Antibody-dependent Cell-mediated Cytotoxicity Activity Research Articles

Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Head Region of Pandemic H1N1 Influenza Virus Play Detrimental Roles in H1N1-Infected Mice.

Engaging the antibody-dependent cell-mediated cytotoxicity (ADCC) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. However, investigation of the ADCC epitopes on the highly immunogenic influenza hemagglutinin (HA) head region has been rarely reported. In this study, we determined the ADCC and antiviral activities of two putative ADCC epitopes, designated E1 and E2, on the HA head of a pandemic H1N1 influenza virus in vitro and in a lethal mouse model. Our data demonstrated that sera from the E1-vaccinated mice could induce high ADCC activities. Importantly, the induction of ADCC response modestly decreased viral load in the lungs of H1N1-infected mice. However, the elevated ADCC significantly increased mouse alveolar damage and mortality than that of the PBS-vaccinated group (P < 0.0001). The phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by ADCC, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of E1-vaccinated mice after H1N1 influenza virus challenge. Overall, our data suggested that ADCC elicited by certain domains of HA head region might have a detrimental rather than protective effect during influenza virus infection. Thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of ADCC.

Analysis of hybridoma and CHO-derived pritumumab for therapeutic applications.

71 Background: Pritumumab, a natural huma IgG1 kappa antibody was obtained from a regional draining lymph node of a patient with cervical carcinoma through traditional hybridoma technology. Methods: Both cell lines and tissues were processed and analyzed for FACS analysis, Western blots, and immunohistochemistry. Results: Specificity analysis of the target antigen, an altered form of vimentin (ecto-domain vimentin (EDV)) shows it to be limited to cell surface expression on cancer cells. Clinically, 249 Japanese brain cancer patients were treated with a low dose pritumumab regimen, either at 1mg once a week or 1mg twice a week, and overall response rates of between 25-30% were seen with several complete and partial responses. A recombinant version of the mAb was made using the GPEx system in CHO cells. In a series of comparable studies the CHO mAb was compared with the original hybridoma. Binding specificity with both flow cytometry and immunohistochemical (IHC) analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activity confirms the comparability of the two versions. IHC analysis suggests restricted binding to tumor cells and not normal cells and tissues. Further studies include establishing a xenograft model with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Conclusions: Overall, these data together suggest pritumumab is suitable for development as an anti-tumor therapeutic.

An “Fc-Silenced” IgG1 Format With Extended Half-Life Designed for Improved Stability

Multiple mutation combinations in the IgG Fc have been characterized to tailor immune effector function or IgG serum persistence to fit desired biological outcomes for monoclonal antibody therapeutics. An unintended consequence of introducing mutations in the Fc (particularly the CH2 domain) can be a reduction in biophysical stability which can correlate with increased aggregation propensity, poor manufacturability, and lower solubility. Herein, we characterize the changes in IgG conformational and colloidal stability when 2 sets of CH2 mutations “TM” (L234F/L235E/P331S) and “YTE” (M252Y/S254T/T256E) are combined to generate an antibody format lacking immune receptor binding and exhibiting extended half-life. In addition to significantly lowered thermostability, we observe greater conformational flexibility for TM-YTE in CH2, increased self-association, and poorer solubility and aggregation profiles. To improve these properties, we dissected the contributions of individual mutations within TM-YTE on thermostability and substituted destabilizing mutations with new mutations that raise thermostability. One novel combination, FQQ-YTE (L234F/L235Q/K322Q/M252Y/S254T/T256E), had significantly improved conformational and colloidal stability, and was found to retain the same biological activities as TM-YTE (extended half-life and lack of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activity). Our engineering approach offers a way to improve the developability of antibodies containing Fc mutations while retaining tailored biological activity.

