The therapeutic efficacy of humanized or chimeric second-generation antitumor antibodies is clearly established, but often limited. In recent years, defined modifications of the glycosylation pattern or the amino-acid sequence of the human immunoglobulin G1 Fc part have resulted in the development of third-generation antibodies with improved capability to recruit Fc receptor-bearing effector cells. The first antibodies of this kind, currently evaluated in early clinical trials, are directed against lymphoma-associated antigens. Fc-engineered antibodies targeting myeloid leukemia are not yet available. We here report on the generation and preclinical characterization of an Fc-optimized antibody directed to the FMS-related tyrosine kinase 3 (FLT3), an antigen expressed on the leukemic blasts of all investigated patients with acute myeloid leukemia (AML). This antibody, termed 4G8SDIEM, mediated markedly enhanced cellular cytotoxicity against FLT3-expressing cell lines as well as blasts of AML patients. FLT3 expression levels on AML cells varied between 300 and 4600 molecules/cell and, in most cases, were substantially higher than those detected on normal hematopoietic precursor cells and dendritic cells (approximately 300 molecules/cell). Antibody-mediated cytotoxicity against these normal cells was not detectable. 4G8SDIEM has been produced in pharmaceutical quality in a university-owned production unit and is currently used for the treatment of leukemia patients.
The chimeric monoclonal antibody Rituximab, directed to the CD20 antigen expressed on normal and malignant cells of the B-lymphocytic lineage, has considerably improved treatment of B-cell-derived lymphomas. In contrast, treatment of non-hematopoietic solid tumors with monoclonal antibodies to different tumor-associated antigens has been less successful. The success of Rituximab has been attributed to several particularities: First, the antibody effectively mediates Fc-dependent effector mechanisms, such as complement- and antibody-dependent cellular cytotoxicity (ADCC). In addition, it induces apoptotic cell death at high concentrations.1 Last, and probably the most important, hematopoietic malignancies, in contrast to solid tumors, are more easily accessible to drugs and immunologic effector cells.
Because of their well-documented -but nevertheless still limited- therapeutic activity, antilymphoma antibodies are currently the first to benefit from newly developed strategies that use antibody engineering technologies to further enhance the therapeutic efficacy of antitumor antibodies.2 Many of these strategies focus on the modulation of the human immunoglobulin (Ig)G1 Fc part that is contained in the majority of antitumor antibodies in clinical use. By defined genetic engineering of the glycosylation pattern or the amino-acid sequence of the Fc part, antibody variants with markedly improved ADCC activity have been generated.3, 4 Different companies use different strategies: Roche (Basel, Switzerland), in cooperation with Glycart (Schlieren, Switzerland), has developed an improved, glyco-engineered CD20 antibody, termed GA101.5 Two other antibodies directed to the lymphoma-associated antigens CD19 (XmAb5574)6, 7 and CD30 (XmAb2513),8 developed by Xencor (Monrovia, CA, USA), carry the amino-acid exchanges S239D and I332E (SDIE modification). All these antibodies were reported to exert markedly enhanced ADCC. At present, these and other1 third-generation antibodies directed to lymphoma-associated antigens are in late preclinical and early clinical developmental stages, respectively.
Leukemias share with lymphomas the advantage of easy accessibility for drugs and immunologic effector cells, and the need for effective new treatment modalities is even more pressing. In addition, the therapeutic activity of antileukemia antibodies is particularly easy to monitor. Nevertheless, efforts to develop antibodies for the treatment of leukemia have been less intense and less successful, compared with those directed to lymphomas: a carefully characterized, humanized antibody directed to the pan-myeloid marker CD33 turned out to be of limited efficacy in clinical trials.9 More recently, humanized antibodies against more specific antigens on leukemic cells, such as the FMS-related tyrosine kinase 3 (FLT3, CD135; clinical trials ID NCT00887926) and the α-chain of the interleukin-3 receptor (CD123),10 have been developed to the stage of early clinical trials. These antibodies, however, have not been optimized by recombinant antibody technology, and the antigens recognized are known to be expressed on dendritic cells (DCs), normal hematopoietic stem cells,11 and —in the case of CD123— cultured mast cells12 and basophils,13, 14 raising concerns about potential toxicity towards these cells.
