RI (CD89) as a Novel Trigger Molecule for Bispecific Antibody Therapy

Fc a Rb, Fc receptor (CD89) isoform Results re-Alternatively spliced forms myeloid Fc receptor ported

M FcgRII (CD32), and FcgRIII (CD16). For IgA, in comparison, one a gene (FcaRI), located on chromosome 19, has ONOCLONAL ANTIBODIES (MoAbs) offer the potential to increase the specificity of oncologic therapy by targeting select tumor-associated antigens. 1,2 Antitumor been characterized that encodes several alternatively spliced transmembrane isoforms of 55 to 110 kD. 13-15 FcaRI (CD89) effects of MoAb may be divided into (1) direct effects on tumor cells (such as blockade of growth factors, inhibition is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but importantly, of proliferation, or induction of apoptosis), or (2) indirect effects. These latter involve recruitment of immune effector not on noneffector cell populations. FcaRI has medium affinity (É5 1 10 7 mol/L 01 ) for both IgA1 and IgA2, 16,17 which mechanisms such as complement-or cell-mediated lysis of target cells and require interaction of therapeutic antibodies is increased on exposure to G-CSF or granulocyte-macrophage colony-stimulating factor (GM-CSF). 18 For signaling, with complement proteins or with immunoglobulin receptors on effector cells. An improved interaction was shown with the a-chain of the IgA receptor requires association with the common FcRg-chain, which results in the phosphorylation chimeric or humanized antibodies relative to their murine counterparts. 3,4 Recent clinical trials with MoAbs in oncol-of an ITAM (immunoreceptor tyrosine-based activation motif) within the g-chain, initiating further downstream signal-ogy focused largely on human IgG1 as therapeutic isotype. 5,6 Human IgG1 effectively activates human complement and ing events. 19 Functions described for FcaRI include ADCC, phagocytosis, induction of a respiratory burst, and inflam-interacts well with human IgG Fc receptors, especially FcgRIIIa (CD16) involved in natural killer (NK) cell-matory mediator or cytokine release. 16,17 In vivo, therapeutic antibodies compete with high concen-mediated antibody-dependent cell-mediated cytotoxicity trations of endogenous immunoglobulins for binding to Fc (ADCC). Data in this report support a role for human IgA receptors. In addition, many Fc receptor-expressing cells are as a promising therapeutic antibody isotype because it effectively recruits neutrophils for ADCC. Neutrophils have tumor-cytolytic potential against a broad spectrum of tumor not cytolytic for tumor cells (eg, B cells, platelets), and some Fc receptor isoforms (eg, FcgRIIb, FcgRIIIb) bind antibodies, but do not trigger cytolytic cascades. However, select cytotoxic Fc receptors on effector cells can be triggered by antibodies reacting with Fc receptors via their variable regions, 20 a process called reverse ADCC. This mechanism is used in the therapeutic concept of bispecific antibodies, where antibodies directed to cytotoxic trigger molecules, such as Fc receptors, are genetically or chemically linked to tumor-directed antibodies. 21 Bispecific antibodies engaging FcgRI or FcgRIII are currently being tested with promising results in clinical trials and may help to overcome some of the above-mentioned limitations of conventional antibodies. 22 Besides their potential as effector cells in antibody-based tumor therapy, neutrophils are the main effector cell population in the host defense against fungi. 23,24 Therefore, we investigated the role of bispecific antibodies directed to FcaRI and Candida albicans. These constructs were found to mediate very effective phagocytosis of Candida by neutrophils. Thus, we identify FcaRI as a promising trigger molecule for bispecific antibody (BsAb)-based therapy and show synergistic effects in combination with G-CSF.

MATERIALS AND METHODS
Blood donors. Experiments reported here were approved by the Ethical Committee of the University of Erlangen, Nürnberg, in accordance with the Declaration of Helsinki. After informed consent, 10 to 20 mL of citrate anticoagulated peripheral blood was drawn from healthy volunteers or from patients receiving G-CSF therapy. Patients were treated with rh-met-G-CSF (Neupogen, 3 to 5 mg/kg of body weight) from Hoffmann La-Roche (Basel, Switzerland), based on clinical indications. As the kinetics of neutrophil recovery during G-CSF treatment vary, the following criteria were defined for analy- kept in RF10 / medium consisting of RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, 100 U/mL streptomycin, and 4 mmol/L L-glutamine (all Life Technologies).
