Antibody-dependent Cellular Phagocytosis Assays Research Articles

CD47 Blockade Enhances Phagocytic Activity of the TCR-Mimic Antibody Hu8F4 Against HLA-A2+ AML

Hu8F4, a humanized monoclonal antibody that recognizes PR1/HLA-A2 complex, is a first-in-class T cell receptor (TCR) - mimic antibody is currently being tested in a phase 1 trial for AML. We previously reported that activity of Hu8F4 primarily depends on its binding to Fcγ receptors (FcγR) on effector cells. Additionally, we identified antibody-dependent cellular phagocytosis (ADCP) as a principal mechanism of Hu8F4 anti-AML activity in vivo. Leukemia cells overexpress the “don't eat me” membrane protein CD47. CD47 on AML binds SIRPα on macrophages and neutrophils to inhibit phagocytosis. Blocking this interaction can allow antibodies to exert powerful phagocytic activity. Importantly, the role CD47 may play in the anti-tumor activity of TCR-mimic antibodies has not yet been investigated. To examine the relationship between CD47 and Hu8F4 activity, we used both in vitro ADCP assays and in vivo NSG mouse models. CD47 blockade was studied as a potential strategy to enhance Hu8F4 activity. To characterize the in vitro phagocytosis, we used an HLA-A2 transfected leukemia cell line, U937 (U937-A2), and an endogenously expressing HLA-A2 cell line THP1, as targets. NSG mouse bone marrow derived macrophages (BMDM) were used as effectors. At an initial E:T=1, Hu8F4 alone (10 mg/ml) was insufficient to prevent growth of U937-A2 and THP1. However, addition of neutralizing anti-CD47 F(ab') 2 antibody significantly slowed leukemia growth and eliminated both target cells by day 5. To further confirm the critical role of CD47 in Hu8F4-mediated ADCP, we created CRISPR/Cas9 CD47 KO U937-A2 and THP1 cells lines. Complete phagocytosis of both CD47KO cell lines was only observed in the presence, but not the absence of Hu8F4. To test if CD47 blockade plays a role in Hu8F4 anti-AML activity in vivo, we injected NGS mice with U937-A2 (5x10^3). Mice were treated with Hu8F4 +/- anti-CD47 F(ab') 2 antibody at 1mg/kg (three times per week for a total of 10 doses starting on day 5, n=5 per group). Control mice, treated with PBS, succumbed to leukemia by day 28. Both Hu8F4 and anti-CD47 F(ab') 2 single-agent treatment slowed leukemia growth, while the combination of Hu8F4 and anti-CD47 F(ab') 2 completely prevented outgrowth of the aggressive U937-A2 cells ( Figure 1), indicating that blocking CD47 significantly increased the activity of Hu8F4 in vivo. Interestingly, CD47 KO U937-A2 did not engraft in NSG mice as expected, (n=5), confirming that CD47 expression is critically important for cell line engraftment in NSG. HuCD47 is known to bind to NSG mouse SIRPα with high affinity providing protection to the human cells from phagocytosis by mouse macrophages. Growth of CD47 KO THP1 in NSG mice was delayed compared to parental THP1, median survival of untreated mice increased from 58 days for THP1 to 99 days for CD47 KO THP1 (p=0.0017, n=5). To facilitate THP1 cell growth in vivo, we used NSGS mice which allows for faster growth of human myeloid cells. Treatment with Hu8F4 for 7 weeks (1 mg/kg three times per week; started on day 2), significantly reduced THP1 leukemia growth and fully eliminated CD47 KO THP1 cells, confirming the role of CD47 in the activity of Hu8F4 ( Figure 2). Finally, to test whether CD47 is also important in Hu8F4-mediated phagocytosis by human effector cells, we generated human macrophages (Mφ) from CD14+ monocytes from healthy donors. We incubated fluorescently labeled human Mφ with CFSE-labelled target cells (U937-A2 or THP1), that had been pre-treated with Hu8F4 or isotype control antibody (Herceptin), at E:T=10, with or without anti-CD47 F(ab') 2. After 3 hours, Hu8F4-mediated phagocytosis, defined as the mean percentage of double-positive target cells, increased from mean 9.7% to 12.3% (n=4, p=0.199) for U937-A2, and from 12.25% to 17.58% (n=4, p=0.005) for THP1 when anti-CD47 F(ab') 2 blocking antibody was added. Anti-CD47 F(ab') 2 alone and in the presence of control Herceptin did not induce phagocytosis. Our data shows that the presence of CD47 on leukemia cells can significantly limit the activity of Hu8F4 both in vitro and in vivo. Importantly, combining Hu8F4 with CD47 blockade significantly increased the Hu8F4 activity and together eliminated AML. The role of other phagocytosis checkpoints, such as SIRPα and LILRB-1 also warrant further investigation.

Serum concentration of antigen-specific IgG can substantially bias interpretation of antibody-dependent phagocytosis assay readout

Because virus neutralization cannot solely explain vaccine-induced, antibody-mediated protection, antibody effector functions are being considered as a potential correlate of protection (CoP). However, measuring effector functions at a fixed serum dilution for high throughput purposes makes it difficult to distinguish between the effect of serum antibody concentration and antibody properties such as epitopes, subclass, and glycosylation. To address this issue, we evaluated antibody-dependent cellular phagocytosis (ADCP) assay against SARS-CoV-2 spike. Adjustment of serum samples to the same concentration of antigen-specific IgG prior to the ADCP assay revealed concentration-independent differences in ADCP after mRNA vaccination in subjects with and without prior SARS-CoV-2 infection not detectable in assay performed with fixed serum dilution. Phagocytosis measured at different concentrations of spike-specific IgG strongly correlated with the area under the curve (AUC) indicating that ADCP assay can be performed at a standardized antibody concentration for the high throughput necessary for vaccine trial analyses.

