Efficacy and Mechanism of Antitumor Activity of an Antibody Targeting Transferrin Receptor 1 in Mouse Models of Human Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy in the United States. In 2017, it is estimated that there were 30,280 new cases of MM with 12,590 estimated deaths (1). MM is a B cell cancer composed of malignant plasma cells and is characterized by the overproduction of a monoclonal Ig (M-protein), immunosuppression, and end-organ damage, including osteolytic lesions (2). Although novel treatment options have led to improved response rates and increased survival, the majority of patients acquire drug resistance and ultimately relapse with more aggressive tumors, developing treatment-refractory disease associated with shortened survival times (2–6). Because MM is a heterogeneous disease (7), it is advantageous to take a broad-spectrum approach to address multiple pathways and targets for sustained efficacy.

Iron is important for multiple cellular processes, including metabolism, respiration, and DNA synthesis (8). Iron in blood is bound by transferrin (Tf), which is transported into cells through the interaction with its receptor, the Tf receptor 1 (TfR1), also known as CD71 (8–10). TfR1 is constitutively internalized and recycled back to the cell surface (9, 10). TfR1 is ubiquitously expressed at relatively low levels on normal cells and at increased levels in rapidly proliferating cells and malignant cells, including hematological malignancies, in which overexpression has been associated with poor prognosis (9–12).

Interestingly, TfR1 has also been reported to be involved in regulating mitochondrial function (13). TfR1 can support mitochondrial respiration and reactive oxygen species production, both of which are key players in tumor cell growth and survival (14). Furthermore, TfR1 mediates NF-κB signaling in malignant cells through the interaction with the inhibitor of the NF-κB kinase (IKK) complex, increasing cancer cell survival (15). Importantly, NF-κB plays an important role in MM cell pathogenesis, and inhibition of this pathway blocks MM cell growth and survival (16). In addition, MM cells are dependent on an increased influx of iron (17). Taken together, the high expression of TfR1 on malignant cells, its extracellular accessibility, and its central role in cancer cell pathology make this receptor an attractive target for Ab-mediated cancer therapy, including therapy for MM.

Targeting TfR1 on malignant cells with an Ab may result in direct cytotoxicity through interference with TfR1 function by inhibiting Tf binding, triggering receptor degradation, or blocking receptor internalization, all of which ultimately impair iron uptake, leading to lethal iron starvation (10, 18). Because the TfR1 may also function in regulating mitochondrial respiration and mediating NF-κB signaling as mentioned above, it is possible that Abs may also interfere with these TfR1-mediated functions as well. Furthermore, Abs may induce cytotoxicity against cancer cells through the induction of these Fc effector functions: Ab-dependent cell-mediated cytotoxicity (ADCC); Ab-dependent cell-mediated phagocytosis (ADCP); and/or complement-dependent cytotoxicity (CDC) (19, 20). In addition to their direct use as anticancer agents, Abs targeting TfR1 can be used as delivery systems for a variety of anticancer agents that are internalized into malignant cells by receptor-mediated endocytosis (9, 18).

ch128.1 is a mouse/human chimeric IgG3 that binds specifically to the human TfR1 and does not interfere with binding of the ligands Tf and hemochromatosis protein (HFE) to TfR1 (21). It promotes degradation of TfR1 (22, 23) and in vitro cytotoxicity against certain malignant B cell lines, such as ARH-77 and IM-9 (22, 24, 25). In addition, a single dose of ch128.1 resulted in significant protection in two disseminated human MM xenograft SCID-Beige mouse models bearing either ARH-77 or KMS-11 cells. Intriguingly, this in vivo antitumor activity was observed even against KMS-11 MM cells that are insensitive to the cytotoxic effects of ch128.1 in vitro (24). ch128.1 also prolonged survival in an AIDS-related human Burkitt lymphoma xenograft model of NOD-SCID mice bearing 2F7 tumors, although no sensitivity was observed in vitro (26). Using the disseminated models of MM, the current study aims to define the mechanism of antitumor activity exhibited by ch128.1 and explore its in vivo efficacy in different therapeutic settings.

