isotype, which requires optimization before immunotherapeutic application. Results: P221R-mutated and wild type IgA2m(1) antibodies were similarly effective in killing tumor cells and in recruiting myeloid effector cells. Conclusion: Improved IgA antibodies constitute promising next generation antibodies for tumor therapy. Significance: These studies support the clinical development of therapeutic IgA antibodies. IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.

IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective Fc␣RI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.
Monoclonal antibodies and their derivatives constitute a rapidly growing class of biotherapeutics for an increasing number of clinical indications (1,2). In tumor therapy, all therapeutic antibodies being approved or in clinical trials, are of the human IgG isotype, with clinical experience being available for IgG1, IgG2, and IgG4. At present, many arguments favor human IgG1 as the preferred isotype: first, human IgG1 effectively recruits NK cells and complement for tumor cell killing (3,4). Second, IgG antibodies display an extended serum half-life, which is mediated by binding to neonatal Fc receptor (FcRn) (5). In addition, production technologies and purification protocols are well established, and regulatory agencies are acquainted with relevant safety issues (6). The importance of antibody stability is underlined by recent observations with human IgG4 antibodies because IgG4 antibodies undergo fragment antibody binding (Fab) arm exchange under experimental and physiological conditions (7,8). Recent data demonstrated that myeloid effector cells were effectively recruited for ADCC by human IgG2 antibodies (9). However, granulocytes and monocytes were particularly cytotoxic for tumor cells with antibodies of the IgA isotype (4, 10 -12), which has not been explored clinically.
IgA antibodies contribute significantly to the humoral arm of the mucosal immune system, which provides important barrier functions to protect the large area of serosal surfaces (13). Three forms of IgA (monomeric, dimeric, and secretory IgA) are distinguished, which have distinct molecular characteristics and serve different functions in the immune system (14). Secretory IgA constitutes a first line of serosal defense against invading pathogens. Secretory IgA is a heavily glysoylated multimeric protein consisting of two monomeric IgA molecules, covalently linked by the joining J-chain and the secretory component (15).
In vivo, secretory IgA is generated when mucosa associated plasma cells produce dimeric IgA, which is transcytosed through epithelial cells via the basolateraly expressed polymeric immunoglobulin receptor (13). Incorporation of the J-chain into dimeric IgA is essential for the covalent interaction of the IgA heavy chain with the secretory component of the polymeric immunoglobulin receptor, which is cleaved proteolytically at the apical side to release secretory IgA (16). The second line of natural defense consists of circulating monomeric IgA, which is produced predominantly by bone marrow-located plasma cells and neutralizes pathogens that passed the mucosal barrier (17). This neutralization is mediated mainly by interacting with the Fc receptor for IgA (Fc␣RI; CD89), which is the best characterized IgA receptor (18). Fc␣RI is expressed on monocytes/ macrophages, granulocytes, subsets of dendritic cells, and Kupffer cells and binds both monomeric and dimeric IgA isoforms with median affinity. Binding of IgA to Fc␣RI mediates effector functions such as phagocytosis, oxidative burst, cytokine release, antigen presentation, and ADCC. Thus, monomeric IgA resembles IgG in many structural and functional aspects, except for the recruited effector cell population, whereas J-chain containing dimeric IgA constitutes a tetravalent molecule that offers the potential to target serosal surfaces (19).