We have focused our efforts on the SDIE modification mentioned above and substituted this modification by adding a C-terminal tag (M-tag) to facilitate antibody detection in vitro and in vivo. After cloning of the respective variable genes, this modification (SDIEM) was used to generate optimized human IgG1 versions of two FLT3 antibodies developed at our institution15 and of the CD19 antibody 4G7 originally generated at the University of California, Stanford.16 In all these cases, we found a markedly enhanced ADCC activity of the SDIEM-modified antibodies compared with that of the unmodified, chimeric versions. Here we report on the preclinical characterization of the two FLT3 antibodies, BV10 and 4G8. FLT3 is a class III receptor tyrosine kinase, which frequently carries mutations in patients with acute myeloid leukemia (AML) resulting in constitutive activation.17 It has an important role in the homeostasis of DCs, and its ligand, FLT3L, has been used in several studies to activate these cells in vitro and in vivo.18, 19, 20, 21, 22 Thus, during characterization of SDIEM-modified FLT3 antibodies we put particular emphasis on the evaluation of antibody specificity and toxicity towards DCs and hematopoietic stem cells in addition to the exploration of ADCC activity. We believe that the results obtained in this study provide a solid basis for clinical pilot studies.
Materials and methods
Production and purification of recombinant and Fc-optimized antibodies
The messenger RNA of mouse FLT3 antibodies BV10 and 4G8, as well as of the CD19 antibody 4G7 (all Igγ1/κ), was isolated from hybridomas with the RNeasy Kit (Qiagen, Hilden, Germany). Unknown variable regions of heavy (VDJ) and light (VJ) chains were identified by sequencing of inverse PCR amplicons generated as previously described,23 using specific primers for mouse constant genes of light (Ck-for: 5′-IndexTermTGTTCAAGAAGCACACGACTGAGGCACCTCC-3′; Ck-back: 5′-IndexTermACTTCTACCCCAAAGACATCAATGTCAAG-3′) and heavy chains (gamma1-for: 5′-IndexTermCAAGGCTTACAACCACAATCCCTGG-3′; gamma1-back: 5′-IndexTermCATATGTACAGTCCCAGAAGTATCATCTG-3′). The sequences for the variable genes of 4G7 were published (GenBank no. AJ555479 and AJ555622). The cloning of the variable genes from the hybridoma 9.2.27 (GenBank no. AJ459796 and AJ459797), producing an Igγ2a/κ antibody directed to the melanoma-associated chondroitin sulfate proteoglycan, has also been described previously.24 For the generation of chimerized and optimized antibodies, the VJ and VDJ elements were re-amplified and cloned into eukaryotic expression vectors. Besides the amino-acid exchanges at S239D and I332E, the optimized γ1 Fc part contains a C-terminal M-tag, which was derived from the amino-acid sequences 455–466 of the human Igα1 tailpiece and the c-myc epitope EQKLISEEDLLR.25 Parental and recombinant BV10, 4G8 and 4G7 antibodies were purified from culture supernatant of hybridoma cells, and Sp2/0 and CHO-transfectants, respectively, using protein A affinity chromatography (GE Healthcare, Munich, Germany). In the case of 4G8SDIEM, a large batch of the antibody (15 g) was produced in GMP (good manufacturing practice)-compliant clean rooms using disposable technology including a 100-l biowave reactor (Sartorius, Goettingen, Germany) for fermentation and an ÄKTAready system for purification by protein A, ion exchange and hydrophobic interaction chromatography (MabSelect Sure and CaptoAdhere columns, GE Healthcare).
A bispecific Fab2 fragment (bsFab2) with FLT3 × CD3 (4G8 × UCHT1) specificity was produced using a previously described conjugation procedure.26
Cells and media
Peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation using LSM 1077 solution (Lonza, Basel, Switzerland), hybridoma cells and NALM-16 cells (ACC 680, DSMZ, Braunschweig, Germany) were kept in RPMI 1640, mouse Sp2/0-Ag14 cells (ATCC, Manassas, VA, USA) in IMDM medium (Lonza). All media were supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, non-essential amino acids, 2 mM L-glutamine and 57 nM β-mercaptoethanol.