produced from hybridomas obtained from the ATCC. For detection of human/mouse chimeric NIP antibodies or murine Fc receptor Monoclonal and bispecific antibodies. Human/mouse chimeric antibodies against the chemical hapten 5-iodo-4-hydroxyl-3-nitro-antibodies, fluorescein isothiocyanate (FITC)-labeled anti-mouse l light chain antibodies or FITC-labeled F(ab) 2 fragments of goat phenacetyl (NIP) consisting of mouse V H , V L , and C L chains of l isotype, and C H chains of human g1, or g3 isotypes were produced anti-mouse were used as staining reagents, respectively (both from Cappel, Cochranville, PA). from Chinese hamster ovary (CHO)-K1 transfectomas and were purified on antigen columns as described. 25 Anti-NIP IgA2 was used BsAb (FcaRI 1 HER-2/neu) was produced as described 31 by chemically cross-linking F(ab) fragments of MoAb A77 (FcaRI; as supernatant from the original transfectoma, 3 which was obtained from the European Collection of Animal Cell Cultures (Porton CD89) with F(ab) fragments of MoAb 520C9 (HER-2/neu). Briefly, F(ab) 2 fragments were produced by limited digestion with pepsin Down, UK).
Antibody 520C9 (mIgG1) against the proto-oncogene product and were then reduced with mercaptoethanol to provide F(ab) with free hinge-region sulfur hydroxyl (SH) groups. The SH groups on HER-2/neu 26 and Fc receptor antibodies A77 (mIgG1) and My43 (mIgM) both to FcaRI, 27,28 22 (mIgG1) and 197 (mIgG2a) both to one of the F(ab) (SH) partners were then fully derivatized with excess o-phenylenedimaleimide (o-PDM) to provide free maleimide FcgRI, 20 whole antibody and Fab fragments of IV.3 (mIgG2b) to FcgRII, 29 and whole antibody and F(ab) 2 fragments of 3G8 (mIgG1) groups. Finally, the F(ab)-o-PDM and F(ab)-SH were combined at a ratio of 1:1 to generate heterodimeric F(ab)-o-PDM-F(ab) to FcgRIII 30 were from Medarex (Annandale, NJ). Antibody MMA (mIgM) to CD15 and negative control antibody 3.6.2 (mIgG2a) were constructs. After purification by size exclusion chromatography and For personal use only. on August 29, 2017. by guest www.bloodjournal.org From  Fig 2. Antibody-mediated cytotoxicity against NIP-haptenized SK-BR-3 breast cancer cells was analyzed using NIP antibodies of human IgG1, IgG3, or IgA2 isotypes. As effector source, whole blood (), fresh human plasma (ᮀ), isolated MNC ( ), or PMN ( ) were compared. All three isotypes induced significant lysis with whole blood. Interestingly, IgA-dependent lysis was predominantly mediated by PMN, whereas IgG1-or IgG3-dependent killing resided mainly in the plasma fraction. Results from five experiments are presented as mean Ô SEM of % specific lysis. Significant (P Ú .05) antibody-mediated lysis is marked by an asterisk ( * ). characterization by high-performance liquid chromatography ies were used at a final concentration of 10 mg/mL. Assays were started by adding target cell suspensions (50 mL), giving a final (HPLC), samples were sterilized by filtration. For production of (FcaRI 1 Candida) BsAb, F(ab) 2 fragments of a polyclonal anti-volume of 200 mL, and an effector to target cell ratio of 80:1 with isolated cells. After 3 hours at 37ЊC, assays were stopped by centrifu-Candida IgG isolated from immunized rabbits (Biodesign, Kennebunk, MA) were treated with sulfo-SMCC, which adds maleimide gation, and 51 Cr release from triplicates was measured. Percentage of cellular cytotoxicity was calculated using the formula: groups to free amino groups. These F(ab) 2 fragments were then reacted with an equimolar concentration of A77 F(ab), and the conjugate was purified from uncoupled molecules by chromatogra-% Specific Lysis Å Experimental cpm 0 Basal cpm Maximal cpm 0 Basal cpm 1 100 phy on Superdex 200 (Pharmacia, Picsataway, NJ). Both BsAb were stored at 4ЊC and showed binding to both effectors and targets consiswith maximal 51 Cr release determined by adding perchloric acid (3% tent with the specificity patterns of the parental antibodies. final concentration) to target cells, and basal release measured in the Isolation of mononuclear and neutrophil effector cells. Isolated presence of sensitizing antibodies without effector cells. For analysis neutrophils were obtained by a method described by Elsässer et al. 9 of effects induced by Fc receptor antibodies, ''% inhibition'' was