IgA antibody immunotherapy targeting GD2 is effective in preclinical neuroblastoma models

BackgroundImmunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab)...

Abstract 6357: Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile

Abstract Background/Purpose: CD47 has been validated to be expressed on various tumor types including acute myeloid leukemia, myelodysplastic syndrome and other hematologic or solid tumors. As primary ‘don’t eat me’ signal, high expression of CD47 on tumor cells interacts with SIRPα negatively regulating the phagocytosis level. However, poor safety profile as well as insufficient combinatory effect remain to be the issues limiting the clinical outcomes of CD47-targeting molecules. Herein, we used rational design strategies to generate SCR9168 with potent affinity improvement, and maintained favorable safety profiles in different animal species, including cynomolgus monkeys. Procedures: Mutagenesis was introduced to the residues critical for CD47/SIRPα binding interface. Affinity was measured by SPR analysis. In vitro activities were determined by biochemical- or cell-based binding, blocking and antibody-dependent cellular phagocytosis (ADCP) assays. The efficacy in vivo was assessed with the OE19 xenograft model. Pharmacokinetics (PK) and safety were monitored in both mice and cynomolgus monkeys. Results: The affinity of SCR9168 achieves 178 pM for human CD47 which is nearly 50-fold increase in comparison with wild-type SIRPα. Enhanced affinity has also been confirmed in binding to OE19 or DLD1 cell lines, as well as in blocking human SIRPα binding to Raji cells. SCR9168 remains strong binding potency to monkey, mouse or rat CD47 which allows the PK and safety assessment using these animal species. The combined effects of SCR9168 and multiple therapeutic antibodies targeting tumor-associated antigens (TAAs) have been evaluated with ADCP assays using human monocyte-derived macrophages. SCR9168 markedly increases the ratio of phagocytic cells combining with antibodies, such as cetuximab. However, due to the inert function of Fc fragment, no phagocytosis of red blood cells or platelets was caused after the incubation in vitro. In addition, SCR9168 treatment in combination with trastuzumab leads to significantly improved suppression of tumor growth in a dose-dependent manner. Moreover, administration of two doses at day 1 and day 11 in cynomolgus monkeys results in no toxicity events related to SCR9168 based on the data from hematological analysis. Conclusion: SCR9168 demonstrates the best-in-class potential among SIRPα mutein molecules. It elicits improved and dose-dependent efficacy in phagocytosis or tumor suppression combining with therapeutic antibodies, such as trastuzumab or cetuximab. Favorable safety profile with no phagocytosis of RBC or platelets in vitro as well as no hematological toxicity observation in cynomolgus monkeys allows broader dose range exploration in early clinical phase. SCR9168 is currently in development stage and IND enabling will be expected in the end of 2023. Citation Format: Yingying Hu, Yayuan Fu, Lei Liu, Wenjing Li, Liting Xue, Zhiyong Yu, Yun Zhang, Meijuan Gao, Yixin Tan, Fudong Wang, Yadan Wu, Jie Li, Zhenzhen Li, Feng Zhou, Wenqing Yang, Zhuoxiao Cao, Renhong Tang. Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6357.

Abstract 1873: D3L-001, a novel bispecific antibody targeting HER2 and CD47, demonstrates potent preclinical efficacy in solid tumors

Abstract Background: HER2 is overexpressed in different solid tumors, including 10-20% breast and gastric cancers. Many HER2-targeted drugs, including trastuzumab, pertuzumab, lapatinib, trastuzumab emtansine (T-DM1), and trastuzumab deruxtecan (T-DXd), have been approved for treatment of HER2-positive early and metastatic breast cancer (BC). However, trials involving pertuzumab, lapatinib and T-DM1 have failed to provide significant clinical improvements in patients with HER2-positive gastric cancer (GC), suggesting differences in HER2 mediated disease biology between HER2-positive BC and GC. Recent studies have suggested efficacy of HER2 antibodies could be further enhanced by antibody-dependent cellular phagocytosis (ADCP) principally regulated by “don’t-eat-me” CD47 signals, which is overexpressed in many HER2-positive cancers and suppresses phagocytosis through binding to SIRPα. Methods: BC and GC cell lines were used. Monocytes were isolated from human PBMC and differentiated into macrophages, which were used for in vitro ADCP assays. In in vivo animal experiments, D3L-001 and comparator antibodies were examined for efficacy in solid tumor xenograft models. Results: D3L-001 was designed to have higher HER2 affinity (KD < 1 nM) than that of CD47 (KD >10 nM). With this unique design, D3L-001 showed preferential binding to HER2/CD47 double positive tumor cells as compared to CD47 single positive cells. D3L-001 showed better anti-tumor effect than trastuzumab in a panel of solid tumor models, including but not limited to BC and GC cell lines or patient derived xenografts. Immunohistochemistry (IHC) analysis was performed in tumor FFPE slides to evaluate expression of HER2 and CD47 in the efficacy tested models, and we observed that D3L-001 showed broad tumor growth inhibition in HER2 or CD47 high/low expression models. In both in vitro and in vivo studies using breast cancer HCC1954, JIMT-1 and gastric cancer N87 models, we observed a synergistic effect in the combination of Trastuzumab with Magrolimab. Moreover, D3L-001 showed a trend of better efficacy than the combination of Trastuzumab and Magrolimab. These results confirm the synergistic effect of dual-targeting HER2 and CD47 simultaneously and suggest advantage of using bispecific antibodies to confer such effect. D3L-001 also displayed potent and comparable in vivo efficacy as Enhertu in gastric N87 model, and it showed synergistic in vivo efficacy, when combining with Pertuzumab or Paclitaxel, in breast HCC1954, JIMT-1 and gastric cancer N87 models. Conclusion: D3L-001, a novel HER2×CD47 bsAb, demonstrated potent anti-tumor effect in a variety of solid tumor models via HER2 guided CD47 co-blocking. It also showed synergistic efficacy when combining with other anti-tumor agents. D3L-001 will be further evaluated in clinic for its potential as a novel treatment option in HER2-positive BC, GC and other solid tumors. Citation Format: Haopeng Rui, Xiaofeng Yang, Jiahao Chen, Jia Wang, Zhiqiang Zheng, Zhiqiang Chen, Jingtao Lu, Peggy Wu, Janet Chen, Allison Wang, George Chen. D3L-001, a novel bispecific antibody targeting HER2 and CD47, demonstrates potent preclinical efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1873.