Full-length complementary DNA of human FLT3 (GenBank no. BC126350) was obtained from ImaGenes (Berlin, Germany). The N-terminally FLAG (DYKDDDDK)-tagged derivatives coding either for the complete FLT3 molecule (aa 1–970) or for variants with truncated ectodomains, Δ1 (aa 162–970), Δ2 (aa 244–970), Δ3 (aa 346–970), Δ4 (aa 435–970), were cloned into the pcDNA3 vector using added BamHI and XbaI restriction sites and transfected into Sp2/0-Ag14 cells by electroporation.
Antibodies and flow cytometry
CD33-PE/Cy5 (clone WM53), CD34-APC (clone 581), CD-45-FITC (clone HI30), CD123-PE/Cy5 (clone 9F5), CD11c-PE (clone B-ly6) and isotype control antibodies were purchased from BD Biosciences (Heidelberg, Germany), the CD303-FITC antibody from Miltenyi Biotec (Bergisch-Gladbach, Germany) and FLAG-FITC (clone M2) from Sigma-Aldrich (Deisenhofen, Germany). All antibodies were incubated with cells for 30 min at 4 °C. For indirect immunofluorescence, PE- or APC-conjugated goat-anti-mouse F(ab)2 fragments and goat-anti-human F(ab)2 fragments, respectively, were used (Jackson ImmunoResearch, West Grove, PA, USA). In several experiments, we combined indirect and direct immunofluorescence for multi-dimensional analysis by adding labeled antibodies in the final step. Cells were analyzed on a FACSCanto II or a FACSCalibur (BD Biosciences).
Beads for the quantitative analysis of indirect immunofluorescence (QIFIKIT) were purchased from Dako (Hamburg, Germany) and used according to the manufacturer's protocol. For quantification of humanized antibodies suitable beads were not available. Thus, a specific fluorescence index was calculated by dividing the mean fluorescence intensity obtained with 4G8SDIEM by that detected with the non-binding, SDIE-modified control antibody 9.2.27. For these experiments, PE-conjugated antibodies generated with the Lynx rapid PE antibody conjugation kit (AbD Serotec, Düsseldorf, Germany) were used. Bone marrow cells were stained using CD34-APC/Cy7, CD38-PE/Cy7 (all from BioLegend, San Diego, CA, USA) and CD45-AmCyan (BD Biosciences) antibodies.
For direct assessment of stem cell toxicity, 5 × 105 bone marrow cells were incubated with 1 μg/ml of 4G8SDIEM, 9.2.27SDIE or FLT × UCHT-1 bsFab2, respectively, in triplicate. After incubation for 24 or 48 h the cells were stained and resuspended in FACS buffer containing 7-AAD (BioLegend) and BD negative control compensation particles (BD Biosciences). Stem cells were gated according to the ISHAGE protocol.27 Absolute numbers of CD34+CD38+ and CD34+CD38− cells were determined by acquiring the same numbers of compensation beads for all samples.
Recombinant FLT3 ligand (rFLT3L) was purchased from Peprotech EC (London, Great Britain, UK). For competition experiments various concentrations of rFLT3L were incubated with NALM-16 cells and BV10SDIEM or 4G8SDIEM (1 μg/ml) for 30 min at 4 °C and analyzed by indirect immunofluorescence and flow cytometry.
3[H]-methyl-thymidine uptake assay
2 × 105 AML blasts were seeded in triplicate in 96-well plates and incubated with various concentrations of optimized antibodies. After 24 h, cells were pulsed for another 20 h with 3[H]-methyl-thymidine (0.5 μCi/well) and harvested on filter mats. Incorporated radioactivity was determined by liquid scintillation counting in a 2450 Microplate counter (Perkin-Elmer, Waltham, MA, USA).
NALM-16 cells and primary AML blasts were used as targets. To separate blasts and effector cells from the PBMC preparations of leukemia patients, cells were labeled with CD34 and CD33 microbeads and separated on LD columns following the manufacturer's (Miltenyi Biotec) protocol. The number of contaminating blast cells in the negatively selected effector cell population was determined by FACS analysis and varied between 1 and 10% depending on the initial blast contamination. In some experiments labeled DCs were used as target cells.