An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity

Background: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. Methods and Results: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. Conclusion: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

P1.07-033 Trastuzumab Emtansine (T-DM1) Suppresses the Growth of HER2-Positive Small-Cell Lung Cancer in Preclinical Models

To overcome chemoresistance is indispensable to bring about better prognosis in small-cell lung cancer (SCLC). We have reported that HER2 is upregulated when HER2-positive SCLC cells acquire chemoresistance. Moreover, HER2-upregulated cisplatin- or etoposide-resistant SCLC cells were sensitive to trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). However, irinotecan-resistant SCLC cells, e.g. SBC-3/SN-38, were refractory to trastuzumab despite high HER2 expression. To address this issue, we studied the antitumor efficacy of trastuzumab emtansine (T-DM1) on trastuzumab-resistant HER2-positive SCLC. SBC-3/SN-38 (HER2-positive) or OS2RA (HER2-negative) SCLC cells were inoculated subcutaneously in the flank of six-week-old male nude mice. Tumor size was measured with a caliper two or three times per week. When tumor volume reached about 150-200 mm3, mice were randomly assigned to three treatment groups. Each group was treated with intravenous injection of T-DM1 15 mg/kg, intraperitoneal injection of trastuzumab 30 mg/kg, or saline as control. To confirm the HER2-specific delivery of T-DM1, in vivo imaging was performed. Namely, several tumor-bearing mice were intravenously administered with fluorescence-labeled T-DM1. Fluorescence images of mice were captured with IVIS® Lumina II system (PerkinElmer). Treatment with T-DM1 significantly suppressed the growth of SBC-3/SN-38 xenografts compared with trastuzumab and saline groups. Histological analysis revealed that T-DM1 remarkably induced apoptosis and inhibited proliferation. Fluorescence-labeled T-DM1 definitely accumulated to the xenografts in a HER2-selective fashion. T-DM1 treatment could be an attractive therapeutic option in trastuzumab-resistant HER2-positive SCLC where trastuzumab cannot induce enough ADCC activity. Delivery of a cytotoxic agent DM1 to the inside of cells via HER2-mediated internalization is expected and crucial to exert antitumor effect in such ADCC-lacking SCLC cells.

Characterization of FcγRIIIA effector cells used in in vitro ADCC bioassay: Comparison of primary NK cells with engineered NK-92 and Jurkat T cells

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) of several therapeutic antibody drugs and evaluation in ADCC bioassays is important in antibody drug development and maintenance. Three types of effector cells now routinely used in bioassay evaluation of ADCC are natural killer cells from human donors (FcγRIIIA+primary NK), FcγRIIIA engineered NK-92 cells and FcγRIIIA/NFAT-RE/luc2 engineered Jurkat T cells. Engineered effector cells were developed to address need for improved precision and accuracy of classic NK cell ADCC bioassays. The main purpose of our study was to rationalize which of these ADCC effector cells best simulate the expected response in human subjects and to identify which effector cells and assays best fit ADCC bioassay needs during antibody drug development. We characterized differences between the effector cells and compared ADCC biological activities using the well-known humanized IgG1 antibody drug, trastuzumab. The three effector cell types studied expressed either V-158 or F-158 allotype of FcγRIIIA, hence six cell preparations were compared. Our results demonstrate highest surface expression of FcγRIIIA in primary NK and engineered NK-92 (V-158) cells with nearly all expressed on the cell surface. In contrast, expression in engineered Jurkat T cells was low with only a small percentage expressed on the cell surface. Studies evaluating binding of trastuzumab to effector cells demonstrated the highest affinity of FcγRIIIA in primary NK and NK-92 (V-158) cells. ADCC cytotoxicity studies showed greatest trastuzumab potency in primary NK and engineered NK-92 (V-158) cells and negligible cell lysis obtained using engineered Jurkat T cells. In contrast, the engineered Jurkat T (V-158) cells responded as effectively as primary NK (V/V) cells to nuclear factor of activated T cells (NFAT2) activation upon binding of trastuzumab to FcγRIIIA, demonstrating similar ADCC pathway activation in these cells despite the low surface expression of FcγRIIIA and its low affinity for trastuzumab. Dose-response range of trastuzumab in activation of NFAT2 (measured as pNFAT2 dephosphorylation) was very similar to response in classic ADCC assay for primary and NK-92 cells and to response in ADCC reporter assay for Jurkat T effector cells, bridging the assays. Trastuzumab potency in ADCC reporter assay using the engineered Jurkat T cells was close to that seen using either primary NK or engineered NK-92 cells in classic ADCC assay. In summary, all three effector cell systems differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, yet only primary NK and engineered NK-92 cells are capable of inducing ADCC-mediated cell lysis. Engineered Jurkat T effector cells have value to assure antibody manufacturing consistency and in other applications where accuracy and precision are important. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells better reflect the physiologically relevant ADCC mechanism of action. As an engineered cell line, NK-92 cells may behave more reproducibly than primary NK, but this must be balanced with the objective for biological relevance in decisions on which NK cells to use in assay.

