Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

HM1.24, an immunological target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance Fc γ R binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via Fc γ R-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM. towards Fc Rs improved in vitro ADCC activity against cells, mediated by NK cells expressing the various Fc γ RIIIa polymorphisms. Fc-engineered therapeutic and anti-CD40 demonstrated enhanced in vitro and in vivo activity against lymphoma and leukemia. Importantly, early clinical data from a phase I trial of the Fc-engineered anti-CD30 antibody XmAb2513 provided encouraging evidence for the safety and antitumor efficacy of this therapeutic strategy. 33 XmAb5592 is a humanized anti-HM1.24 mAb with a similarly engineered Fc-domain that specifically increases affinity for Fc γ receptors expressed on various effector cells, and associated cytotoxicity. Here, we evaluate the preclinical activity of XmAb5592 in MM and demonstrate that, compared to an anti-HM1.24 mAb with normal Fc γ R binding (IgG1 analog), it has much greater anti-MM activity in vitro and in vivo, mediated via superior induction of NK cell activation and degranulation. The anti-MM activity of XmAb5592 shows synergism when combined with lenalidomide pretreatment of effector cells. Its potential for clinical efficacy was also demonstrated by the ability to deplete plasma cells from both blood and bone marrow in non-human primates. XmAb5592 represents a promising next-generation immunotherapeutic for MM and several other malignancies. mouse and human immune systems. In a recent study in cynomolgus monkeys, with an immune system closely homologous to that of humans, we have shown that no depletion. These observations point to the potential therapeutic benefits of effector function enhanced antibodies for treatment of human malignancies.


Introduction
30μL/minute for 2 minutes followed by dissociation for 5 minutes. Data were fit and analyzed to obtain K d values as described. 32

ADCC & Phagocytosis Assays
ADCC for MM cell lines was measured using a lactate dehydrogenase (LDH) activity assay with purified peripheral blood mononuclear cells (PBMCs) as effector cells as described. 29,30 Target cells were plated at 10,000 cells per well with 500,000 PBMCs (E/T ratio 50:1). In some experiments, PBMCs were pretreated with 2μM lenalidomide (Selleck Chemicals) for 48 hrs before using in ADCC assays. Calcein-AM release assay was used to measure the ADCC activity against CD138 + purified patient MM cells, and/or when purified NK cells were used as effector cells. 4,5,10 Macrophage driven antibody-dependent cell-mediated phagocytosis (ADCP) was determined by flow cytometry as described, 30 using RPMI8226 or U266B1 cells as targets.

In vivo tumor xenograft models
Six-to twelve-week old female C.B-17 severe combined immunodeficient (SCID) mice (Taconic) were injected subcutaneously in the right flank with 10 7 RPMI8226 human MM cells. Twenty-one days later, mice bearing tumors between 30-100 mm 3 were selected and placed into groups with similar mean tumor volume (± 10%, n = 5-15/group). For the dose response study, mice were injected intraperitoneally with 0.9, 3, or 9 mg/kg XmAb5592 or vehicle (PBS) twice per week for 7 doses; and in the comparison study mice were injected intraperitoneally with 9 mg/kg antibody (XmAb5592, anti-HM1.24 IgG1 or anti-HM1.24 Fc-KO) or vehicle twice per week for 7 doses. For the lenalidomide combination study, lenalidomide (25 mg/kg in PBS) was injected intraperitoneally twice a week for a total of 4 doses in 2 of the groups as indicated. 6 mg/kg of XmAb5592 or PBS was injected in the groups as indicated twice a week for 8 doses. Tumors were measured twice per week in all the studies with calipers and volumes (mm 3 ) calculated as π/6(length x width x height). The differences between groups were calculated using the nonparametric Mann-Whitney U test with P values less than 0.05 considered significant. Xencor Institutional Animal Care and Use Committee approved all experiments.