Chromium release assays were performed as previously described.28 Briefly, labeled target cells and PBMCs were incubated at 37 °C for 4 or 8 h in 96-well flat-bottom plates in the presence of various concentrations of antibodies at a PBMC : target ratio of 50:1. Percentage of specific 51[Cr] release was calculated according to the formula (c.p.m. (test)–c.p.m. (spontaneous)/(c.p.m. (triton lysis))–c.p.m. (spontaneous)).
NALM-16 cells or AML blasts were incubated with various concentrations of 4G8SDIEM or BV10SDIEM in RPMI 1640 medium. After 24 or 48 h the cells were washed with ice-cold FACS buffer, incubated with a saturating concentration of 4G8SDIEM (2 μg/ml) for 30 min at 4 °C, stained with PE-conjugated goat-anti-human F(ab)2 fragments and analyzed by FACS. Relative surface expression of FLT3 was calculated, defining the mean fluorescence intensity of cells preincubated without antibody as 100%.
DC isolation and maturation
DCs were isolated from buffy coat preparations of healthy individuals using the blood DC isolation kit II according to the protocol of the manufacturer (Miltenyi Biotec). Myeloid DC and plasmacytoid DC subsets were stained with a mixture of CD11c-PE, CD303-FITC and CD123-PE/Cy5 antibodies. For in vitro generation of myeloid DC, 1 × 108 PBMCs of healthy individuals were seeded in 10 ml X-Vivo15 medium (Gibco, Darmstadt, Germany). After 2 h at 37 °C adherent cells were cultured in RPMI 1640 medium supplemented with 50 ng/ml GM-CSF and interleukin-4 (20 ng/ml) for 5 days. On day 6 lipopolysaccharide (100 ng/ml) was added. Cells were harvested on day 7 and analyzed by flow cytometry.
Colony-forming unit assay
Bone marrow cells were obtained by lavage of the femoral head from patients undergoing hip surgery. The cells were purified by density gradient centrifugation and seeded at 1 × 107/ml in RPMI 1640 medium containing 5 μg/ml of 4G8SDIEM or 9.2.27SDIE. After 24 h of incubation, cells were transferred into antibody-containing (5 μg/ml) methylcellulose medium (10 000 cells/ml, MethoCult H4434 classic, Stemcell Technologies, Grenoble, France). The assay was performed in triplicate. After 12 days, colonies were counted and classified.
The parental mouse antibodies 4G8 and BV10 were originally described and characterized as recognizing the FLT3 protein,15 a class III receptor tyrosine kinase with five extracellular immunoglobulin-like domains.29 Epitope mapping using different deletion variants of the molecule revealed binding of BV10 and 4G8 antibodies to domain 2 and 4, respectively (Figure 1a). This is consistent with previous observations that both antibodies do not cross-block each other in competition experiments (data not shown). Both antibodies saturated FLT3 molecules on NALM-16 cells at concentrations below 1 μg/ml. Binding of the chimerized 4G8 antibody was stronger than that of BV10. This is not due to chimerization or optimization as a similar difference was observed when binding of the mouse parental versions of 4G8 and BV10 was compared. No differences in binding between the chimeric and the SDIEM-modified chimeric versions of the antibodies were detected (Supplementary Figure 1). A SDIE-modified antibody, termed 9.2.27SDIE, directed to the melanoma-associated surface antigen chondroitin sulfate proteoglycan, did not bind to NALM-16 cells and was used as a negative control in this and several subsequent experiments.
Competition with binding of the FLT3 ligand
In general, interference with binding of the natural ligand may contribute to the therapeutic activity of an antibody. Figure 1b shows that recombinant FLT3L partly inhibits binding of 4G8SDIEM, but not of BV10SDIEM to NALM-16 cells. Therefore, we evaluated the effect of 4G8SDIEM on the spontaneous proliferation of the leukemic blasts of three different patients in vitro using the non-binding SDIE-modified 9.2.27 antibody as a control. Whereas spontaneous proliferation of the primary AML cells varied substantially, significant effects of the antibodies on cell proliferation were not observed up to a concentration of 10 μg/ml (Figure 1c).