In humans, two IgA isotypes, IgA1 and IgA2, and three allotypes, IgA2m(1), IgA2m(2) and IgA2n, have been distinguished (14). The major differences between IgA1 and IgA2 are located (i) in the hinge region, with IgA2 lacking the 13-amino acid elongation with up to five O-glycosylation sites existing in IgA1, (ii) in the N-glycosylation, with IgA2 being more than twice as heavily glycosylated than IgA1, and (iii) in the heavy and light chain linkage, where the Caucasian IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and in the IgA2m(2) allotype of those of African and Asian descent (14,20). In previous reports, we demonstrated that IgA antibodies were able to trigger polymorphonuclear cell (PMN)-mediated ADCC more efficiently than IgG antibodies, with IgA2 being more effective than IgA1 (11) and dimeric being more effective than monomeric IgA1 (12). However, the noncovalent linkage of heavy and light chains in the Caucasian IgA2m(1) makes this isotype less attractive for biotechnological production and for a therapeutic antibody as it might lead to an unstable recombinant molecule under certain conditions. This appears particularly relevant for dimeric IgA2, which constitutes a complex molecule consisting of four light and four heavy chains linked by a single J-chain. Thus, dimeric IgA2 may be particularly difficult to produce if the light and heavy chain linkage is not stable. To solve this issue and to create a fully predictable and covalently linked IgA2 molecule, a point mutation from proline to arginine at position 221 was introduced into the ␣2 heavy chain of the IgA2m(1) molecule, which is derived from the IgA2m(2) allotype sequence. This mutation sterically allows the formation of an alternate disulfide bond between the heavy (Cys-241) and the light chain cysteines (Cys-214) (21,22), similar to that found in non-Caucasian IgA2 allotypes, IgA2m (2) and IgA2n. In the present study, we demonstrate that this mutation results in monomeric and dimeric isoforms of an IgA2m(1) antibody, which demonstrate similar functionality as the respective wild type proteins but possess covalent heavy and light chain linkage. Thus, mutated monomeric or dimeric IgA2m(1) antibodies appear better suited for clinical applications.
The epidermal growth factor receptor (EGFR) 2 constitutes a well established target molecule for tumor therapy, which can be targeted by small molecule tyrosine kinase inhibitors and monoclonal antibodies (23,24). In contrast to tyrosine kinase inhibitors, which only inhibit EGFR signaling, monoclonal antibodies have a different, dual mode of action against cancer cells (25). Thus, monoclonal EGFR antibodies, similar to cetuximab, zalutumumab, or panitumumab mediate Fab-mediated effector mechanisms, such as blocking ligand binding or signal transduction, growth inhibition or EGFR down-modulation. In addition, their fragment crystallizable (Fc) part can trigger effector mechanisms such as complement-dependent cytotoxicity, phagocytosis, or ADCC. Increasing evidence suggests that ADCC may be a particularly important effector mechanism of EGFR antibodies in patients, whereas the predominant effector cell type (natural killer cells or myeloid cells) is controversial (25). For the CD20 antibody rituximab, the innate mononuclear phagocyte network was demonstrated to be the predominant effector cell population in vivo (26). In tumor biopsies, macrophages constitute a major component of the leukocyte infiltrate, where monocyte-derived M0 macrophages are thought to polarize into the tumor-inhibiting M1 or the tumor-promoting M2 phenotypes (27,28). In vitro, both M1 and M2 macrophages phagocytosed human IgG1 antibody opsonized tumor cells (29), but the potential of these subpopulations as effector cells for IgA antibodies has not been investigated previously. Here, we demonstrate that M0, M1, and M2 macrophages as well as monocytes and PMN are recruited effectively for ADCC by monomeric and dimeric IgA2 antibodies against EGFR. Both monomeric and dimeric isoforms of a P221R-mutated IgA2m(1) antibody were similarly effective as the respective wild type constructs and constitute promising molecules for next generation antibodies.

EXPERIMENTAL PROCEDURES
Experiments reported here were approved by the Ethical Committee of the Christian Albrechts University (Kiel, Germany) in accordance with the Declaration of Helsinki.
Flow Cytometry and EGFR Down-modulation-Binding to EGFR and to Fc␣RI was analyzed by flow cytometry as described previously (12). Down-modulation of EGFR was analyzed on murine BaF3 cells transfected with human EGFR, which were incubated with 225-IgA and control IgA (6 nM) for 4 or 24 h. Residual surface EGFR was detected with Alexa Fluor 488-labeled murine 425 antibody using the DyLight Fluor antibody labeling kit (Pierce). Results were calculated as "% EGFR down-modulation ϭ 100 Ϫ (relative fluorescence intensity (RFI) m425-FITC/RFI sample) ϫ 100. All samples were analyzed on a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter), collecting 1 ϫ 10 4 events for each experimental value. Data were analyzed using XL-System II software (version 3.0, Beckman Coulter). Relative fluorescence intensities were calculated as the ratio of mean linear fluorescence intensity of relevant to irrelevant isotype-matched antibodies.