Briefly, citrate anticoagulated blood was layered over a discontinucalculated: ous percoll (Seromed, Berlin, Germany) gradient. After centrifugation, neutrophils were collected at the interphase between the two % Inhibition percoll layers and mononuclear cells from the percoll/plasma interphase. Remaining erythrocytes were removed by hypotonic lysis. Å % Lysis Without 0 % Lysis With FcR Antibody % Lysis Without FcR Antibody 1 100 Purity of neutrophils was determined by cytospin preparations and exceeded 95%, with few contaminating eosinophils, and less than 1% mononuclear cells. Viability of cells tested by Trypan blue exclu-Negative values determined by this formula are reported as ''% sion was always greater than 95%. stimulation'' in the presence of Fc receptor antibody. Immunofluorescence analysis. Isolated neutrophils, mononu-Candida phagocytosis. C albicans (strain ATCC 448585) was clear cells (MNC) or SK-BR-3 breast cancer cells were incubated cultured overnight at 37ЊC in Sabouraud Maltose Broth (DIFCO, with MoAb. During incubation of effector cells with MoAb, poly-Detroit, MI), harvested by centrifugation, washed three times with clonal human IgG (4 mg/mL) was added to inhibit nonspecific bindphosphate-buffered saline (PBS), and labeled by incubation with ing to FcgRI. After staining with FITC-labeled reagents, fluores-FITC (Sigma, St Louis, MO) at a concentration of 0.1 mg/mL in cence was analyzed on an EPICS Profile flow cytometer (Coulter). 0.1 mol/L NaH 2 PO 4 /Na 2 HPO 4 buffer (pH 9.6) for 30 minutes at 4ЊC. For each cell population, relative fluorescence intensity (RFI) was After three washes with PBS, 5 1 10 5 yeast particles were incubated calculated as the ratio of mean linear fluorescence intensity of relefor 30 minutes at 37ЊC with 2 1 10 5 isolated PMN without or with vant to irrelevant, isotype-matched antibodies.
10 mg/mL (A77 1 aCandida) BsAb. C albicans phagocytosis was Antibody-dependent cellular cytotoxicity assays. ADCC assays quantitated by measuring FITC-fluorescence intensities of cells on a were performed as described. 10  cytotoxicity assays were established, which are schemati-effector cells. In contrast, IgG1-or IgG3-mediated killing resided predominantly in the plasma fraction, with signifi-cally represented in Fig 1. First, SK-BR-3 breast cancer cells were covalently coated with the hapten NIP and sensi-cant cell-mediated lysis only observed with IgG1. Antibodymediated lysis in the presence of plasma was completely tized with NIP-specific chimeric antibodies of human IgA2, IgG1, or IgG3 isotypes (Fig 1A). All three antibodies gave abrogated when plasma was heat-inactivated at 56ЊC for 30 minutes (n Å 3), indicating complement-mediated lysis to high levels of target cell sensitization (RFI Å 130, 276, or 238, respectively) and did not induce direct target cell kill-be the underlying effector mechanism. In accordance with previous reports, 17 IgA was found to be less effective than ing. In the presence of whole blood, all three mediated effective target cell lysis. For further analysis of effector mecha-IgG1 in complement-dependent target cell killing. However, uncontrolled complement activation induced by therapeutic nisms, whole blood was fractionated into plasma, isolated PMN, or MNC effector cells, which were then compared for antibodies in vivo may cause serious side effects in patients. 32 On the other hand, IgA proved considerably more their capacity to mediate lysis via NIP antibodies (Fig 2). Remarkably, IgA2-induced killing was predominantly medi-effective in recruiting PMN for ADCC than IgG1. To our knowledge, no therapeutic human IgA MoAb has yet been ated by PMN, and to a smaller extent, by plasma or MNC

Fig 4. Increasing concentrations of (A77 Ì 520C9) BsAb directed to FcaRI and HER-2/neu were analyzed in ADCC against SK-BR-3 breast cancer cells. As effector cell source, unfractionated whole blood (A, q) was compared with human plasma (᭝), isolated MNC (᭺), or PMN effector cells () (B) at an E:T ra-
tio of 80 to 1. Significant ADCC (P Ú .05 indicated by an asterisk [*]) was observed with BsAb concentrations higher than 0.08 mg/mL in whole blood and with PMN, but not with plasma or MNC. Results from three experiments are presented as mean Ô SEM of % specific lysis.