Antibody-dependent cellular cytotoxicity
The hallmark of various SDIE-modified antibodies evaluated previously was their impressively increased activity in ADCC assays. Figure 2a shows that the ADCC activity of PBMCs against NALM-16 cells is markedly enhanced in the presence of the SDIEM-modified antibodies as compared with that of the unmodified chimeric antibody versions. In several experiments, the concentrations required to achieve comparable lysis by unmodified and SDIEM-modified antibodies differed by a factor of at least 100. Killing by the 4G8SDIEM antibody was significantly better than that achieved by BV10SDIEM, in particular at low concentrations. This may be due to the moderately lower binding avidity of BV10 (Supplementary Figure 1) and/or the different FLT3 epitopes recognized (Figure 1a). The superior activity of 4G8SDIEM prompted us to focus on this antibody for further characterization. In Figure 2b the ADCC activity of 4G8SDIEM is compared with that of the optimized CD19-antibody 4G7SDIEM. After 4 h a significant difference was observed in favor of the CD19 antibody corresponding to the fourfold-higher expression level of CD19 on NALM-16 cells (Supplementary Figure 2). After 20 h, however, the activity of both antibodies appeared to be comparable. In Figure 3a the ADCC activity of 4G8SDIEM is depicted using PBMCs of three additional healthy donors (PBMC I–III). In these experiments, the SDIE-modified monoclonal antibody 9.2.27 was used as a negative control. The cytolytic activity in the presence of this reagent did not exceed that of natural killer (NK) cells in the absence of antibodies, which varied between 0 and 20%.
In Figure 3b the ADCC activity of PBMCs from a healthy donor (PBMC II) against leukemic blasts of three different patients is shown. ADCC activity mediated against these blasts (AML 1, AML 2, AML 11), carrying 3750, 4000 and 2600 FLT3 molecules per cell, respectively, was less pronounced than that against cultured NALM-16 cells. It required 8 rather than 4 h to become clearly detectable. Generally, the ADCC as well as the NK activity against NALM-16 cells and leukemic blasts continued to rise after 8 h (see Figure 2b).
We next wanted to evaluate the ADCC activity of PBMCs isolated from the blood of AML patients against autologous blasts. To this end, we depleted leukemic blasts from PBMC preparations and used the depleted PBMCs as effector cells against the positively selected blasts (see Materials and Methods). Under these conditions, we detected significant lysis in two (AML 1, 21) out of five independent experiments with blasts and autologous PBMCs of the respective patients (Figure 3c). It should be noted that this procedure has possible technical shortcomings: (i) contamination of effector cells with residual blasts may result in the deviation of E:T (effector cell to target cell) ratios in favor of the blasts and (ii) lysis may not be complete after 4 and potentially even 8 h (Figure 2b). To overcome the latter restriction, we tried to establish a long-term cytolytic assay covering 24 rather than 4 or 8 h. These experiments failed because of an exceedingly high spontaneous death rate of AML blasts. The extraordinary fragility of AML blasts under standard culture conditions became apparent already after 8 h of culture, when added allogeneic effector cells in most cases inhibit the spontaneous release of chromium rather than enhancing it by exerting NK-mediated lysis. This stabilizing effect resulted in a formally negative NK effect in 51[Cr]-release assays (Figure 3b).
Binding to normal and leukemic cells, antigen shift
Figures 4a and c show binding of the parental mouse 4G8 antibody and 4G8SDIEM, respectively, to a panel of leukemic cells obtained from patients suffering from the indicated subtypes of AML. Gated CD33+CD45dim or CD34+CD45dim cells were analyzed. FLT3 was detected in all 24 patient samples. The number of molecules per cell determined by indirect immunofluorescence and quantitative flow cytometry varied from 350 to 4700. On NALM-16 cells approximately 6800 molecules/cell (mpc) were detected (Figure 4a). Our panel contained only four samples with confirmed positive internal tandem repeat status (Figure 4b), two of them with an intermediate and two with a low expression of FLT3. Thus, these data are not conclusive regarding a correlation between the expression level and the mutation status of the FLT3 molecule. In Figure 4c, 4G8SDIEM-PE rather than mouse 4G8 was used for staining. In this case, a specific fluorescence index value was calculated to quantify antibody binding. For blasts from 7 of the 24 donors, this index was not determined because of high, unspecific reactivity with the control antibody 9.2.27SDIE-PE. As expected, specific fluorescence index values of the evaluable samples closely matched the numbers of molecules determined by quantitative FACS (Figure 4c).