Generation and Polarization of Macrophages-Monocytes were isolated from peripheral blood using CD14-magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and seeded into tissue culture flasks in RPMI 1640 supplemented with 10% FCS, 1% penicillin-streptomycin, 1% nonessential amino acids (Invitrogen), 0.1% sodium pyruvate (Sigma) containing 25 ng/ml human recombinant macrophage-colony stimulating factor (M-CSF), 10 ng/ml IL-1␤ (both Peprotech, Hamburg, Germany), and 1% (v/v) human sera for 7 days to differentiate into adherent macrophages (29). Macrophages were gained by scrapping. To polarize macrophages into M1 or M2 phenotype, IFN␥ (100 units/ml) and LPS (100 ng/ml) or IL-4 (20 ng/ml) (cytokines from Peprotech, LPS from Sigma) were added as indicated 24 h before harvesting (29). Polarized macrophages were washed three times with medium before being used in ADCC assays to remove cytokines. With the purpose to analyze chemokine and cytokine release, polarized and nonpolarized macrophages were cultured for further 24 h in fresh medium. Content of cytokines and chemokines in supernatants was analyzed using the human cytokine array panel A array kit (R&D Systems, Abingdon, UK) according to manufacturer's protocol. Pixel density (PD) of resulting dot plots was analyzed using ImageJ software (National Institutes of Health), and relative PD was calculated as % PD ϭ (PD PC Ϫ PD NC )/ (PD PC Ϫ PD Sample ) ϫ 100, where PC indicates positive control, and NC indicates negative control.
ADCC Assays-ADCC was measured using a 51 chromium ( 51 Cr) release assay (12). Briefly, citrate-anticoagulated blood from healthy volunteers was layered over a discontinuous Percoll (Biochrom, Berlin, Germany) gradient consisting of 70 and 63% Percoll. After centrifugation, mononuclear cells were collected from the plasma/Percoll interface and PMN from the interface between the two Percoll layers. Monocytes were isolated from mononuclear cells by CD14-positive selection using magnetic beads. Macrophages were generated as described above. Whole blood (25% v/v) or effector cells and antibodies were added to round-bottomed microtiter plates (Wallac, Turku, Finnland). Assays were started by adding effector and target cells at an effector:target ratio of 80:1 (40:1 in the case of macrophages) or as indicated. IgA-Fc␣RI interaction on effector cells during ADCC was blocked using My43, a murine IgM antibody specific for human Fc␣RI (Medarex, Annandale, VA). After incubation at 37°C (3 h for whole blood and GM-CSFactivated PMN (11) assays, 16 h for monocytes/macrophages), aliquots of supernatants were transferred into 96-well plates containing a scintillation mixture (OptiPhase Scintillator Supermix, PerkinElmer Life Science). 51 Cr release was measured in cpm using a scintillation counter (MetaBase TriLux, PerkinElmer Life Science). Percentage of cellular cytotoxicity was calculated as % specific lysis ϭ (experimental cpm Ϫ basal cpm)/(maximal cpm Ϫ basal cpm) ϫ 100, with maximal 51 Cr release determined by adding Triton X-100 (Merck, 1% final concentration) to target cells and basal release as measured in the absence of sensitizing antibodies and effector cells. Antibody-independent cytotoxicity (effectors without target antibodies) or effector-independent (target antibodies without effectors) was not observed.
Data Processing and Statistical Analyses-Data are displayed graphically and analyzed statistically using GraphPad Prism (version 5.0, GraphPad Software, San Diego, CA). Group data are reported as means Ϯ S.E. Differences between groups were analyzed by unpaired (or, when appropriate, paired) Student's t tests. EC 50 values were calculated from dose-response curves, reported as means Ϯ S.E. and compared by paired Student's t test to calculate significant differences between data groups. Significance was accepted when p values were Յ 0.05.