The KMS-11 human MM cell line was a kind gift from Lawrence H. Boise (Emory University, Atlanta, GA) and was cultured in IMDM (Life Technologies, Carlsbad, CA). ARH-77, an EBV-transformed human B lymphoblastoid cell line, was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Life Technologies). All cell lines were cultured in media supplemented with penicillin, streptomycin (Thermo Fisher Scientific, Canoga Park, CA), and 10% heat-inactivated FBS (Atlanta Biologicals, Atlanta, GA) in 5% CO₂ at 37°C.

ch128.1 (IgG3/κ) and the ch128.1 triple mutant L234A/L235A/P329S were produced in murine myeloma cells and affinity-purified as described (27, 28). Mutations were previously generated on the ch128.1 H chain γ3 expression vector to disrupt the binding to FcγRs and the complement component C1q (27). An IgG3/κ isotype control Ab specific for the hapten dansyl (5-dimethylamino naphthalene-1-sulfonyl chloride; referred to as IgG3) (22) was produced with the expression vectors and methods used to develop ch128.1.

ARH-77 cells were incubated with various concentrations of the Abs for a total of 96 h. Proliferation was monitored using the [³H]-thymidine incorporation assay as described (25). The cells were incubated with [³H]-thymidine for the final 16 h of the treatment period.

All experimental protocols were approved by the University of California, Los Angeles, Institutional Animal Care and Use Committee, and all local and national guidelines on the care of animals were strictly adhered to. C.B-17 SCID-Beige mice were obtained and housed in the Defined Flora Mouse Facility in the Department of Radiation Oncology at UCLA. Female mice (8–12 wk old) were exposed to 3 Gy total-body sublethal irradiation (MARK 1-30 irradiator [¹³⁷Cs]; J.L. Shepherd & Associates, San Fernando, CA) 1 d before the tumor challenge. To establish the disseminated disease, 5 × 10⁶ ARH-77 or KMS-11 cells were injected i.v. into the lateral tail vein, as described (24). Mice were randomized into treatment groups. A single dose of ch128.1, its triple mutant, the IgG3 isotype control, or the buffer alone was injected i.v. 2 or 9 d after the tumor challenge. All animals are littermates and animals in the same treatment groups were cohoused. Survival was based on the time from the tumor challenge to development of hindlimb paralysis. Survival plots were generated using GraphPad Prism Version 5 (GraphPad Software, La Jolla, CA). Significant differences in survival were determined by the log-rank test using the same software. Results were considered significant if p < 0.05.

The interaction of ch128.1 and its mutant with the murine neonatal Fc receptor (FcRn) was monitored by surface plasmon resonance (SPR) detection on a Biacore 3000 instrument using a CM5 sensor chip (Biacore; GE Healthcare Life Sciences, Pittsburgh, PA) as described (29), with modifications. Recombinant mouse FcRn (100 μg/ml; R&D Systems, Minneapolis, MN) was amine-coupled to flow cell 2 of the sensor chip and flow cells were blocked with 1 M ethanolamine-HCl (pH 8.5). Flow cell 1 without FcRn was used as a control surface. ch128.1 or its mutant (10–400 nM) were flowed over FcRn in PBS/Tween 20 (50 mM sodium phosphate pH 6, 150 mM NaCl, 0.02% NaN₃, 0.01% Tween 20) at 25°C, 20 μl/min for 10 min. Flow cells were regenerated using PBS (pH 8) containing 0.05% Tween 20. Sensograms were generated and analyzed, and equilibrium K_D values were determined using the steady-state affinity model included in the BIAevaluation software v4.1 (29). Murine FcRn, which binds human IgG (30), was used to reflect binding in the in vivo model.