A Phase 1B Study of CC-122 in Combination with Obinutuzumab (GA101) in Relapsed or Refractory Diffuse Large B-Cell Lymphoma and Indolent Non-Hodgkin Lymphoma

A Phase 1B Study of CC-122 in Combination with Obinutuzumab (GA101) in Relapsed or Refractory Diffuse Large B-Cell Lymphoma and Indolent Non-Hodgkin Lymphoma

Engineering Human Pluripotent Stem Cell-Derived Natural Killer Cells to Prevent CD16a Shedding for Enhanced Anti-Tumor Killing

Natural killer (NK) cells are lymphocytes able to recognize and lyse transformed cells without HLA-restriction and without prior sensitization, mediated through a repertoire of cell surface receptors. Solid tumors are typically more refractory to NK cell-mediated killing compared to hematologic malignancies. To improve NK cell-mediated killing of solid tumors, we have engineered NK cells to express a more stable form of CD16a. The IgG Fc receptor CD16a represents one of the most potent activators of NK cell anti-tumor activity. This receptor binds antibodies attached to target cells to initiate antibody-dependent cell-mediated cytotoxicity (ADCC). Importantly, CD16a undergoes a rapid down-regulation in expression by ADAM17 (A Disintegrin And Metalloprotease-17) mediated proteolytic cleavage upon cell activation. This loss of CD16a expression leads to reduced ADCC activity and decreased anti-tumor killing. Previous studies by our group identified cleavage sites in CD16a that are targeted by ADAM17 activity. Substitution of the serine at position 197 for a proline (S197P) in the middle of the cleavage region effectively blocks ADAM17-mediated CD16a cleavage. To evaluate the ability of this cleavage-resistant CD16a(S197P) to improve ADCC, we have used a system previously developed by our group to efficiently produce NK cells from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). These hESC/iPSC-derived NK cells have a phenotype and function similar to NK cells isolated from peripheral blood (PB-NK cells). However, unlike PB-NK cells, it is relatively easy to genetically-modify hESC/iPSC-derived NK cells by engineering at the stem cell level. Here, we use two strategies to generate iPSC-derived NK cells with stabilized CD16a expression. First, we used the Sleeping Beautytransposon system to stably express the cleavage-resistant CD16a(S197P) in iPSCs. Second, we have used the CRISPR/Cas9 system to delete the ADAM17 gene. This strategy allows us to compare specifically engineering stabilized CD16a expression to the broader effect of ADAM17-deletion which cleaves multiple cell-surface proteins.NK cells derived from the CD16a(S197P) iPSCs are resistant to CD16a shedding upon activation with IL-12 and IL-18 as 97.5% of iPSC-NK cells are CD16+ before stimulation and 95.2% of cells are CD16+ after stimulation. In contrast, PB-NK cells demonstrate a decrease from 85% CD16+ cells before stimulation to 67% CD16+ cells after stimulation. Next, we demonstrate improved ADCC and NK cell degranulation (measured by CD107a expression) in this engineered iPSC-derived NK cell compared to non-engineered cells upon addition of the anti-Her2 antibody (ab) Herceptin to ovarian cancer cells with differing levels of Her2 expression. Specifically, CD16a(S197P) iPSC-NK cells stimulated with the Her2-high SKOV3 ovarian carcinoma cells were 26.7% CD107a+ cells with addition of anti-Her2 and 10.8% CD107a+ cells without ab. In contrast, unmodified iPSC-NK cells were 9.6% vs. 6.9% CD107a+ with and without anti-Her2; and PB-NK cells were 12.4% vs. 9.4% CD107a+ with and without anti-Her2. However, when co-cultured with the Her2-low MA-148 ovarian carcinoma none of the three NK cell populations demonstrated increased CD107a+ cells with anti-Her2 treatment compared to no ab controls. Similar results were obtained when testing the ADCC of these cells using the Cr-51 release cytotoxicity assay. Killing of SKOV3 cells by CD16a(S197P) iPSC-NK cells was markedly increased upon addition of anti-Her2 ab from 32.2% cytoxicity with ab vs. 11% cytotoxicity without ab. In contrast, unmodified iPSC-NK cells had less killing of the SKOV3 cells (8.1% vs. 3.7% with or without anti-Her2 ab) and PB-NK cell killing of SKOV3 cells was 20.5% vs. 9.2% with or without anti-Her2 ab. Currently, we are comparing anti-tumor activity of the CD16a(S197P) iPSC-NK cells to the ADAM17-deleted iPSC-NK cells, both as resting NK cells and IL12+IL18-stimulated NK cells. Overall, this strategy will allow us to improve the ability of readily available anti-tumor antibodies to improve NK cell-mediated killing of solid tumors and use engineered iPSC-derived NK cells as an "off-the-shelf" approach for allogeneic cell-based immunotherapies. DisclosuresKaufman:Fate Therapeutics: Consultancy.