Production and Purification of Mutant 225-IgA2-P221R
Antibody-CHO-K1 cells, growing under serum-free suspension culture conditions, were transfected with vectors coding for the appropriate heavy and light chain genes by seeding them in DMEM-select medium to gain re-adherence (30). Twentyfour hours after transfection, the cells were placed under L-methionine sulfoximine selection and re-adapted to serum-free conditions by growing in CD-CHO-select medium (30). During the next weeks, single clones were produced by limiting dilution cloning and screening for antibody concentration in supernatants by IgA-specific ELISA. To produce dimeric IgA2 antibodies, well producing 225-IgA2-WT and 225-IgA2-P221R transfectomas were transfected additionally with a plasmid encoding the human His-tagged J-chain (12). Best producing clones were cultured in special cell line CL1000 production flasks. This system allowed a production of 5.9 Ϯ 2.8 and 4.7 Ϯ 2.7 mg/week/flask and a median antibody concentration of 296 Ϯ 141 and 239 Ϯ 127 g/ml for the monomeric mutant and wild type IgA2, respectively. For dimeric 225-IgA2-WT and 225-IgA2-P221R a median yield of 4.6 Ϯ 1.8 and 4.6 Ϯ 2.5 mg/week/flask and a median antibody concentration of 229 Ϯ 91 and 231 Ϯ 125 g/ml were obtained, respectively. Purification of monomeric and dimeric IgA2 was performed as described previously (12,30). In Fig. 1A, affinity-purified IgA2m(1) antibodies were subjected to size exclusion chroma-tography to isolate monomeric 225-IgA2m(1)-WT (left) and -P221R (right). Elution profiles of both preparations are presented, and relative areas under the curve of each peak compared with overall AUC were calculated (Fig. 1A, table). A significantly lower content of aggregated high molecular weight molecules was detected for the mutated IgA2m(1) antibody. Aggregates of both antibodies consisted of mixtures of covalently and noncovalently linked molecules (data not shown).
Long term stability and content of aggregates or decay products were analyzed on analytical size exclusion columns. The elution profiles of monomeric wild type and mutant 225-IgA2m(1) antibodies displayed single peaks for both batches, either freshly prepared ( Fig. 1E1 and E3) or stored for 1 year (Fig. 1, E2 and E4, respectively). Both batches of P221R-mutated dimeric 225-IgA2m(1) displayed only one peak representing dimeric antibodies (Fig. 1, F3 and F4). After 1 year of storage, dimeric 225-IgA2m(1)-WT displayed several decay products, including heavy and light chain homodimers as well as monomeric antibodies and heavy and light chain fragments (Fig. 1, F1 and F2).

Effector Cells for a Stabilized IgA2m(1) Antibody
Effector Cell Recruitment by EGFR-directed IgA2 Antibodies-The ability of 225-IgA2 antibodies to bind the IgA Fc receptor, Fc␣RI, was analyzed by indirect immunofluorescence analyses using Fc␣RI/FcR␥-chain-co-transfected BHK cells (31). On these transfectants, the mutant 225-IgA2-P221R antibody demonstrated significantly lower binding than the wild type 225-IgA2 (p Յ 0.05), in monomeric as well as in dimeric form, whereas a control IgA2 antibody showed intermediate binding (Table 1, Fig. 3A).
In ADCC assays with human whole blood as effector source, both monomeric and dimeric 225-IgA2-WT performed similarly effective ADCC of A431 tumor cells as their respective P221R mutants and outperformed the respective IgG1 antibody (p Յ 0.001, Table 1, Fig. 3B). Next, we isolated Fc␣RI-expressing mono-and polymorphonuclear effector cells from blood of healthy donors to assess their contribution to ADCC. In ADCC assays with GM-CSF-activated PMN effector cells, which mainly consist of neutrophils, monomeric and dimeric 225- IgA2-P221R triggered similar effective ADCC than their respective wild type counterparts but significantly higher levels of killing than 225-IgG1 (p Յ 0.001, Table 1, Fig. 3C). With isolated monocytes as effector cells, both monomeric and dimeric 225-IgA2-WT were similarly effective in triggering tumor cell killing compared with their respective P221R mutant forms and to 225-IgG1 (Table 1, Fig. 3E). Next, we investigated whether wild type and mutant IgA2m(1) were equally resistant to denaturizing temperature conditions (Fig.  2E). Both monomeric 225-IgA2 antibodies were incubated for 5 min at different temperatures, and functionality was analyzed in ADCC assays using freshly isolated PMN as effector and A431 as target cells. Wild type 225-IgA2m(1) and the respective 225-IgG1 were significantly less efficient in mediating ADCC after incubation at 72-84°C than the 225-IgA2m(1)-P221R (Fig. 4A). In addition, both 225-IgA2 antibodies as well as the IgG1 were incubated at 78°C for different time intervals (Fig.  4B). Also, in this case, the P221R-mutated 225-IgA2m(1) displayed higher resistance under these denaturizing conditions than its wild type IgA2m(1) and the IgG1 counterpart. However, above 84°C, also the P221R-mutated 225-IgA2 was denaturized and failed to induce ADCC.