Evaluation of immunotherapies improving macrophage anti-tumor response using a microfluidic model

Tumor-on-a-chip models are rapidly coming to the fore as a promising platform to accurately mimic tumor biology. These models overcome ethical concerns of animal usage in research, and are of particular use in the field of immuno-oncology, as there are substantial interspecies differences in how cells of the immune system operate. Additionally, they allow a human-centered investigation of novel immunotherapeutic approaches. Here, we report a new application of a microfluidic tumor-on-a-chip system and show its utility by investigating macrophage responses in the context of a promising therapeutic approach that combines anti-epidermal growth factor receptor (EGFR) IgA with an anti-CD47 innate immune checkpoint inhibitor. We report a novel on-chip microscopy-based antibody-dependent cellular phagocytosis (ADCP) assay with human M1-like pro- or M2-like anti-inflammatory macrophages and tumor cells in a collagen matrix. The tumor microenvironment was further characterized by ELISA for secreted factors in the culture medium and through endpoint analyses of gene expression by RT-qPCR. Employing the novel on-chip assay, we show for the first time that the combination of anti -EGFR IgA and a CD47 checkpoint inhibitor synergistically activate macrophage phagocytic function to specifically kill cancer cells, especially for M2-like macrophages. We further demonstrate that the checkpoint inhibition is responsible for elevated secretion of inflammatory cytokines such as TNFα and IL-6, and tends to elevate expression of genes regulating both inflammation (IL-1β) and phagocytic function (CD209), particularly in M2-like macrophages. Taken together, we demonstrate a novel on-chip ADCP assay compatible with multi-parameter characterization of the tumor microenvironment on-chip and demonstrate its utility for yielding novel insights regarding innate immunotherapy combinations. • Microscopic imaging-based antibody-dependent cellular phagocytosis assay on-chip. • Multiparameter analysis of tumor microenvironment on-chip. • Anti -EGFR IgA and CD47 blocker synergistically activate macrophages on-chip. • Stronger therapeutic responses in 2D vs. on-chip 3D cultures.