Modulation of target antigen expression upon antibody binding is a phenomenon often observed during antibody therapy. In particular, a sustained and complete loss has been reported upon treatment of AML patients with a saturating dose of the CD33 antibody Lintuzumab.9 Figure 4d depicts the antigen shift induced after incubation of NALM-16 cells or primary leukemic blasts of two patients (AML 1 and 2) with various concentrations of 4G8SDIEM for 48 h. On all these cells, a moderate antigen shift was observed, which was already completed after 24 h of incubation (data not shown).
Figures 5a–c show that binding of mouse 4G8 to CD11c-positive myeloid DCs and to CD303-positive plasmacytoid DCs purified from normal PBMCs was marginal. The numbers of FLT3 molecules expressed on these cells were below 300/cell. In addition, we generated DCs from normal PBMCs. Although these cells expressed large amounts of the DC-associated markers CD80, CD86 and CD123, binding of 4G8 antibodies was again barely detectable (data not shown). Next, we evaluated binding of mouse 4G8 to CD34-positive cells in normal bone marrow. Again, binding of the antibody to bone marrow cells of three different donors was marginal, corresponding to less than 600 molecules per cell (Figures 5d and e). In summary, binding of FLT3 antibodies to normal DCs and bone marrow cells was significantly lower than that to most FLT3-expressing leukemic cells examined (see Figures 5f–h). In addition, we did not observe binding of FLT3 antibodies to thrombocytes, erythrocytes and granulocytes (data not shown). This is in accordance with our failure to detect FLT3 on a wide array of normal and malignant cryopreserved tissues by immunohistology using 4G8 as well as BV10 antibodies (data not shown).
Toxicity in vitro
Despite the relatively low levels of 4G8SDIEM binding to normal bone marrow precursor cells and DCs, we attempted to assess the potential toxicity of this antibody towards such cells. To this end, we incubated bone marrow cells with saturating concentrations of 4G8SDIEM and 9.2.27SDIE, and determined the influence of these antibodies on the capacity of the bone marrow cells to give rise to colonies (colony forming units) in semi-solid medium. No significant influence of the antibodies on the capacity to form colony-forming units was detected in two experiments with bone marrow cells from different healthy donors (Figure 6a). To assess toxic effects of 4G8 SDIEM on human hematopoietic precursor cells more directly, we incubated bone marrow cells of healthy donors with various antibodies at 1 μg/ml for 24 and 48 h and determined the percentage of the CD34+CD38− as well as CD34+CD38+ subpopulations by flow cytometry. In these experiments, a chemically hybridized antibody with FLT3 × CD3-specificity served as a positive control. This antibody activates T cells in the presence of FLT3-expressing target cells (data not shown), resulting in a significant reduction of the CD34+CD38− as well as the CD34+CD38+ population. The control antibody 9.2.27SDIE as well as 4G8SDIEM did affect hematopoietic progenitor cells in these experiments neither after 24 (Figure 6b) nor after 48 h (data not shown).
Finally, we incubated human DCs with autologous PBMCs as effector cells. Although 4G8SDIEM mediated effective ADCC against NALM-16 cells, when used as positive control, no killing of autologous DCs was observed (Figure 6c).
Despite their undisputed success in lymphoma therapy, up to date, effective monoclonal antibodies for the treatment of myeloid leukemias have not been developed. The FMS-like receptor tyrosine kinase FLT3 (CD135), expressed on the leukemic cells of most AML patients, has been considered a promising target for small drug molecules,30 such as tyrosine kinase inhibitors, as well as for monoclonal antibodies.31 Antibodies have the potential advantage of superior efficiency due to induction of immune effector mechanisms. In addition, their activity does not rely on the FLT3 mutation status.