Macrophages Are a Potent Effector Cell Population for IgA2 Antibodies-Next, we investigated whether macrophages could be recruited by EGFR-directed IgA2 antibodies to mediate ADCC against A431 cells. Both monomeric and dimeric EGFRspecific 225-IgA2 antibodies and 225-IgG1 were similarly effective in mediating macrophage-dependent ADCC of A431 tumor cells (Table 1, Fig. 5, A1 and A2). In ADCC assays with different effector:target ratios, both wild type and mutant IgA2 antibodies were similarly effective in mediating ADCC by macrophage recruitment even at low effector:target ratios (Fig.  5B). Maximal effectiveness of macrophages-mediated ADCC was reached at effector:target ratios of 40:1 and higher. For the respective IgG1, similar levels of cytotoxicity were detected. Subsequently, we investigated whether IgA-mediated ADCC was dependent on Fc␣RI interaction. Therefore, we performed ADCC assays with a constant concentration of EGFR-specific IgA2 or IgG in the presence of My43 to specifically block IgA-Fc␣RI interactions (Fig. 5C). Addition of My43 blocked ADCC mediated by both 225-IgA2-WT and 225-IgA2-P221R antibodies in a dose-dependent manner, whereas IgG-mediated ADCC was not affected. Specific lyses decreased from initially 48.9 Ϯ 7.4 and 46.0 Ϯ 7.3% to 7.4 Ϯ 4.7 and 6.8 Ϯ 2.7%.
Effects of Macrophage Polarization on Cytokine and Chemokine Release and ADCC-To investigate the influence of macrophage polarization on their capability to mediate ADCC, macrophages were polarized into M1 or M2 phenotypes by supplementing medium with IFN␥ and LPS or IL-4 for 24 h, respectively. In response to cytokine treatment, macrophages changed their cytokine and chemokine expression profile: M1 macrophages, treated with IFN␥ and LPS, strongly expressed chemokines (CXCL10, CXCL11, CCL2, CCL5), whereas M2 macrophages, treated with IL-4, and M0 (untreated) macrophages did not (Fig. 6B). Concerning cytokines, M1 macrophages expressed IFN␥ at high levels, TNF␣ at higher and IL-1ra and IL-8 at lower levels compared with M2 and M0 macrophages. M0 macrophages displayed lower expression of cytokines (IL-1ra, IFN␥) and of chemokines (CXCL10, CCL2). Next, we analyzed whether treatment of macrophages with cytokines influences their capability to mediate ADCC of human A431 tumor cells by EGFR-specific antibodies. Interestingly, M1 and M2 macrophages showed similarly efficient killing by all three EGFR antibodies compared with M0 macrophages (Fig. 6C).

Advantages and Disadvantages of IgA Iso-and Allotype
Selection-IgA antibodies serve important functions in the mucosal immune system (13)(14)(15) and have been demonstrated to mediate potent tumor cell killing in vitro (11,12). During these in vitro evaluations, monomeric IgA2m(1) antibodies proved more effective in triggering ADCC than IgA1 antibodies (11), which might be related to the different Fab arm orientation of IgA1 versus IgA2 antibodies (31). In view of a clinical application, the IgA2 isotype has other potential advantages. First, IgA1 antibodies contain an elongated heavily O-glycosylated hinge region (32), which is more susceptible to bacterial proteases than the IgA2 hinge (33). This may cause stability issues in vivo, particularly at bacterially colonized sites. Additionally, O-glycosylation is typically diverse and difficult to control during biomolecule production, which limits regulatory and safety experience (6). Importantly, aberrantly hypogalactosylated natural IgA1 antibodies are critically involved in the development of IgA nephropathy, one of the most common causes leading to renal failure (34). Thus, IgA2 represents the preferred isotype for further immunotherapeutic development.