Plasma cell depletion study in non-human primates
Cynomolgus monkey studies were conducted at SNBL USA, and all protocols were approved by their Institutional Animal Care and Use Committee. The study used 2 male and 2 female naïve cynomolgus monkeys, 3-5 years old and weighing 2.5-4.5 kg; animals were acclimated to the study rooms for 21 days before study initiation. XmAb5592 was formulated in 10 mM sodium phosphate, 150 mM sodium chloride, and 0.01% polysorbate 20 (pH 7.0), and administered at 20 mg/kg as a single 1-hour intravenous infusion to the left saphenous vein. Blood samples drawn from the cephalic or femoral veins were collected pre-dose and throughout the duration of the study (1, 2, 3, 4, 7, 14, 21 and 28 days post XmAb5592 injection). Bone marrow aspirates were collected from the humerus pre-dose, and at day 7, 14 and prior to necropsy at day 28. Immunophenotyping was performed on all samples using cynomolgus monkey cross-reactive, anti-human mouse monoclonal antibodies against CD20 (PE-Cy7 conjugated 2H7, BD Biosciences) and CD38 (FITC conjugated clone AT-1, StemCell Technologies).
Cynomolgus cross-reactive, anti-human CD19 IgG1 antibody 29 was PerCP conjugated using labeling kit (Prozyme). Antibodies were added to the samples, incubated, and washed prior to flow cytometry analysis by gating on live lymphocytes based on forward and side scatter (FSC vs SSC), and then determining the counts of CD38 ++ CD19 low/-CD20cells, expressed as a percentage of the total lymphocytes.  (Table 1), thus serving as a control devoid of effector function.

XmAb5592 enhances ADCC and ADCP against MM cells
We next determined whether enhanced binding to FcγR-bearing effector cells could be translated to increased XmAb5592 cytotoxic activity compared to the anti-HM1.24 IgG1 analog. The ADCC activity was evaluated against a panel of MM cell lines using PBMCs isolated from healthy donors as effector cells. Relative to its IgG1 analog, XmAb5592 markedly enhanced lysis of MM cell lines ( Figure 1A), significantly increasing both efficacy (maximal lysis) and potency (EC 50 ) ( Figure 1B). EC 50 values for XmAb5592 ranged from 5-27 ng/ml, indicating increased potency up to 9-fold. Maximal lysis by  Figure 3).
The ADCC activity of XmAb5592 against MM patient derived CD138 + primary MM cells was next evaluated, using NK cells derived from the same patient (autologous). This more closely mimics the in vivo clinical setting in patients. XmAb5592 induced significantly enhanced ADCC compared to the IgG1 analog in a dose dependent manner; maximal lysis seen with XmAb5592 was 40 ± 2.2 % vs only 5 ± 2.5 % for the IgG1 version at 1μg/ml ( Figure 1C). XmAb5592 similarly induced autologous lysis against additional MM patient cells ( Figure 1D), with no ADCC seen for the XmAb isotype control. Primary tumor cells are generally more resistant, and ADCC activity seen with XmAb5592 therefore underscores the utility of this Fc engineered therapeutic compared to the lack of significant activity seen with the IgG1 analog.
We also assessed the impact of XmAb5592 on macrophage phagocytosis, as it is an important contributor to the cytotoxic activity of therapeutic antibodies. 30,36 ADCP assays were done with monocytederived macrophages as effectors, and using RPMI8226 or U266B1 MM cell lines as target cells. With both cell lines, XmAb5592 displayed approximately 2-fold greater potency relative to the IgG1 analog ( Figure 1E). Maximal phagocytosis increased from 55% to 67% for RPMI8226 cells, and from 28% to 56% for U266B1 cells, when using XmAb5592 vs. the IgG1 analog. As expected, the XmAb isotype control did not induce any macrophage phagocytic activity. Both XmAb5592 and the IgG1 analog For personal use only. on August 24, 2017. by guest www.bloodjournal.org From showed no detectable complement-dependent cytotoxic (CDC) activity against RPMI8226 cells when incubated in the presence of human serum complement for 2 hours (data not shown).