Here we report on the optimization of two FLT3 antibodies using recombinant antibody technology. Initially, we considered two different strategies for optimization of the Fc part: modifications of the glycosylation pattern and the amino-acid sequence, respectively. We chose the latter strategy because (i) the stable fermentation of producer cells with altered glycosylation patterns is delicate; (ii) we found in previous experiments that the ADCC activity of a SDIEM-modified CD19 antibody was comparable to that of an otherwise identical reagent with optimized, low-fucose glycosylation (data not shown). Thus, for the experiments described here, we focused on the SDIEM modification described above and generated (i) chimeric versions of the parental mouse antibodies carrying constant regions of the human IgG1 subtype and (ii) SDIEM variants of these versions with two additional amino-acid modifications in the CH2 domain of the human Fc part to enhance its interaction with Fc-receptor-expressing effector cells.
As expected, the chimerized and SDIEM-modified antibodies showed no difference in binding avidity; however, their capability to mediate ADCC against FLT3-expressing target cells differed markedly: the SDIEM-modified versions of both antibodies were active in vitro at concentrations as low as 1 ng/ml, whereas the unmodified chimeric versions rapidly lost activity below 10 μg/ml. The modified 4G8 antibody was consistently superior to its BV10 counterpart with respect to target antigen binding and ADCC induction. Whether the close proximity to the membrane of the epitope recognized by the 4G8 antibody is —at least in part—responsible for its superior activity remains at present unclear. In any case, the remarkable ADCC activity of 4G8SDIEM may be properly ranged if (i) its activity against NALM-16 cells is compared with that of a SDIEM-optimized antibody directed to CD19 (4G7-SDIEM), an antigen expressed with a fourfold excess (6000 vs 25 000 molecules per cell) on this particular cell line. It should also be noted in this context that the optimized CD19 antibody is significantly more effective than the well-established CD20 antibody rituximab in mediating ADCC against Raji cells expressing comparable amounts of CD19 and CD20 (data not shown). In general, the expression levels of FLT3 on cultured cells as well as leukemic blasts as reported here (<5000 molecules per cell) are clearly lower than the levels reported for CD19 and CD20 antibodies (10 000–35 000 and 65 000–300 000 molecules per cell, respectively).32
When leukemic blasts isolated from the peripheral blood of patients rather than cultured NALM-16 cells were used as targets for allogeneic and autologous PBMCs, killing was less pronounced. We do not think that this is merely due to the lower expression of FLT3 on leukemic blasts but rather reflects, at least in part, a heightened resistance of these primary cells towards antibody-mediated killing. In addition, detection of ADCC against autologous AML blasts is hampered by an exceedingly high spontaneous death rate of these cells in long-term (>8 h) assays. In any case, therapeutic activity of ADCC-inducing antibodies may be limited to situations where favorable target:effector ratios prevail, such as in minimal residual disease.
One of our antibodies, 4G8, partly blocks binding of the physiological FLT3 ligand. However, it does not affect proliferation of cultured NALM-16 cells or AML blasts at concentrations up to 10 μg/ml. This may be due to incomplete blocking. Alternatively, FLT3 signaling may not be crucial for proliferation of the respective cells in vitro. The latter possibility is supported by a report that also highlights the importance of ADCC for the in vivo activity of FLT3 antibodies. It describes two humanized, but not Fc-optimized FLT3 antibodies originally developed by ImClone. Although both of these antibodies interfere with FLT3 signaling in primary AML blasts, only one of them, termed EB10, mediates ADCC in vitro and prolongs survival of SCID mice injected with leukemic cells.31 It is difficult, however, to compare the ADCC activity of EB10 with that of the antibodies described in our study as, for EB10, cytotoxicity data at concentrations lower than 10 μg/ml were not provided. Despite our impression that therapeutic effects of antibody-mediated FLT3 blockade may be limited, we have chosen 4G8SDIEM, the antibody partly blocking binding of the FLT3 ligand, for further characterization, mainly because of its superior binding and ADCC activity.