Stabilizing IgA2m(1) by a Single Mutation-In humans, three different allotypes have been described for IgA2: IgA2m(1), m(2), or IgA2n (14). Although immunogenicity of allotypes has not been an important issue with IgG1 antibodies (35), this may be different for IgA antibodies, which target more immunologically active sites such as the mucosal tissue. Thus, the ethnicity of potential patient populations may impact the selection of appropriate antibody allotypes. However, the most common Caucasian allotype, IgA2m(1), contains an unusual pairing of  (2) and IgA2n are covalently assembled as in IgG1 or IgA1 antibodies, we decided to improve the well characterized and functionally well performing 225-IgA2m(1) antibody by a single point mutation instead of developing a completely new antibody by changing the allotype. Mutations in the C␣1 domain of the IgA2 heavy chain (proline to arginine at position 221 and proline to serine at position 212) were described pre- viously to prevent the protein from degradation (21,22). These mutations sterically enable the formation of an alternate disulfide bridge between the heavy and light chains in the IgA2m(1) allotype, similar to the IgA2m(2) allotype. Our results show that a single P221R amino acid exchange derived from the IgA2m(2) sequence is sufficient to prevent dissociation into heavy and light chain homodimers in both monomeric and dimeric isoforms. Furthermore, the P221R mutation enhanced the stability of the protein as its resistance to long term storage and denaturizing temperature was increased significantly. Thus, a P221R-mutated 225-IgA2m(1) antibody might be better suited for bioindustrial production and for clinical applications. Functional Evaluation of Mutated 225-IgA2m(1) Antibody-Next, we investigated whether the P221R mutation influenced the capability of the IgA2 antibodies to activate Fab-and Fcmediated effector functions. The 225-IgA2-P221R antibody was equally efficient as 225-IgA2-WT in Fab-mediated functions, such as binding to EGFR, growth inhibition, and downmodulation of EGFR in tumor cells. Although mutant 225-IgA2-P221R was less efficient than wild type IgA2 in binding to Fc␣RI in transfected BHK cells, it was as effective as its wild type counterpart in killing assays using unfractionated human whole blood, activated PMN, isolated monocytes, or monocyte-derived-macrophages. This observation might be related to Fc␣RI splice variants differently expressed by human leukocytes (36,37) and upon cellular activation (38). Furthermore, the functionality of Fc␣RI is dependent on the interaction with the FcR␥ chain and sensitive to inside-out signaling (39,40). Thus, the Maintenance of functionality was analyzed in ADCC assays using PMN as effector and A431 as target cells. Relative specific lysis was reduced for both wild type and mutant IgA2m(1) antibodies, with the latter demonstrating a significantly slower reduction. Results (n ϭ 5) are presented as mean Ϯ S.E. of relative specific lysis (%). Significant differences (p Յ 0.001) between wild type and mutant IgA2m(1) are indicated by number. In B, curves for IgG1 and IgA2(m1)-WT are superimposed.  (1) antibodies as well as 225-IgG1 induced similar specific lysis in macrophage-mediated ADCC assays of A431 tumor cells. In B, different ratios of macrophages (effectors) and A431 cells (targets) were used in ADCC assays. In ADCC assays using monocyte-derived macrophages (C), specific lysis of A431 tumor cells induced by monomeric 225-IgA2-WT and 225-IgA2-P221R was reduced if Fc␣RI-directed antibody My43 was supplied in a dose-dependent manner, whereas ADCC mediated by the respective 225-IgG1 was not affected. Results (n Ն 5) are shown as mean Ϯ S.E. of % specific lyses. Asterisks indicate significant differences (p Յ 0.01) of EGFR-specific to control IgA (A and B) or between IgA and IgG antibodies (C) using black asterisks for mutant IgA2m(1) and gray asterisks for wild type IgA2m(1) antibodies. E:T, effector: target ratio. JULY 20, 2012 • VOLUME 287 • NUMBER 30 BHK double transfectants might not fully reflect all dynamic states of the Fc␣RI receptor on human leukocytes. Unfortunately, binding of IgA antibodies to Fc␣RI on human effector cells could not be reliably investigated by immunofluorescence analyses due to the low Fc␣RI expression on these cells and the low affinity interaction with the ligand. Both wild type and mutated 225-IgA2 antibodies demonstrated higher efficiency in Fab-and Fc-mediated assays when dimeric IgA2 was compared with equimolar concentrations of the respective monomeric molecules, which was also the case for the respective 225-IgG1. Similar results have been reported previously for monomeric versus dimeric IgA1 (12) and are probably explained by the tetravalency compared with bivalency of dimeric versus monomeric IgA.