Hosu-53, a Novel Potent Dihydroorotate Dehydrogenase Inhibitor Accentuates CD47 Blockade Therapy Resulting in Long Term Survival in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a complex disease characterized by leukemia stem cells in a state of differentiation arrest leading to suboptimal innate immune function and immunosuppression. The effectiveness of allogeneic hematopoietic stem cell transplantation, NK cell directed therapies, and recently CD47 blockade highlights the importance of immune surveillance as potentially curative therapies for AML. Dihydroorotate dehydrogenase inhibitors (DHODHi) have been reported to have both cytotoxic and differentiation properties which propelled their clinical development for AML. We developed HOSU-53, a novel DHODHi which is described in detail in another abstract at this meeting. HOSU-53 has (i) nanomolar potency against human DHODH biochemical activity, (ii) in vitro anti-proliferative activity toward AML with a potent tumor DNA damage response, (iii) favorable oral bioavailability, (iv) a monitorable on-target pharmacodynamic biomarker predictive of efficacy and toxicity, and (v) superior in vivo survival outcome in disseminated AML cell line derived xenograft models. Prior anti-viral studies using DHODHi have shown that deprivation of pyrimidine synthesis and associated DNA damage response can enhance innate immune activation. Therefore, we hypothesized that HOSU-53 could enhance phagocytosis and antibody dependent cellular phagocytosis (ADCP) and thereby synergize with myeloid immune checkpoint blockade therapies. Herein, we report compelling preclinical data demonstrating curative potential in AML xenograft models. Treating AML cells with HOSU-53 resulted in restored ability to phagocytose E.coli suggesting enhanced innate immunity. Given our previous reports that DHODHi could induce cell surface markers such as CD38, we next validated that HOSU-53 can increase expression of calreticulin (CALR) and CD47. In vitro treatment with HOSU-53 demonstrated increased tumor surface expression of the pro-phagocytic signal, CALR and a dose dependent increase in CD47 expression. We performed ADCP assays using murine bone marrow derived macrophages and observed enhanced phagocytosis when AML cells were pretreated with HOSU-53, prompting us to conduct in vivo validation using the MOLM-13 disseminated model. Mice were enrolled four days post-engraftment to receive single agent anti-CD47 monoclonal antibody at 0.5mg daily for 21 days and either daily 4 or 10 mg/kg HOSU-53, based on our prior dose optimization studies. We also investigated the combination of CD47 antibody with each dose level of HOSU-53. All monotherapy cohorts resulted in significant prolonged survival, but still eventually succumb to AML disease. In contrast, the combination cohorts resulted in disease-free survival as determined by the absence of measurable human CD45+ cells in spleen or bone marrow cells harvested from mice at the end of treatment on day 80, or on day 106 after remaining on study for 25 days without continued treatment. The curative potential in this model prompted us to repeat our study in a model with higher tumor burden. In this follow-up study, mice were enrolled ten days post engraftment to receive daily HOSU-53 (4 or 10 mg/kg) or B6H12 anti-CD47 (0.5mg daily or every three days for 21 days) or combinations of these treatments. We observed that anti-CD47 monotherapy was ineffective with this more established tumor model, but HOSU-53 monotherapy was comparable to our previous study and both 10 mg/kg combination cohorts significantly prolonged survival (Fig. 1, ongoing study). Further mechanistic studies of HOSU-53-mediated synergy of CD47 therapy are ongoing. Herein, using our DHODHi HOSU-53, we describe that pyrimidine depletion can enhance innate immunity to potentiate CD47 blockade therapy, representing a novel treatment approach. We show HOSU-53 increased tumor innate immune function, CALR expression and subsequently increased in vitro phagocytosis with CD47 blockade. Furthermore, in vivo combination of HOSU-53 with anti-CD47 antibody promoted dramatic synergy exhibiting a potentially curative approach in an otherwise very aggressive tumor model. HOSU-53 is currently in the IND-enabling studies phase with plans for filing an IND application in 2023 for phase 1 clinical trials in AML and MM. This data and our previously reported combination of HOSU-53 with anti-CD38 antibodies warrants the clinical translation of DHODHi in combination with immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Sumoylation Inhibitor TAK-981 in Combination with the CD19-Targeting Antibody Tafasitamab Shows Enhanced Anti-Tumor Activity in Preclinical B-Cell Lymphoma Models

IntroductionTAK-981 is a first-in-class small molecule inhibitor of the SUMO activating enzyme currently in Phase I/II clinical trials. TAK-981 has been shown to increase NK cell activation and M1 macrophage polarization via upregulation of Type I interferon (IFN) signaling, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in combination with rituximab (Nakamura 2019, AACR).Tafasitamab (MOR208) is a CD19-targeting antibody with enhanced Fc effector function mediating ADCC, ADCP and direct cytotoxic activities against B-lymphoma cells. Based on the Phase II clinical study L-MIND (Salles et al., 2020 and Duell et al., 2021), tafasitamab in combination with lenalidomide received accelerated approval by the Food and Drug Administration for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL).Due to the potential for TAK-981 to enhance the activity of tafasitamab via activation of innate effector cells, we aimed to investigate the effects of this drug combination on ADCC, ADCP and tumor cell viability in vitro. Additionally, combinatorial activity of TAK-981 plus tafasitamab was evaluated in lymphoma xenograft models.MethodsA panel of 9 aggressive lymphoma cell lines was analyzed (7 DLBCL and 2 Burkitt lymphoma). For ADCC, PBMC effector cells from healthy human donors were pre-treated with 0.1 or 1 µM TAK-981 or dimethyl sulfoxide (DMSO) control for 24 hours. Tumor cells were incubated with/without 1 nM tafasitamab in the presence of TAK-981 pretreated PBMCs at effector-to-target (E:T) ratios of 5:1 to 10:1 for 2 hours. Degranulation of NK cells was determined via CD107a surface expression after co-incubation of TAK-981 pre-treated PBMCs with tumor cells and 0.1 or 10 nM tafasitamab for 3 hours. Cytokine levels in the supernatant were investigated upon incubation of PBMCs with lymphoma cells, 1 µM TAK-981 and/or 10 nM tafasitamab for 24 hours. For the ADCP assays, in vitro differentiated macrophages were treated with 1 µM TAK-981 for 24 hours. Next, macrophages were incubated with lymphoma cells and 1 or 10 nM tafasitamab at an E:T ratio of 2:1 for 3 hours. For cell viability assays, tumor cells were treated with 1-1000 nM TAK-981 and/or 5 nM tafasitamab for 24 hours in the absence of effector cells. Cytotoxicity, phagocytosis, degranulation and cytokine release were analyzed by flow cytometry. Cell viability was assessed by determination of ATP levels. For in vivo analysis, effects of TAK-981 (7.5 mg/kg IV twice weekly) in combination with tafasitamab (3, 10 or 20 mg/kg IP twice weekly) on tumor growth were evaluated in Daudi and WSU-DLCL2 xenograft models of Burkitt lymphoma and DLBCL grown in SCID mice.ResultsIn ADCC experiments, increased cytotoxicity was observed upon combination treatment with TAK-981 and tafasitamab compared to the respective mono treatments in 5/8 tested lymphoma cell lines (Daudi, SU-DHL-2, SU-DHL-6, TMD8, OCI-LY10). Moreover, TAK-981 plus tafasitamab enhanced degranulation of NK cells and cytokine release compared to mono treatments. In ADCP assays, combination of TAK-981 and tafasitamab resulted in increased phagocytosis rates in comparison to mono treatments in 2/2 tested cell lines (Daudi, Ramos). Cell viability analysis revealed a combination benefit by increased direct cytotoxic effects against SU-DHL-6 cells. Finally, TAK-981 and tafasitamab were investigated in Daudi and WSU-DLCL2 xenograft models with 3 weeks of dosing. In the Daudi model, the combination treatments of TAK-981 with 10 or 20 mg/kg tafasitamab performed better than either treatment alone, and in the WSU-DLCL2 model, the combination treatments of TAK-981 with 3, 10 or 20 mg/kg tafasitamab performed better than the single agent treatments.ConclusionsThe combination of TAK-981 with tafasitamab significantly enhanced anti-tumor effects compared to the respective monotherapies in vitro and in vivo. These preclinical data support a clinical evaluation of this drug combination in patients with lymphoma including aggressive subtypes such as Burkitt lymphoma and DLBCL.The study was funded by MorphoSys AG and Takeda Development Center Americas, Inc. DisclosuresPatra-Kneuer: MorphoSys AG: Current Employment. Nakamura: Takeda Development Center Americas, Inc.: Current Employment. Song: Takeda Pharmaceuticals International Co.: Current Employment. Grossman: Takeda Development Center, Cambridge MA: Current Employment. Polzer: MorphoSys: Current Employment. Ginzel: MorphoSys: Current Employment. Steidl: MorphoSys AG: Current Employment. Berger: Takeda Development Center Americas, Inc.: Current Employment. Proscurshim: Takeda Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company. Heitmüller: MorphoSys AG: Current Employment.