XmAb5592 induces potent ADCC and activates NK degranulation in the co-culture of MM-BMSCs
Since the bone marrow (BM) microenvironment protects MM cells against cell death, we next determined whether XmAb5592 still triggered ADCC lysis against MM cells in the presence of BMSCs. Strong XmAb5592-induced ADCC activity was seen against MM1S or MM1R target cells in the absence or presence of BMSCs (Figure 2A), suggesting that it will be effective against MM cells in the BM microenvironment. Significantly, XmAb5592 retains ADCC activity against IL6-dependent INA-6 cells in the presence of BMSCs, although overall lysis is reduced. This apparent reduction in killing in the presence of BMSCs is likely due to a generalized effect of these cells on MM survival.
During granzyme-B/perforin dependent NK cell degranulation process, CD107a (lysosomeassociated membrane protein 1) becomes transiently mobilized to the cell surface, serving as a functional marker for NK activity. 37 We assessed surface expression of CD107a on NK cells in the presence of target MM1S cells and XmAb5592 or other control antibodies, with or without BMSCs. XmAb5592 induced approximately 10-fold more NK degranulation than the IgG1 analog, regardless of the presence of BMSCs ( Figure 2B). Calcein-AM release ADCC assays performed simultaneously with serial dilutions of XmAb5592 and the IgG1 analog show a similar lysis pattern, both in the presence or absence of BMSCs ( Figure 2C). The enhanced CD107a-mediated NK degranulation triggered by XmAb5592 significantly correlated with increased ADCC against MM cells, regardless of the presence of BMSCs.
XmAb5592 also induced >10-fold greater NK degranulation against primary CD138 + MM cells relative to the IgG1 analog, using NK cells from the same patient ( Figure 3A). Pre-treatment of MM cells with 0.1 μM dexamethasone did not affect the NK degranulation, suggesting that the XmAb5592's therapeutics benefits would be combinable with conventional treatment regimens ( Figure 3A).
Additionally, pretreatment of effector cells with IL-2 enhanced XmAb5592-induced cytotoxicity against MM patient cells ( Figure 3B). IL-2 did not enhance NK degranulation in the presence of anti-HM1.24 Fc-KO, further confirming the importance of the engineered Fc-domain of XmAb5592 for improving therapeutic efficacy.

XmAb5592 strongly inhibits growth of established myeloma tumors in vivo
The in vivo activity of XmAb5592 was next examined in an established human MM tumor model. SCID mice bearing RPMI8226 subcutaneous tumors were treated with 0.9, 3.0 or 9.0 mg/kg of XmAb5592 twice a week to determine the optimal treatment dose. XmAb5592 inhibited RPMI8226 tumor growth in a dose-dependent manner ( Figure 4A). On day 41, after 3 weeks of treatment, 0.9, 3.0 or 9.0 mg/kg of XmAb5592 decreased RPMI8226 tumor growth by 23%, 75% and 80%, respectively, relative to vehicle XmAb5592 also inhibited tumor growth to a greater extent than the IgG1 analog in another SCID mice xenograft study of established human OPM-2 MM tumors (supplemental Figure 4). Significantly improved in vivo activity of XmAb5592 relative to its IgG1 analog across multiple xenografts, coupled with the ineffectiveness of the anti-HM1.24 Fc-KO, indicates that the anti-tumor activity of XmAb5592 is mediated via FcγR-dependent mechanisms.