ch128.1 or its mutant (100 μg) were injected i.v. into the lateral tail vein of SCID-Beige female mice 8–12 wk old. Blood was collected from the lateral tail vein at 2, 24, 48, 72, and 168 h after injection from two alternating groups of mice. Serum Ab levels were quantified by ELISA and Ab integrity was confirmed by immunoblotting. Briefly, ELISA Immulon 2HB plates (Thermo Fisher Scientific, Waltham, MA) were coated with 1 μg/ml anti-human IgG (SouthernBiotech, Birmingham, AL). Pooled serum from four to five mice was diluted 1:500 into 1% FBS/PBS. Two-fold serial dilutions were tested for all sera. Recombinant ch128.1 was used to generate a standard curve. Binding was detected with alkaline phosphatase (AP)–conjugated goat anti-human κ (MilliporeSigma, St. Louis, MO) and AP substrate (MilliporeSigma) dissolved in diethanolamine buffer (9.6% diethanolamine [v/v], 0.24 mM MgCl₂ in water, pH 9.8). Plates were read at absorbance 405 nm using a FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). Immunoblot analysis was performed with pooled serum (diluted 1:500) separated by SDS-PAGE under nonreducing conditions and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences). The human IgG3 Abs were detected using rabbit anti-human IgG (1:1000; Bethyl Laboratories, Montgomery, TX) and HRP-conjugated donkey anti-rabbit IgG (1:5000; GE Healthcare Life Sciences). The ChemiGlow West Chemiluminescent Substrate Kit (ProteinSimple, San Jose, CA) was used for detection. Recombinant Abs (ch128.1 or its mutant) were used as positive controls, and serum from untreated mice was used as a negative control. Digital images of blots were obtained using a FluorChem Megapixel High Performance Fluorescence, Chemiluminescence, and Visible Imaging System (ProteinSimple).

Macrophages in female SCID-Beige mice 8–12 wk old were depleted by i.p. injection of 200 μl Clodrosome (clodronate-encapsulated liposomes, 5 mg/ml; Encapsula NanoSciences, Brentwood, TN) 4 h after tumor inoculation, followed by a second dose (100 μl) 5 d later. Clodronate phagocytosed by macrophages inhibits mitochondrial ATP/ADP translocase (31) and induces death by apoptosis (32). PBS-encapsulated liposomes were used as a negative control. Depletion was confirmed by immunohistochemistry (IHC) of the spleen using F4/80 as a marker (33). Spleens of mice injected with the same doses of Clodrosome were excised 5 d after the second dose. Briefly, spleen tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Ag retrieval was performed using citrate buffer and macrophages were visualized using rat anti-mouse F4/80 Ab (Bio-Rad Laboratories, Hercules, CA), rabbit anti-rat secondary Ab (Vector Laboratories, Burlingame, CA), Dako EnVision⁺ System-HRP labeled polymer anti-rabbit kit (Dako, Carpenteria, CA), and 3,3′-diaminobenzidine (BioCore Medical Technologies, Elkridge, MD). Tissue sections were counterstained with Harris hematoxylin. Serial sections were stained with H&E. Images were obtained using an Olympus BX51 light microscope at original magnification ×20. Staining intensity was quantified using the reciprocal intensity method and the Fiji Software (ImageJ) as described (34). The reciprocal intensity is derived in the image after color deconvolution and is directly proportional to the amount of chromogen present in the image.

For complement depletion, female SCID-Beige mice 8–12 wk old were given 25 U cobra venom factor (CVF; Quidel, San Diego, CA) i.p. on the day of the tumor challenge and at 3, 6, and 9 d postchallenge. CVF is a structural and functional analog of the complement component C3 that forms a stable C3/C5 convertase resistant to the regulatory complement proteins, resulting in exhausted complement activation and ultimately complement depletion (35). Depletion was assessed via the detection of C3a in mouse serum by ELISA, as described (36) with modifications. Immulon H2B plates (Thermo Fisher Scientific) were coated overnight at 4°C with 4 μg/ml rat anti-mouse C3a (BD Biosciences, San Jose, CA) in PBS (pH 6.5). Plates were washed with PBS then blocked with 3% BSA in PBS. Mouse serum samples from the same time point were pooled and diluted 1:50–1:500 in 1% BSA in PBS. Diluted mouse serum and purified mouse C3a (used as a positive control; BD Biosciences) were incubated overnight at 4°C. Plates were washed and incubated with biotinylated rat anti-mouse C3a (BD Biosciences) for 1 h at room temperature. The presence of C3a was detected with streptavidin–AP and AP substrate (MilliporeSigma) dissolved in diethanolamine buffer and read at absorbance 405 nm using a FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices).

