Michael Dechant

Recombinant Dimeric IgA Antibodies against the Epidermal Growth Factor Receptor Mediate Effective Tumor Cell Killing

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti–His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.

Serum-free production and purification of chimeric IgA antibodies

Natural IgA antibodies are abundantly produced in vivo to protect serosal surfaces from invading infectious organisms. However, the immunotherapeutic potential of IgA has hardly been explored, although there is evidence that recombinant IgA antibodies may broaden the armentarium to combat certain infectious or malignant diseases. One of the limitations for exploring IgAs therapeutic activity has been the difficulty to obtain enough recombinant material with desired specificity for in vivo studies. Here, we describe the production and purification of monomeric recombinant IgA1 and IgA2 antibodies under serum-free conditions. For antibody production, suspension adapted CHO-K1 cells and a glutamine synthetase selection vector were used, which resulted in specific production rates of up to 2.2 pg/cell/day. Purities of >95% of monomeric antibodies were obtained by a combination of affinity chromatography-using an anti-kappa-light chain matrix-and size exclusion chromatography. Purified antibodies displayed the expected biochemical characteristics and were functionally fully active. Importantly, all required reagents and methods are commercially available and not dependent on the specificity of the desired antibody. In addition, all employed technologies and methodologies are similar to those used for the production of therapeutic IgG antibodies - thus allowing further up-scaling and streamlining according to existing antibody production technologies. In conclusion, the described methodology may assist in the development of recombinant IgA antibodies for therapeutic applications.

IgA EGFR antibodies mediate tumour killing in vivo

Currently all approved anti‐cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti‐tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti‐tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10‐EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short‐term syngeneic peritoneal model using EGFR‐transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

Effector mechanisms of therapeutic antibodies against ErbB receptors

ErbB1 and ErbB2 constitute validated target antigens for tumor therapy-as documented by the approval of antibodies and tyrosine kinase inhibitors (TKIs) against both antigens. However, their complex biology in development and tumorigenesis poses significant challenges on the optimization of this targeted approach. Crystallographic studies have significantly improved concepts about structure/function relationships of these receptors, and may assist in improving the efficacy of ErbB-directed therapy over the following years. Here, we will review these recent advances and their implications for ErbB-directed therapies. Although we will focus on the mechanisms of action of ErbB therapeutic antibodies, we will also briefly discuss TKIs.

Fc‐engineered EGF‐R antibodies mediate improved antibody‐dependent cellular cytotoxicity (ADCC) against KRAS‐mutated tumor cells

Oncogenic mutations of the KRAS gene have emerged as a common mechanism of resistance against epidermal growth factor receptor (EGF‐R)‐directed tumor therapy. Mutated KRAS leads to ligand‐independent activation of signaling pathways downstream of EGF‐R. Thereby, direct effector mechanisms of EGF‐R antibodies, such as blockade of ligand binding and inhibition of signaling, are bypassed. Thus, a humanized variant of the approved EGF‐R antibody Cetuximab inhibited growth of wild‐type KRAS‐expressing A431 cells, but did not inhibit KRAS‐mutated A549 tumor cells. We then investigated whether killing of tumor cells harboring mutated KRAS can be improved by enhancing antibody‐dependent cellular cytotoxicity (ADCC). Protein‐ and glyco‐engineering of antibodies’ Fc region are established technologies to enhance ADCC by increasing antibodies’ affinity to activating Fcγ receptors. Thus, EGF‐R antibody variants with increased affinity for the natural killer (NK) cell‐expressed FcγRIIIa (CD16) were generated and analyzed. These variants triggered significantly enhanced mononuclear cell (MNC)‐mediated killing of KRAS‐mutated tumor cells compared to wild‐type antibodies. Additionally, cells transfected with mutated KRAS were killed as effectively by ADCC as vector‐transfected control cells. Together, these data demonstrate that KRAS mutations are not sufficient to render tumor cells resistant to ADCC. Consequently Fc‐engineered EGF‐R antibodies may prove effective against KRAS‐mutated tumors, which are not susceptible to signaling inhibition by EGF‐R antibodies.

Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC.