Optimisation and validation of a new method for antibody dependent cellular phagocytosis in hepatitis C virus infection

Lack of a simple, high throughput antibody-dependent cellular phagocytosis (ADCP) assay has limited our understanding of its potential role of in hepatitis C (HCV) infection. Here, we optimised a flow-cytometry based ADCP assay using HCV envelope (E2)-protein coated microbeads that were opsonised with anti-E2 monoclonal IgG antibody (αE2 mAb) and the THP-1 monocyte cell line as effector cells. We found 1.5 × 109/ml microbeads opsonised with 5 μg/ml αE2 mAb and 1.6 × 106/ml THP-1 cells were optimal conditions to distinguish between healthy controls and patients with HCV. This optimised assay was then used to investigate ADCP in plasma obtained from 72 patients with chronic HCV infection and 15 healthy controls. We found that 75% of patients with genotype 1 and 87% of patients with genotype 3 HCV infection had significantly higher levels of ADCP compared to healthy controls. In patients, there was a significant correlation between increase in ADCP and higher concentrations of anti-E2 IgG antibodies in the plasma. Taken together, we established a simple, quick and high throughput ADCP assay for HCV infection that can readily be used for screening of large cohorts of patients and investigation of the role of ADCP in the pathogenesis or protection from this disease.

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p<0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

The Combination of Tafasitamab and Rituximab Increases Cytotoxicity Against Lymphoma Cells In Vitro

Introduction Tafasitamab (MOR208) is a CD19-targeting, Fc effector function-enhanced antibody that has shown promising clinical activity in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). For patients newly-diagnosed with DLBCL, R-CHOP regimens combining the anti-CD20 antibody, rituximab (RTX) with chemotherapy is the standard of care; however, 30-40% of patients experience relapse with a high mortality rate. CD20 is not as broadly expressed as CD19 on B-cell lymphomas, and CD19 expression is preserved on CD20-negative tumor sub-populations and following CD20-targeting therapy (Horna, et al. EHA 2020). These findings support the evaluation of immunotherapies combining anti-CD19 and anti-CD20 antibodies to target all malignant lymphoma cells. To further explore the rationale for adding tafasitamab to a RTX-containing regimen, we analyzed the effect of this combination on antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and tumor B-cell viability in vitro. Additionally, protein expression of the oncogenic transcription factor c-Myc was investigated to gain deeper insight into the molecular effects mediated by RTX and tafasitamab. Methods A comprehensive panel of 11 aggressive lymphoma cell lines was analyzed (9 DLBCL and 2 Burkitt's lymphoma). For the ADCC assays, tumor cells were incubated with tafasitamab and/or RTX at saturating concentrations in the presence of natural killer cells from healthy human donors at effector-to-target (E:T) ratios of 0.5:1 to 3:1 for 2 hours. Cytotoxicity was assessed by quantification of DAPI-positive, CFSE-labeled target cells. For the ADCP assays, lymphoma cells were incubated with tafasitamab and/or RTX at saturating concentrations in the presence of in vitro differentiated macrophages derived from healthy human donors at an E:T ratio of 2:1 for 3 hours. Phagocytosis was assessed by quantification of double-positive events (CFSE-labeled macrophages and CellTrace Violet-stained target cells). C-Myc protein levels were measured following the treatment of tumor cells with tafasitamab and/or RTX for 17-48 hours. CD19/CD20 surface expression, ADCC and ADCP rates, as well as intracellular c-Myc protein levels were analyzed by flow cytometry. Direct effects of antibody treatment on cell viability were assessed by the determination of ATP levels upon incubation of lymphoma cells for 24-96 hours. Results Increased ADCC activity for the combination of tafasitamab and RTX compared with the respective monotherapies was observed in 4/11 cell lines. For the remaining cell lines, the combination activity was similar to the most active monotherapy (tafasitamab: 5/11, RTX: 2/11). Furthermore, in 5/11 cell lines, the combination of tafasitamab and RTX resulted in enhanced ADCP activity compared with the monotherapies. Similar to ADCC, the combination activity in the other cell lines was comparable to the most active monotherapy (tafasitamab: 2/11, RTX: 4/11). Interestingly, a slight correlation between ADCC/ADCP activity and CD19/CD20 surface expression levels was observed. Treatment of tumor cells with the respective antibodies without effector cells showed a beneficial combinatorial effect of tafasitamab with RTX on the reduction of cell viability in 3/8 cell lines, whereas 2 cell lines were primarily sensitive to tafasitamab and 3 were sensitive to RTX. This observed difference in the activity profile of tafasitamab versus RTX suggests different mechanisms for induction of direct cytotoxicity (Figure 1). To further elucidate the molecular basis of the observed direct effects on cell proliferation, expression levels of c-Myc were assessed. A correlation between decreased cell viability and reduction of c-Myc expression was demonstrated. This finding is in line with the role of c-Myc in the regulation of cellular processes, such as proliferation and apoptosis. Conclusions The co-treatment of tafasitamab with RTX demonstrated superiority compared with the respective monotherapies for at least one and up to all three of the modes of action tested (ADCC, ADCP, or direct impact on cell viability) in 9/11 lymphoma cell lines. These data demonstrate that the combination of tafasitamab and RTX mediates increased tumor cell death in vitro via different mechanisms of action and support the clinical evaluation of this antibody combination in patients with DLBqCL. Disclosures Patra: MorphoSys AG: Current Employment. Augsberger:MorphoSys AG: Current Employment. Ginzel:MorphoSys AG: Current Employment. Polzer:MorphoSys AG: Current Employment. Landgraf:MorphoSys AG: Current Employment. Bartel:MorphoSys AG: Current Employment. Ness:MorphoSys AG: Current Employment. Steidl:MorphoSys AG: Current Employment. Heitmüller:MorphoSys AG: Current Employment. Schanzer:MorphoSys AG: Current Employment. Endell:Morphosys: Current Employment.