Macrophages were derived from bone marrow (BM) as described (37) with modifications. Briefly, BM was flushed from femurs and tibias of BALB/c mice bred in-house (6–12 wk old) in RPMI 1640 supplemented with 10% FBS, glutamine, and antibiotics. Cells were passed through a 40 μm cell-strainer (Corning Life Sciences, Tewksbury, MA). RBC were lysed with ammonium chloride for 30 min. Cells were cultured in Petri dishes in RPMI 1640 with 10% FBS and 20–30 ng/ml of recombinant M-CSF (PeproTech, Rocky Hill, NJ) for 7 d with replenishment every 3 d. Cells were confirmed to be macrophages via flow cytometry using fluorescence-conjugated Abs against CD11b (Bio-Rad Laboratories) and F4/80 on a BD LSR II cytometer (BD Biosciences).

A three-color flow cytometric assay was performed as described (38) with modifications. Briefly, KMS-11 target cells were labeled with CFSE at 250 nM/10⁶ cells/ml (Life Technologies), coated with Abs (10 μg/ml) for 30 min on ice, and incubated with bone marrow–derived macrophage (BMDM) effector cells (E:T ratio of 3:1) at 37°C for 2 h in duplicates. Cells were washed in buffer (5% FBS in PBS) and incubated for 30 min on ice with a PE-conjugated anti-F4/80 Ab (Bio-Rad Laboratories) to visualize macrophages. DAPI (1 μg/ml) was added to identify dead cells. At this concentration, only dead cells are stained with DAPI. Cells treated with saponin (0.3% final concentration; MilliporeSigma) were used as a positive control for dead cells. Fifty thousand events were acquired by flow cytometry. Acquisition and measurement of single-cell events were monitored by forward-scatter versus side-scatter dot plots and compared with control single- and mixed-population samples. ADCC and ADCP were calculated as described (38), with CFSE⁺/PE⁺ cells classified as ADCP and CFSE⁺/DAPI⁺ cells classified as ADCC. Results were considered significant if p < 0.05 as determined by the Student t test.

The ADCC/ADCP assay was performed as described above. After the 2 h incubation, the cells were fixed using 2% paraformaldehyde in PBS. Samples were run on an ImageStreamX Mark II (Amnis, part of MilliporeSigma) Imaging Flow Cytometer using the INSPIRE acquisition software (Version 2; Amnis). Brightfield and fluorescent images were acquired at original magnification ×40. During event acquisition, and excluding cell debris, 10,000 events were recorded with the following settings: brightfield LED intensity 25.81 mW; 405 nm laser 10 mW; and 488 nm laser 2.0 mW. The IDEAS software (Version 6.2; Amnis) was used for data analysis. Single stained samples were used to construct a compensation matrix that was then applied to subsequent flow analyses as well as to draw positive and negative signal gates. Events that were best in focus (determined using the gradient root mean square value of 40 or greater on the brightfield channel) and single cells (determined from the brightfield area versus aspect ratio) were selected via gating for further analysis. From this population gate, a scatterplot was drawn graphing fluorescence intensity of the CFSE signal versus fluorescence intensity of the PE signal. Gates were drawn containing PE⁺/CFSE⁻ (macrophage population), PE⁻/CFSE⁺ (KMS-11 tumor cell population), and PE⁺/CFSE⁺ subpopulations using the single stained controls.

To examine the mechanism of protection conferred by ch128.1, we used a previously generated mutant with impaired binding to FcγRs and C1q (27). The triple mutant (L234A/L235A/P329S) retains the antigen-binding activity of ch128.1 and exhibits a lack of ADCC and CDC activity in vitro (27). We now show that this mutant also retains the in vitro cytotoxic activity of ch128.1. Exposure to the ch128.1 mutant led to the reduced proliferation of ARH-77 cells, to levels comparable to those observed in cells treated with ch128.1 (Fig. 1A). ARH-77 cells were used because they previously showed sensitivity to ch128.1 in vitro and in vivo, in contrast to KMS-11 cells, which only showed sensitivity in vivo (24). These results show that, as expected, these mutations do not alter the in vitro antiproliferative effect of ch128.1.