Effector Cells for a Stabilized IgA2m(1) Antibody
Macrophages as Effector Cell Population for 225-IgA2m(1) Antibodies-Myeloid cells such as monocytes and PMN have been described previously to be recruited effectively for ADCC by EGFR antibodies of IgA1 isotype in vitro (12). In tumor sections, myeloid cells, including tumor-associated macrophages, constitute the major component of the leukocyte tumor infiltrate (27,28); however, their potential role for tumor progression or surveillance is controversial. For example, experiments with cytokine-transfected tumor cells demonstrated inhibitory effects of the recruited myeloid cells on tumor growth (41,42).
More recently, tumor-associated macrophages were divided into the tumor growth inhibiting M1 and the tumor promoting M2 phenotype, which are both thought to derive from a common M0 precursor (43). Also for tumor-associated neutrophils, corresponding N1 and N2 phenotypes have been proposed (44). However, a programmatic change of myeloid cells, e.g. on IL-12 exposure has been reported (45), and most of these studies investigated the role of tumor associated myeloid cells in the absence of tumor-directed antibodies. In the presence of antibodies, both M1 and M2 macrophages were demonstrated to kill rituximab-opsonized tumor cells in vitro (29), relating to the contribution of myeloid cells for the therapeutic efficacy of rituximab in vivo (26). Recently, cetuximab was demonstrated to activate M2 macrophages in vitro (46), but the contribution of this activation on in vivo growth of EGFR-expressing tumors was not investigated in these experiments. To the best of our knowledge, our study is the first to investigate the impact of IgA antibodies on tumor cell killing by selectively differentiated macrophages. Interestingly, M0, M1, and M2 macrophages were similarly effective in killing of IgG-and IgA-coated tumor cells (Fig. 5C). Because macrophages are phagocytes internalizing rapidly cross-linked IgA, the results of the cytotoxicity assays might underestimate the potency of IgA (47). Whether these in vitro experiments also relate to in vivo efficacy of EGFR FIGURE 6. Polarized macrophages display different cytokine and chemokine profiles but similar capacity to trigger IgA-mediated ADCC. Monocytes were treated with M-CSF for 7 days to induce differentiation into macrophages. Next, cells were left untreated (M0) or treated for 24 h with IFN␥ϩLPS or IL-4 for final polarization into M1 or M2 phenotypes, respectively. A, cytokines and chemokines released into fresh medium were analyzed using a commercial array kit. Pixel density of dot plots was calculated using ImageJ software. Results (n Ն 2) are shown as mean Ϯ S.E. of pixel density (% to PC). B, both 225-IgA2 antibodies and the respective 225-IgG1 induced similar specific lysis by recruiting M0, M1, and M2 macrophages to mediate ADCC of A431 tumor cells. Results (n Ն 4) are shown as mean Ϯ S.E. of % specific lysis. Significant differences (p Յ 0.01) between 225-IgA2m(1) and control IgA2 antibody were indicated with respectively colored asterisks.
antibodies of IgA isotype needs to be determined in further studies. However, these studies are complicated by the lack of a Fc␣RI orthologue in mice (48), a limitation that can be overcome by using human Fc␣RI transgenic mice (17). First, in vivo studies in these mice demonstrated an unexpectedly short serum half-life of our IgA antibodies, which may be related to the lack of sialylation. Thus, exposed terminal galactose may mediate binding of both IgA2m(1) antibodies to the hepatically expressed asialoglycoprotein-receptor (49), which rapidly internalizes these antibodies. These in vivo data are indicating the requirement for further improvements of our current molecules.
Conclusion-In conclusion, we have demonstrated that macrophages, in addition to monocytes and PMN, may constitute another potent effector cell population for EGFR-directed IgA antibodies. Interestingly, M0, M1, and M2 macrophages displayed similar ADCC activity, suggesting that also tumorassociated M2 macrophages can be recruited against cancer cells by tumor-specific IgA antibodies. Furthermore, a P221Rmutated version of the Caucasian IgA2m(1) allotype demonstrated the typical heavy and light chain pairing of other antibody isotypes and proved functionally as effective as its wild type counterpart. Together, these results further promote the concept of employing IgA antibodies in tumor therapy.