Abstract 2199: Improvement of ADCP response by re-education of tumor-associated macrophages with Resiquimod-encapsulated liposomes

Abstract Background: Tumor-associated macrophages (TAMs) are the major part of the tumor microenvironment 1, which are classified as two groups - the tumor-suppressing M1 type and the tumor-supporting M2 type 2. Thus, TAMs could act as a double-edged sword. Reprogramming TAMs from M2 to M1 type would improve the efficacy of anti-tumor therapies 3. TAMs may influence the efficacy of therapeutic anti-tumor antibodies. One of the modes of action of therapeutic antibodies is an antibody-dependent cellular phagocytosis (ADCP) 1. However, it is still controversial whether the status of TAMs influences ADCP response 4,5. Resiquimod (R848) is an agonist for Toll-like receptor 7 and 8 (TLR7/8) that can reprogram TAMs from M2 to M1 6. In this study, we evaluated whether R848-induced M1 type TAMs could enhance ADCP response. Therefore, we encapsulated R848 into liposomes, intending to achieve TAM-specific R848 delivery, TAMs re-education, ADCP improvement, and enhancement of therapeutic efficacy of antibodies. Methods: For ADCP assay, mouse bone marrow derived macrophages (BMDM) were differentiated into M1 and M2 as TAM models. Phagocytosis of EGFP-expressing cancer cells (Raji, A431, and SKBR3) by TAMs was evaluated by FACS. For TAM re-education, M2 TAMs were incubated with R848. Expression level of macrophage markers (CD80 and CD206) was measured by FACS. Biodistribution of liposomal R848 (R848-LPs) in WiDr cell-bearing nude mice was evaluated by in vivo imaging and FACS. In vivo TAM re-education ability, ADCP enhancement ability, and tumor growth inhibition ability of R848-LPs combined with therapeutic antibody were investigated in WiDr cell-bearing nude mice. Results: In vitro ADCP assay showed that M1 macrophages elicited the more efficient ADCP response than M2 macrophages towards Raji, A431, and SKBR3 using Rituximab (α-CD20 antibody), α-EGFR antibody (528), and Trastuzumab (α-HER2 antibody), respectively. Upon treatment with R848-LPs, macrophages showed an M1 polarization and an enhanced ADCP response in vitro. In vivo imaging and FACS analysis showed that R848-LPs rapidly accumulated in TAMs at tumor site. R848-LP treatment efficiently enhanced the ADCP response towards WiDr using α-EGFR antibody (528). By the treatment of R848-LPs, a significant increased ratio of CD80/CD206 was observed. WiDr-bearing nude mice treated with R848-LP combined with α-EGFR antibody (528) suppressed tumor growth compared with control groups (non-treatment and α-EGFR antibody without R848-LPs). Conclusions: Liposomal R848 is a potential TAM re-education agent, which could significantly enhance the therapeutic effect of antibodies by improving ADCP. Keywords: tumor-associated macrophage; therapeutic antibodies; antibody-dependent cellular phagocytosis; Resiquimod; liposomes

Anti-CD20 Therapy Reliance on Antibody-Dependent Cellular Phagocytosis Affects Combination Drug Choice