Interestingly, the in vivo protective effect of ch128.1 was not observed with the mutant in the two disseminated xenograft models of MM. ARH-77 B lymphoblastoid cells have been used as a model of MM because their i.v. injection in SCID mice leads to the development of a disease that mimics human MM (39, 40). KMS-11 human MM cells have been used to evaluate the in vivo efficacy of MM therapeutics (41, 42). In both models, significant prolonged survival was observed with ch128.1-treated animals (p < 0.0001 compared with animals treated with buffer alone; Fig. 1B, 1C). In contrast, the mutant did not show significant antitumor activity and mice succumbed to disease at rates similar to those seen in control animals. An isotype control Ab tested previously showed no antitumor effect in these models (24), confirming that TfR1 targeting was necessary for antitumor protection.

FcRn, also known as the Brambell receptor or salvage receptor, is critical to maintaining the bioavailability of IgG Abs in blood and preventing their degradation (20, 43). Thus, we determined if the binding of the ch128.1 triple mutant to mouse FcRn was impaired, as its lack of antitumor activity might be due to less bioavailability. However, SPR analysis showed similar binding affinity between mouse FcRn and ch128.1 or its mutant (Table I). To determine if the blood bioavailability of the mutant Ab was affected, Ab levels in the serum of SCID-Beige mice were examined after i.v. administration. The mutant ch128.1 Ab did not show decreased serum levels; in fact, the levels found were slightly higher than those of wild type ch128.1 (Fig. 2A). Additionally, we found that both Abs remain intact in mouse serum (Fig. 2B), showing bands consistent with the molecular mass of human IgG3 (165 kDa) (20). Together, these data indicate that the Fc region of ch128.1 is required for the antitumor activity in these models and that the lack of antitumor activity of the mutant is not due to a decrease in blood bioavailability (or increased clearance) in vivo.

The SCID-Beige model used is deficient in B and T cells (44) and has impaired NK cell and neutrophil activity (45–47). However, this model has macrophages capable of eliminating tumor cells (48–50) and an active complement pathway (51). To further define the importance of the Fc region in ch128.1-mediated antitumor activity, the role of macrophages was examined. Mice challenged with KMS-11 cells were treated with Clodrosome to deplete macrophages. The depletion of macrophages in ch128.1-treated mice significantly reduced the antitumor effect (p < 0.001 compared with that of ch128.1 administered with the liposomal control; Fig. 3A). However, a statistically significant survival benefit was still observed in these mice, compared with similarly depleted mice without ch128.1 treatment (p < 0.01). Depletion of macrophages in the absence of ch128.1 did not affect tumor growth, and mice succumbed to disease at the same rate as the buffer control group. Depletion of macrophages (F4/80⁺ cells) was confirmed in the spleen of mice using IHC (Fig. 3B). Even though macrophage depletion was not complete (approximately a 63% reduction in macrophages was observed), it was sufficient to significantly decrease ch128.1-induced antitumor activity. These results suggest that macrophages play a crucial role in the antitumor activity of ch128.1 in the KMS-11 xenograft model.

The role of complement in ch128.1-induced antitumor activity was assessed using CVF to deplete C3. Mice challenged with KMS-11 cells were treated with CVF or buffer. ch128.1 treatment alone or combined with CVF prevented MM in over 80% of the animals, with no significant difference in survival between the two groups (Fig. 4A). In contrast, CVF- or buffer-treated mice that did not receive the therapeutic Ab succumbed to disease at the same rate and were both significantly different compared with mice treated with ch128.1 alone (p < 0.0001). A substantial decrease of C3a levels in the sera of CVF-treated mice was observed within 24 h and persisted up to 8 d, confirming complement inactivation (Fig. 4B). The Fc region of human IgG1 and IgG3 is able to bind mouse complement and induce CDC, as shown with rituximab, a mouse/human chimeric anti-CD20 IgG1 (52), as well as with mouse/human chimeric anti-glucuronoxylomannan IgG1 and IgG3 Abs (53). Even though ch128.1 is capable of activating complement in vitro (27), our studies indicate that this pathway is not relevant in the mouse model used.