Protein- or glyco-engineering of antibody molecules can be used to enhance Fc-mediated effector functions. ScFv-Fc fusion proteins (scFv-Fc) represent interesting antibody derivatives due to their relatively simple design and increased tissue penetration. Here, the impact of protein- and glyco-engineering on ADCC potency of a panel of human IgG1-based scFv-Fc was tested. Three matched sets of scFv-Fc variants targeting CD7, CD20 or HLA class II and optimized for CD16a binding by mutagenesis, lack of core-fucose, or their combination, were generated and functionally tested in comparison to the corresponding wild type scFv-Fc. Antigen binding activity was not compromised by altered glycosylation or Fc mutagenesis, whereas Fc binding to CD16a was significantly enhanced in the order: non-core fucosylated/Fc-mutated double-engineered≫Fc-mutated≥non-core-fucosylated>wild-type IgG1-Fc. All engineered variants triggered potent ADCC with up to 100-fold reduced EC50 values compared to non-engineered variants. Interestingly, double-engineered variants were similarly effective in triggering ADCC compared to single-engineered variants irrespective of their 1 log greater CD16a binding affinity. Thus, these data demonstrate that protein- and glyco-engineering enhances NK-cell mediated ADCC of scFv-Fc similarly and show that enhancing CD16a affinity beyond a certain threshold does not result in a further increase of NK-cell mediated ADCC.

Prevalence and specificity of immunoglobulin G and immunoglobulin A non-complement-binding anti-HLA alloantibodies in retransplant candidates

The role of complement-binding donor-directed anti-human leukocyte antigen (HLA) antibodies in graft rejection is well established, whereas the prevalence and relevance of non-complement-binding (NCB) anti-HLA antibodies are less well defined. The aim of our study was to establish a sensitive and reliable test system for the detection and the specification of these NCB anti-HLA antibodies. Sera from 60 patients awaiting retransplantation were analysed for the presence of anti-HLA class I alloantibodies with complement-dependent cytotoxicity (CDC) tests. Immunoglobulin (Ig)G(all) anti-HLA class I and class II alloantibodies were differentiated on generic level by plate-based solid phase enzyme-linked immunosorbent assay. Subsequently, a modified bead-based (Luminex) assay was applied, allowing the investigation of IgG(2/4) NCB isotypes as well as IgA(1/2). The anti-HLA specificities of the NCB alloantibodies were determined and compared with known mismatches from previous transplants. Seventeen of the 60 sera (28%) were positive in the CDC increasing to 26 of 60 (43%) in the class I and 33 of 60 (55%) in the class II plate-based assay. Using the modified bead-based system 24 of 60 sera (40%) contained NCB IgG(2/4), which were mostly donor specific. In addition, a high prevalence of NCB IgA antibodies was detected (26 of 60 sera), which occurred independently of IgG(2/4) NCB, and half of which were donor specific. NCB anti-HLA alloantibodies, including the IgA isotype, can reliably be detected using the modified bead-based test system. These NCB alloantibodies had a high prevalence in retransplant candidates and were mostly donor specific.

A Recombinant Bispecific Single-Chain Fragment Variable Specific for HLA Class II and FcαRI (CD89) Recruits Polymorphonuclear Neutrophils for Efficient Lysis of Malignant B Lymphoid Cells

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against FcαRI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. FcαRI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at KD values of 148 ± 42 nM and 113 ± 25 nM for the anti-FcαRI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (FcαRI × HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an FcαRI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (FcαRI × HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

Tumor cell killing mechanisms of epidermal growth factor receptor (EGFR) antibodies are not affected by lung cancer-associated EGFR kinase mutations.

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.

Complement‐mediated tumor‐specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII)

Monoclonal antibodies (mAb) against variant III of epidermal growth factor receptor (EGFRvIII) hold promise for improving tumor selectivity of EGFR‐targeted therapy. Here, we compared Fc‐mediated effector functions of three mAb against EGFRvIII (MR1‐1, ch806, 13.1.2) with those of zalutumumab, a high affinity EGFR mAb in advanced clinical trials. MR1‐1 and ch806 demonstrated preferential and 13.1.2 exclusive binding to EGFRvIII, in contrast to zalutumumab, which bound both wild‐type and EGFRvIII. All four human IgG1κ mAb mediated antibody‐dependent cellular cytotoxicity (ADCC) of EGFRvIII‐expressing cells with mononuclear cells and isolated monocytes, while only zalutumumab in addition triggered ADCC by polymorphonuclear cells. Interestingly, combinations of zalutumumab and EGFRvIII mAb specifically mediated complement‐dependent cytotoxicity (CDC) of EGFRvIII‐transfected but not wild‐type cells. Moreover, EGFRvIII‐specific CDC was significantly enhanced when zalutumumab was combined with a Fc‐engineered variant of MR1‐1 (K326A/E333A). These observations confirm the immunotherapeutic potential of antibody combinations against EGFR, and demonstrate that tumor selectivity can be improved by combining therapeutic EGFR mAb with an antibody against EGFRvIII. (Cancer Sci 2011; 102: 1761–1768)