The efficacy of many therapeutic unconjugated monoclonal antibodies (mAbs), including those targeting CD20 in CLL, requires immune cell-mediated cytotoxicity. mAbs have often been optimized for natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC) activity. However, in vivo mouse studies have shown that antibody-dependent cellular phagocytosis (ADCP) by macrophages is the major mechanism of clearance of circulating B cells by anti-CD20 mAbs. To directly compare ADCC versus ADCP, we previously used a panel of anti-CD20 mAbs (rituximab, ofatumumab, obinutuzimab, ocaratuzumab) to test cytotoxicity of paired human NK cells and monocyte-derived macrophages (hMDM) against CLL cells in vitro. All mAbs demonstrated ADCP activity at least 10-fold greater than ADCC as measured by CLL cell depletion per effector cell. Moreover, ADCC and ADCP activity levels did not correlate, meaning that ADCC cannot be used as a surrogate measure of ADCP for these mAb. This could explain why mAb optimization for ADCC activity has often failed to translate into more efficacious treatment. Thus, ADCP may be an effective translational measurement of anti-CD20 mAb performance. Because of the clinical interest in combining anti-CD20 mAb with targeted small molecule inhibitors, we began studying the effects of the Bruton tyrosine kinase (BTK) inhibitors on anti-CD20 mAb-mediated ADCP. Our initial studies showed that ibrutinib (IBR), but not acalabrutinib (Acala), significantly decreased anti-CD20 ADCP as measured by a flow cytometry-based assay that measures single timepoint cell collections. These types of assays cannot easily determine the kinetics and individual effector cell activity of ADCP. Thus, to more fully study the BTK inhibitor effects on ADCP, we developed a live cell time-lapse imaging method for measuring ADCP, utilizing recent advances in microscopy, cellular dye labeling, digital imaging, imaging software and computing. Whole-cell labeling of macrophages enabled visualization of internalized CLL cells as regions of dye exclusion or "voids". Because of the vast number of images acquired during live cell time-lapse imaging, we utilized computer software-aided image recognition and enumeration to measure the number of macrophages and voids inside each macrophage. As a measure of phagocytic engulfment, we developed a void index, which provides a relative measure of phagocytic engulfment per macrophage. Measuring ADCP in this manner replicates clinical observation of mAb therapeutic activity. Clinically, intravenous anti-CD20 mAb therapy typically induces a rapid decrease in circulating CLL cells (within hours), followed by a long period (days) of stable to increased levels of circulating cells. Similarly, our live cell time-lapse video assay shows initial rapid ADCP over the first 2 hours followed by a prolonged period of "hypophagia" with little ADCP for the remainder of the assay (imaged every 2 minutes for 8 hours). This "hypophagia" phenomenon may explain the resistance to therapeutic mAb observed clinically. With these new tools for quantitation of ADCP, we compared the effects of serial dilutions of IBR or Acala on ADCP. Overall, as measured by Area Under the Curve (AUC) analysis, IBR decreased phagocytic capacity of anti-CD20 mediated CLL cell ADCP at concentrations of 0.41 μM and above. By contrast, Acala did not begin to decrease AUC measurements until 3.7 μM, and subsequent AUC values were higher in Acala versus IBR-treated ADCP assays up to the highest tested drug concentration (100 μM). Similarly, the initial ADCP kinetics (void index / min over the first hour) reflected a decrease with IBR treatment at 0.41 μM that continued until a nadir was reached at 33 μM. In contrast, Acala did not induce a decrease in this kinetic measurement until 3.7 μM and a nadir was not reached (up to 100 μM). Thus, IBR significantly decreases ADCP by hMDM at concentrations much lower than a more specific BTK inhibitor, Acala. This result suggests that BTK inhibition has little to no effect on ADCP and furthermore suggests that IBR off-target effects decrease ADCP. IBR off-target candidates include other tyrosine kinases in the TEC (tyrosine kinase expressed in hepatocellular carcinoma) family. These data suggest that a a highly selective BTK inhibitor with little effect on ADCP could be a more suitable drug to combine with therapeutic mAb(s). Disclosures Chu: Pfizer: Equity Ownership; Acerta Pharma: Research Funding. VanDerMeid:AstraZeneca: Research Funding. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents; AstraZeneca: Equity Ownership. Munugalavadla:Acerta Pharma: Employment; AstraZeneca, Gilead Sciences: Equity Ownership. Barr:TG Therapeutics: Consultancy, Research Funding; Celgene: Consultancy; Pharmacyclics LLC, an AbbVie company: Consultancy, Research Funding; Seattle Genetics: Consultancy; Merck: Consultancy; Genentech: Consultancy; Verastem: Consultancy; Gilead: Consultancy; Astra Zeneca: Consultancy, Research Funding; Janssen: Consultancy; AbbVie: Consultancy. Elliott:Astra Zeneca: Research Funding. Zent:Mentrik Biotech: Research Funding; Astra Zeneca: Research Funding.

A SUPPORT VECTOR-BASED PREDICTIVE MODEL TO REVEAL THE RELATIONSHIPS AMONG ANTIBODY FEATURES AND THEIR EFFECTIVE FUNCTIONS AGAINST HIV