Antigen shift, that is, loss of surface expression due to internalization or shedding after antibody binding, is frequently observed upon application of therapeutic antibodies in vitro and in vivo. Internalization of antigen/antibody complexes may be desirable during therapy with immunotoxins. However, for strategies relying on antibody-mediated effector mechanisms such as complement activation or ADCC, antigen loss by whatever mechanism may be detrimental. In fact, a profound and long-lasting antigen shift after therapeutic application of saturating antibody doses may have been a major reason for the limited therapeutic efficiency of the CD33 antibody Lintuzumab in clinical trials.9 Very recently, it was reported that this phenomenon may also limit the therapeutic activity of the widely used CD20 antibody Rituximab.33 Although a ‘shaving’ mechanism on chronic lymphocytic leukemia cells has been postulated,34 CD20 has been described in several reports as relatively resistant towards shifting. However, in most cases, the respective in vitro assays have been terminated after several hours, which is possibly too early to detect delayed shifting.33 The antibody 4G8SDIEM described here clearly modulates the surface expression of FLT3 on NALM-16 cells and on leukemic blasts. However, this modulation, which is completed after 24 h, does not reduce expression levels below 50%. Thus, we do not expect this phenomenon to seriously hamper the therapeutic activity of the antibody, although it should certainly be monitored carefully during clinical application.
With respect to antibody toxicity, the specificity of the target antigen recognized is a central issue. FLT3 has been reported to be expressed on normal hematopoietic precursor cells as well as on DCs. In our experiments, FLT3 expression on DCs was hardly detectable (<300 molecules per cell) and was marginal on CD34+ bone marrow cells (<500 molecules per cell). The low expression of FLT3 on DCs is particularly surprising. A number of articles describe successful activation of these cells by recombinant FLT3 ligand resulting in increased antitumor effects mediated by the activated DCs.18, 22, 35 We are aware that our results are not easily reconciled with these findings and propose the hypothesis that activation of DCs via FLT3 requires only few molecules on the surface of the cells. In any case, given the low number of FLT3 molecules on normal DCs and hematopoietic precursor cells, the lack of cytotoxic activity mediated by 4G8SDIEM against such cells in 51[Cr]-release and colony-forming unit assays is not surprising. With respect to the potential toxicity of the 4G8SDIEM antibody these findings are confirmative. Of note, the expression of the interleukin-3 receptor, an alternative target on leukemic cells, on DCs, was found to be considerably higher than that of FLT3, which implies that the specificity of FLT3 for leukemic cells might be superior to that of CD123. In conclusion, we believe that the results discussed above provide a solid basis for the clinical evaluation of 4G8SDIEM.
Recently, we succeeded in the production of 4G8SDIEM in pharmaceutical quality and quantity in a university-owned, GMP-compliant production unit. At present we treat selected AML patients with this antibody on a compassionate need basis. In the first patient we observed NK cell activation and complete but transient clearance of leukemic blasts from the peripheral blood. A human anti-human response was not detected up to 3 months after completion of the therapy. We envisage to start a phase I study with 4G8SDIEM in the second half of 2012. Thus, it appears possible to develop innovative therapeutic antibodies within university institutions up to the stage of clinical pilot studies. We strongly feel that substantial participation of academic institutions will improve the speed and efficiency of antibody development. In any case, studies performed with 4G8SDIEM and other third-generation antibodies, designed for increased ADCC-activity, might soon enable us to judge more reliably the role of this activity for the therapeutic efficiency of antitumor antibodies in humans.
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We thank Sabrina Grimm and Sandra Drescher for expert technical assistance. This work was supported by the GO-Bio programme of the Federal Ministry for Education and Research (BMBF), Germany, and in part by a grant from the Deutsche Forschungsgemeinschaft (SFB 685, project C10).
LG-H, SA, H-GR and GJ are co-owners of a recently founded company, Synimmune GmbH. The company will develop antitumor antibodies optimized by gene technology. Its foundation is a prerequisite for further funding by the GO-Bio programme of the Federal Ministry for Education and Research (BMBF), Germany.
Supplementary Information accompanies the paper on the Leukemia website
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Hofmann, M., Große-Hovest, L., Nübling, T. et al. Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia. Leukemia 26, 1228–1237 (2012). https://doi.org/10.1038/leu.2011.372
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