To confirm the ability of ch128.1 to induce effector functions mediated by macrophages, an in vitro assay to assess ADCC and ADCP was performed. Murine macrophages were used for these studies because human IgG3 has been shown to bind all four murine FcγRs (54), and these cells were identified as a major player in the in vivo mechanism of action of the Ab. Significant ADCC and ADCP were exhibited in the presence of 10 μg/ml ch128.1 (Fig. 5A) (p < 0.05 compared with negative control), which was also observed with 5 μg/ml (data not shown). As expected, the levels of ADCC and ADCP induced by the ch128.1 mutant were similar to those of the buffer control. To confirm the occurrence of phagocytosis induced by ch128.1 treatment, ImageStream imaging flow cytometry was performed. This method has previously been used to visualize macrophage phagocytosis of tumor cells mediated by rituximab (55). Phagocytosis was observed in the CFSE⁺/PE⁺ population, as indicated by the presence of tumor cells within macrophages (Fig. 5B). Together, these results show that murine macrophages can mediate tumor cell death via ADCC and ADCP.

The in vivo antitumor activity of ch128.1 in an early disease setting has been examined in disseminated MM models using ARH-77 and KMS-11 cell lines, as shown previously (24) and in Fig. 1B, 1C. We now show that a single dose of 50 μg ch128.1 (∼2.5 mg/kg), administered 2 d after tumor inoculation (early-stage), is sufficient for protection against MM in SCID-Beige mice, with over 85% of mice tumor-free at the termination of the experiment (120 d), compared with mice treated with buffer alone (p < 0.0005; Fig. 6A). This is similar to studies using a 100 μg dose in the KMS-11 disseminated-disease model, as shown previously (24) and in Fig. 1C. Importantly, statistically significant survival benefit was still observed when treatment was delayed to 9 d after tumor cell challenge (p ≤ 0.001 compared with buffer treated mice; Fig. 6A). A higher dose of ch128.1 (400 μg) was also tested in this late-stage disease model, but increased protection, compared with the 100 μg dose, was not observed (Fig. 6B; p < 0.05 compared with buffer-treated mice). The antitumor effects observed in these studies require targeting of the TfR1 because, as expected, an isotype control Ab did not confer protection in either the early-stage or late-stage disease setting (Fig. 6C). In both cases, the isotype control, whether given at day 2 or day 9, did not show an improvement in survival. In this study, ch128.1 showed significant tumor protection when compared with the IgG3 isotype control or buffer alone, when animals were treated 2 d after tumor challenge. Mice treated with ch128.1 in the late-stage disease setting (9 d post–tumor challenge) also showed significant protection when compared with the IgG3 isotype control.

We have previously shown that ch128.1 exhibits cytotoxic activity against certain hematopoietic malignant cell lines, including ARH-77 cells, in vitro through TfR1 degradation and iron deprivation (22, 24). However, KMS-11 cells were not sensitive to this direct in vitro activity of ch128.1 (24). We have also shown that ch128.1 induces ADCC and CDC in vitro against Ramos human non-Hodgkin lymphoma B cells (27) and that it confers similar antitumor protection in two disseminated MM xenograft models, ARH-77 and KMS-11, in an early-stage disease setting (24). This serves as a cautionary note that in vitro models may not always predict in vivo outcomes, at least in the case of Abs targeting TfR1. In this report, we examined the mechanism of the in vivo antitumor activity of ch128.1. For these studies, mutations were generated at sites important for FcγR and complement C1q binding (L234, L235, and P329), which resulted in a disrupted interaction with effector cells and the inability to fix complement, leading to impaired ADCC and CDC (27). As expected, this triple mutant Ab retains binding to the Ag (27) as well as the in vitro antiproliferative activity against sensitive malignant B cells (ARH-77), as we now report. It was used in this study to evaluate the relevance of the ch128.1 Fc region in its in vivo antitumor activity.