Lenalidomide further enhances XmAb5592 induced anti-myeloma activity both in vitro and in vivo
Lenalidomide is an immunomodulatory drug that has been used very effectively for treatment of MM, 38 and is known to increase the activity of NK cells. Lenalidomide pretreatment also increased the ADCC activity of the IgG1 analog against some of the cell lines, notably MM1R cells. The increased XmAb5592 ADCC with lenalidomide pretreated effector cells was also further augmented with IL-2 pre-treatment of NK cells (supplemental Figure 5).
The potential for enhancing the therapeutic activity of XmAb5592 in vivo through combination with lenalidomide treatment was evaluated in a murine xenograft model of RPMI8226 human MM cells.
Tumor-bearing SCID mice were treated with 25 mg/kg of lenalidomide (by i.p. injections twice a week for 2 weeks), alone or in combination with 6 mg/kg of XmAb5592 (by i.p injections twice a week for 4 weeks).
The doses of both treatments were chosen to be suboptimal in order to see a synergistic effect.
Combination treatment with XmAb5592 and lenalidomide was more efficacious than either of the treatments alone ( Figure 5B). On day 56, the mean tumor volumes were 46, 127, and 411 mm 3 for the combination, XmAb5592 alone and lenalidomide alone groups, respectively. The in vitro and in vivo therapeutic enhancement seen with lenalidomide and XmAb5592 support a combination strategy for their clinical evaluation.

XmAb5592 depletes blood/bone marrow plasma cells in cynomolgus monkeys
The murine anti-HM1.24 antibody, from which XmAb5592 was derived, cross-reacted with the HM1.24 antigen on lymphocytes from cynomolgus monkeys, but not with those from rats, mice, guinea pigs, rabbits or dogs. 39 In immunohistochemistry studies with normal tissues, XmAb5592 showed a similar cross-reactivity pattern with human and cynomolgus monkey tissues (data not shown), supporting the use of cynomolgus monkey for assessing the biological activity of the antibody. To investigate the impact of XmAb5592 on depletion of plasma cells in blood and BM, cynomolgus monkeys (2 males and 2 females) were given a single intravenous infusion of the drug at 20 mg/kg, and followed for 28 days. Based on the staining of pre-dose blood and BM samples from monkeys for CD19, CD20, CD38 and HM1.24, a strong co-incidence was seen for CD38 high /CD19 low/-/CD20plasma cells with HM1.24 expression (supplemental Figure 6), and this population was quantified following XmAb5592 injection. XmAb5592 depleted blood plasma cells an average of 80% after 1 day of injection, with recovery to starting levels by day 8 ( Figure   6A). In BM, XmAb5592 depleted an average of 60% of the initial plasma cells by day 7 post-injection, For personal use only. on August 24, 2017. by guest www.bloodjournal.org From which did not recover fully by day 28 ( Figure 6B). These data clearly indicate that a single dose of XmAb5592 depletes plasma cells in both blood and BM compartments in cynomolgus monkey.
Moreover, the XmAb5592 administration was well tolerated, and no changes were identified in food consumption, body weight, serum chemistry, or in anatomical pathology parameters, suggesting a favorable therapeutic index for clinical application.

Figure 5. Lenalidomide further enhances XmAb5592 induced anti-myeloma activity both in vitro
For personal use only. on August 24, 2017. by guest www.bloodjournal.org From and in vivo. (A) PBMCs were pre-incubated with or without lenalidomide (2 μM) for 48 hrs prior to LDH release-based ADCC assays against target MM cells. Percent specific lysis is shown, as mean ± SD from triplicate measurements. Lenalidomide pre-treatment significantly enhanced improved ADCC activity of XmAb5592 than the IgG1 analog. (B) RPMI8226 tumor-bearing mice (n=5) were treated with PBS, lenalidomide alone, XmAb5592 alone and XmAb5529 plus lenalidomide. Lenalidomide (25 mg/kg) was injected i.p. twice weekly for 4 doses ( ). XmAb5592 (6 mg/kg) was injected i.p twice weekly for a total of 8 doses (↓). Combination treatment with XmAb5592 and lenalidomide was more efficacious than either of the treatments alone (day 56 MTV = 46, 127 and 411 mm 3 for the combination, XmAb5592 alone and lenalidomide alone, respectively). In bone marrow (B), the plasma cells were reduced an average of ~60% at day 7 post injection; however the levels did not recover completely by the study end on day 28.
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