Despite 4 decades’ effort, an effective HIV-1 vaccine has not been produced owing to the inevitable antigenic diversity of the virus and millions of people around the world have lost their lives due to HIV. Increasing the knowledge of adaptive immune response to vaccination would ultimately lead to an effective HIV cure. Antibodies, which are responsible for protection and fighting against antigens, are vital parts of immune system response. In order to identify discriminative antibodies, which provide protection against HIV, and to disclose the associations between antibody features and their functional outcomes, computational methods, such as feature selection, regression and classification can be used to construct predictive models. Here we used our unsupervised K-Means Based Feature Selection (KBFS) method which is presented in our previous study, to identify functional antibodies that fight against HIV. The accuracy results for the proposed KBFS framework are compared with those presented in a recent study and are also compared with results from four different state-of-the-art unsupervised feature selection methods, namely MCFS, InFS, LapFS, and SPFS, along with the entire feature set. Then, support vector based systems are utilised to predict the associations between antibody features and their functional activities, namely gp120-specific antibody dependent cellular phagocytosis (ADCP), antibody dependent cellular cytotoxicity (ADCC) and cytokine release activities on RV144 vaccine recipients. Pearson Correlation Coefficient (PCC) metric is used to evaluate the prediction accuracy of the predictive models and to be consistent with the previous study. Our SVR based KBFS framework presented higher accuracy than the original study by improving prediction performance 16% for ADCP assay, 200% for the ADCC assay.

CD47 limits antibody dependent phagocytosis against non-malignant B cells.

Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases.

CD47 Blockade Enhances Therapeutic Activity of TCR Mimic Antibodies to Ultra-Low Density Cancer Epitopes through Cytokine Feed Forward Mechanisms

INTRODUCTIONT Cell Receptor mimic (TCRm) antibodies to low-density peptide epitopes from undruggable intracellular proteins presented in the context of major histocompatibility (MHC) molecules are therapeutically effective in mouse models of human cancers. CD47 blockade by use of a high affinity SIRPα variant protein (CV1) has been shown to improve the effects of monoclonal antibodies to high-density antigens in tumor models by enhancement of antibody dependent cellular phagocytosis (ADCP). We asked if combination therapy with a TCRm antibody to Preferentially Expressed Antigen in Melanoma (PRAME) could enhance activity of both drugs in vitro and in vivo. Additionally, we explored the role of macrophage-secreted cytokines in the enhanced in vivo activity.METHODSWe performed in vitro ADCP assays with human acute myeloid and acute lymphoid leukemia cell lines containing antigens of interest using the two agents alone and in combination. We performed therapy experiments in NSG mice using the same leukemia cell lines transformed with a luciferase vector and measured tumor burden through bioluminescent imaging. Survival was measured. We examined cell-surface expression of epitopes of interest and HLA on cell lines in vitro after incubation with IFNγ and TNFα using flow cytometry and performed in vitro ADCP assays with the leukemia cell lines after pretreatment with IFNγ.RESULTSCV1 and TCRm antibody showed additive effects in vitro with a statistically significant increase in phagocytosis in both antigen positive cell lines with combination therapy versus single agent therapy. CV1 and TCRm antibody showed greater than additive therapeutic effects in vivo with a 3-log reduction in leukemia burden relative to control untreated mice and a 5-10 fold reduction relative to single agent groups. After therapy was stopped, mice treated with the combination had statistically significant increases in survival (p<0.0001). IFNγ and TNFα led to up-regulation of cell surface HLA-A*02:01. Additionally, the cytokines led to up-regulation of the PRAME derived epitope of interest. Pretreatment of human leukemia cell lines with IFNγ led to statistically significant increases in ADCP in vitro.CONCLUSIONSThe elimination of anti-phagocytic signal produced by CD47 blockade with the high affinity SIRPα variant CV1, combined with the pro-phagocytic signal of Fc receptor engaging TCRm was effective even with an ultra-low density epitope (700-3000 sites per cell) in vitro and in vivo. A greater than additive effect was seen in two tumor models. These results support the potency of this drug combination. The large enhancement in activity in vivo vs. in vitro may be explained by macrophage-released cytokines leading to increased presentation of epitopes of interest and increased tumor kill. DisclosuresLiu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Garcia:Alexo Therapeutics: Equity Ownership.

Macrophages are required for anti-CD19 mAb mediated B cell depletion in mice (50.16)

Abstract Anti-CD20 mediated B cell depletion is an effective treatment for rheumatoid arthritis. An anti-CD19 mAb is under investigation as a new B cell depleting reagent. Studies have demonstrated that the interaction between the mAb Fc and Fc? receptors on effector cells is important for efficient target cell depletion. To enhance effector function, the anti-CD19 mAb was engineered with BioWa's Potelligent® technology to remove fucose from the Fc portion. The fucose-free mAb has increased affinity to human FCγRIIIa and mouse FCγRIV as well as improved cytotoxicity. The fucose-free anti-CD19 antibody (anti-CD19-aFuc) depleted &amp;gt;90% of murine B cells in human CD19 Transgenic mice. Further studies were carried out to investigate the mechanisms of action of anti-CD19-aFuc. B cell depletion was not affected in mice which were treated with to deplete NK cells or neutrophils. Similarly, depletion of B cells was not impaired in mice in which complement activation was blocked. In contrast, B cell depletion by anti-CD19-aFuc was significantly inhibited in mice in which macrophages were eliminated. Furthermore, anti-CD19-aFuc showed robust activity in an antibody dependent cellular phagocytosis assay with freshly isolate mouse macrophages as effector cells. These results suggest that anti-CD19-aFuc depletes B cells in mice through a macrophage dependent mechanism. This study is funded by MedImmune, LLC.

Augmentation of antitumour activity of defucosylated chimeric anti‐CCR4 monoclonal antibody in SCID mouse model of adult T‐cell leukaemia/lymphoma using G‐CSF

Augmentation of antitumour activity of defucosylated chimeric anti‐CCR4 monoclonal antibody in SCID mouse model of adult T‐cell leukaemia/lymphoma using G‐CSF