The ch128.1 mutant Ab failed to provide a survival benefit in vivo, indicating the relevance of the Fc fragment in ch128.1-induced antitumor activity. We explored the possibility that this loss of activity was due to a loss of bioavailability in mouse serum. Importantly, ch128.1 and its mutant showed similar binding affinity to mouse FcRn. Thus, the mutations at residues L234, L235, and P329 did not affect the binding of ch128.1 to this receptor, consistent with a human IgG1 mutated at the same sites that similarly exhibited abolished FcγR and C1q interactions but retained binding to FcRn (56). The residues mutated are located at sites distinct from those residues that are known to play a crucial role in the binding of human IgG Abs to mouse or human FcRn (especially S254, H310, H435, I253, and Y436) (57, 58). Because the FcRn protects IgG from degradation and in so doing is crucial in maintaining the Ab bioavailability in blood (20, 43), and because binding to this receptor is not affected in the ch128.1 triple mutant, its lack of antitumor activity is not expected to be due to a decrease in bioavailability. In fact, we showed that the mutations introduced in ch128.1 did not result in a decrease in its serum bioavailability.

Importantly, the ch128.1 mutant retains the direct in vitro cytotoxic effect against sensitive (ARH-77) cells. However, both sensitive (ARH-77) and resistant (KMS-11) cells respond similarly in vivo to ch128.1 treatment; thus, TfR1 degradation followed by iron deprivation does not appear to be the mechanism of action in these mouse models. Macrophages are important for this activity, whereas the complement pathway did not appear to contribute to ch128.1-mediated antitumor activity in our model, even though it binds C1q and induces CDC in vitro (27). There are two possible and non–mutually exclusive ways in which the Fc fragment and interaction with macrophages could potentially elicit antitumor activity. One is through Fc effector functions, including ADCC and/or ADCP. Even though TfR1 is constitutively internalized, significant ADCC has been observed in vitro by us and others (27, 59, 60), and tumor growth inhibition induced by Abs targeting TfR1 in oral squamous cell carcinoma and adult T cell leukemia/lymphoma murine xenograft models has been reported (59, 60). In this study, we also show that murine BMDM mediate ADCC and ADCP in the presence of ch128.1 but not its mutant. The other possibility is that ch128.1 simultaneously binds TfR1 on cancer cells and FcγRs on the surface of effector cells such as macrophages, leading to extensive cross-linking of the TfR1, resulting in the inhibition of receptor internalization and iron uptake. This has been observed with rat IgM anti-TfR1 Abs RI7 208 and REM 17.2, which as multivalent Abs induce the cross-linking of TfR1, interference with TfR1 internalization, and growth inhibition of AKR1 thymic lymphoma cells and SL-2 leukemic cells in vitro (61, 62). RI7 208 also exhibited antitumor activity against SL-2 cells in an AKR/J syngeneic model (62).

SCID-Beige mice are deficient in B and T cells (44) and have impaired NK cell and neutrophil activity (45–47) but retain functional macrophages and complement activity (45, 48–50). Even though the depletion of macrophages is incomplete in our studies, the antitumor activity induced by ch128.1 was significantly reduced. We have also shown previously that ch128.1 prolonged the survival of NOD-SCID mice bearing xenograft tumors of 2F7 AIDS-related non-Hodgkin lymphoma cells (26), which are tumors that are infiltrated with F4/80⁺ cells (63). NOD-SCID mice are deficient in B and T cells and have impaired NK cell and complement activity (64), supporting the importance of macrophages for ch128.1-induced antitumor activity. Macrophages are a particularly relevant component of the stroma in MM, providing support to the malignant plasma cells and vice versa (65). Such cross-talk protects the malignant cells from drug-induced apoptosis (66). However, it is expected that the presence of an Ab targeting the malignant cells in the MM microenvironment would result in macrophage-mediated MM cell killing as our studies suggest. It is also possible that the in vivo antitumor activity of ch128.1 may be magnified in the presence of the complete repertoire of functional immune effector cells, such as NK cells and neutrophils.

Consistent with our results, phagocytosis by human monocyte-derived macrophages of human MM cell lines and MM cells isolated from patients has been observed in vitro in the presence of the anti-CD38 Ab daratumumab (67). Additionally, macrophages contribute to the in vivo antitumor activity of daratumumab in SCID-Beige mice bearing Daudi Burkitt lymphoma (67). Furthermore, the recruitment of macrophages and the mediation of ADCP by an Ab specific for the B cell maturation Ag in s.c. and disseminated MM models in SCID-Beige mice has also been demonstrated (68). Together, these studies support the notion that macrophages play an important role in mediating the antitumor effects of various therapeutic Abs in mice bearing human malignant B cells.