For personal use only. on August 29, 2017. by guest www.bloodjournal.org From given to patients. Together with our previous observation isotype control antibodies or by Fcg receptor antibodies, which were documented to block IgG-mediated ADCC 11,12 that neutrophil-mediated ADCC against malignant B cells was strongly target antigen restricted, 9 results reported here ( Fig 3A). Reverse ADCC, induced by antibodies directed against suggest IgA antibodies to be particularly promising when ADCC by neutrophils is a major effector mechanism for surface molecules on effector cells, represents a sensitive and specific approach to identify tumor-cytolytic trigger mol-MoAbs. Further studies are in progress to investigate whether neutrophils' target antigen restriction can be over-ecules. To compare different Fc receptors for their capacity to mediate reverse cytotoxicity, we set-up a novel assay come by using therapeutic antibodies of IgA isotype.
To identify cellular receptors involved in IgA-mediated using rat HB58 hybridoma cells as targets in ADCC ( Fig  1B). These cells were selected for high membrane expression ADCC, we analyzed PMNs' expression of FcaRI (CD89), FcgRI (CD64), FcgRII (CD32), and FcgRIII (CD16) by of their surface immunoglobulin, a rat antibody directed to mouse k light chains. In the presence of murine effector indirect immunofluorescence with MoAb A77, 22, IV.3, or 3G8, respectively. Thus, PMN expressed FcaRI, FcgRII, cell-directed antibodies, these target cells are sensitized for reverse ADCC. Importantly, healthy donor PMN very effec-and FcgRIII (RFI 11.0 { 1.1, 16.5 { 2.2, and 99.1 { 20.5, respectively, n Å 8), but very low levels of FcgRI (RFI Å tively lysed HB58 hybridoma cells in the presence of FcaRI antibody A77 (Fig 3B). Furthermore, they were effective 1.6 { 0.2, n Å 8). Blocking antibodies to FcgRI (197, whole antibody), FcgRII (IV.3, F(ab) fragments), FcgRIII (3G8, with FcgRII antibody IV.3, but not with FcgRI or FcgRIII antibodies (22, and 3G8, respectively). G-CSF primed PMN F(ab) 2 fragments), or FcaRI (My43, whole antibody) served to identify trigger molecules on PMN involved in IgA-medi-were additionally effective with FcgRI antibody 22 (not shown). Data with this broadly applicable assay establish ated killing. IgA-mediated ADCC by PMN was significantly (P õ .05) inhibited by FcaRI antibody My43, but not by FcaRI on PMN as novel cytotoxic trigger molecule. For  Fcg receptors, they are in agreement with results using Fc isolated MNC or PMN effector cells ( Fig 4B). As shown, (A77 1 520C9) BsAb induced significant target cell lysis at receptor-specific hybridoma targets in reverse ADCC assays, which were selected for high surface expression of their concentrations higher than 80 pg/mL, with optimal killing at 2.0 mg/mL, by whole blood or PMN. Isolated MNC or immunoglobulins. 9, 33 The mechanism of reverse ADCC is exploited in the thera-human plasma had no activity with this BsAb, indicating that reverse ADCC by PMN represents the predominant effector peutic concept of bispecific antibodies (Fig 1C). We, therefore, tested a BsAb constructed by chemically cross-linking mechanism in these whole blood assays. G-CSF is known to stimulate IgG-dependent ADCC by F(ab) fragments of antibodies A77 and 520C9, specific for FcaRI (CD89), or the proto-oncogene protein HER-2/neu, PMN via induction of FcgRI expression 34 and to increase the affinity of IgA binding. 18 Therefore, we compared iso-respectively. This BsAb showed the expected binding characteristics and was tested in ADCC against HER-2/neu posi-lated PMN from healthy donors and from patients during G-CSF therapy (Fig 5) for FcaRI expression (Fig 5A), IgA-tive SK-BR-3 breast cancer cells (Fig 4) using either whole blood (Fig 4A), or fractions consisting of human plasma, mediated ADCC against haptenized targets (Fig 5B), reverse   6 (IL-6), and an acceptable toxicity profile. 38