A similar survival benefit in the KMS-11 model is achieved even with less than half of the previously reported dose of ch128.1 (125 μg) (24), indicating that this Ab is particularly effective against disseminated B cell malignancies. We also examined the efficacy of ch128.1 in a late-stage disease setting. As expected, less protection was detected compared with treatment at an earlier time point. However, significant prolongation of survival was still observed using a single dose of ch128.1. Interestingly, both 100 and 400 μg of ch128.1 comparably prolonged survival in this setting, suggesting that saturating conditions were reached.

Normal cells expressing high levels of TfR1 may be targeted by anti-TfR1 Abs, including ch128.1. Cells that are of particular concern include activated immune cells that are rapidly dividing as well as hematopoietic progenitor cells, including erythroblasts that require iron for heme synthesis (10). The xenograft models used in this study do not adequately address the toxicity of ch128.1 to these cell populations because this Ab does not cross-react with the murine TfR1 (69). However, this is a common problem for many Abs developed for clinical use, and despite this fact, these models have still shown predictive value for Abs that are currently used in the clinic (70–72). The toxicity of human anti-TfR1 Abs to normal cells has previously been evaluated. Toxicity to normal erythroblasts and PHA-activated T cells has been reported in vitro upon treatment with the fully human anti-TfR1 IgG1 Ab JST-TFR09, whereas no significant cytotoxicity was observed with this Ab in resting T cells (60). Similar effects against activated cells were observed with the mouse/human chimeric IgG1 Ab D2C (73). Two studies in cynomolgus monkeys (Macaca fascicularis) have been reported using anti-TfR1 Abs and are consistent with these in vitro findings. Monkeys given the murine IgG2b A24 Ab did not show significant toxicity; however, they showed decreased hemoglobin levels, decreased serum iron levels, and increased apoptosis in lymph node germinal centers that contain highly proliferative B and T cells (74). In the second report, monkeys given multiple doses of 30 mg/kg JST-TFR09 (also known as PPMX-T003) human IgG1 Ab demonstrated moderate anemia, but no other toxicities were observed (60, 75). Even though committed, hematopoietic progenitor cells express high levels of the TfR1 and are vulnerable to the effects of anti-TfR1 Abs, hematopoietic pluripotent stem cells lack TfR1 expression (10, 76–78). In fact, we have shown that this cell population is not affected by an immunotoxin consisting of an Ab-avidin fusion protein that is composed of ch128.1 genetically fused to chicken avidin and conjugated to the plant toxin saporin (79). One phase I clinical trial using an anti-TfR1 Ab has been reported (80). The 42/6 murine IgA Ab was generally well tolerated in this study; however, an allergic-type response that was associated with the human anti-mouse Ab response was observed in one patient that received two doses of the IgA (80). Further studies using Abs with human constant regions are needed to fully define the toxic effects and therapeutic potential of these Abs. Because of the expression of TfR1 on a variety of normal cells, toxicity must be carefully monitored.

In summary, we show that the Fc fragment of ch128.1 is crucial for its antitumor activity in the disseminated MM xenograft models examined. We have shown that macrophages can mediate both ADCC and ADCP in vitro in the presence of ch128.1. Additionally, our studies are consistent with the conclusion that macrophages play a major role in ch128.1-induced in vivo antitumor activity in our mouse models. Importantly, a single low dose of ch128.1 is protective in models at different stages of MM disease. Our results suggest that ch128.1 is a promising therapeutic against human B cell malignancies such as MM.

We thank Dr. Fu Li (Seattle Genetics, Inc.) for technical advice on Clodrosome depletion of macrophages in SCID mice and Dr. Donald M. Lamkin (University of California, Los Angeles) for technical advice on BMDM isolation and differentiation. We thank Salem Haile (UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility) for technical assistance running the ImageStreamX Mark II and Dr. Tim C. Chang (Amnis, part of MilliporeSigma) for assistance with the analysis of these data.

M.L.P. is a shareholder of Klyss Biotech, Inc. The Regents of the University of California are in discussions with Klyss to license M.L.P.’s technology to this firm. The other authors have no financial conflicts of interest.