Herpes Simplex Virus (HSV)-2 Candidate Vaccine Virus Deleted in Glycoprotein D (ΔgD-2) Elicits High-Titer Immunoglobulin (Ig)G2 Antibodies With Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activity, Protects Mice From Skin and Vaginal Challenge With Clinical Isolates of HSV-1 and HSV-2, and Prevents the Establishment of Latency

Herpes Simplex Virus (HSV)-2 Candidate Vaccine Virus Deleted in Glycoprotein D (ΔgD-2) Elicits High-Titer Immunoglobulin (Ig)G2 Antibodies With Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activity, Protects Mice From Skin and Vaginal Challenge With Clinical Isolates of HSV-1 and HSV-2, and Prevents the Establishment of Latency

975P - Individual ADCC capability predict treatment outcome under cetuximab-based therapy in head and neck cancer?

Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for the treatment of local advanced-head and neck squamous cell carcinoma (LA-HNSCC). A working mechanism of cetuximab include the ability to develop antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell, mainly on Natural Killer (NK) cells. Invariant NKT (iNKT) cells play an important role in the activation of immune cell subset, including NK cells, and their deficiency is related to poor clinical outcome in LA-HNSCC. The aim of this work was to investigate the predictive role of ADCC and iNKT in LA-HNSCC patients treated with cetuximab-based therapy. Twenty-eight LA-HNSCC patients treated with curative intent with cetuximab-based radio-chemotherapy were analysed. Peripheral blood samples were collected at start of therapy. ADCC was evaluated as ex vivo NK-dependent activity measuring LDH release as previously standardized in our laboratory. EGFR tumoral expression was analyzed by immunohistochemistry. ADCC activity above the median value (high) correlated with a better OS as compared to ADCC below the median (low) in LA-HNSCC (p = 0.033). We observed a correlation also between high ADCC and response rate (p = 0.038). By univariate analysis on OS there were ADCC activity tumor size and HPV status as significant pronostic indicators. According to multivariate log-rank analysis only ADCC remained a significant predictor for OS (p = 0.03). If we stratified the EGFR 3+ patients into high and low ADCC performers, we observed an impressive increased OS in those with high ADCC (p = 0.024). iNKT cells number didn't significantly correlate with OS: nonetheless, we observed a trend to a longer survival after 10 months in patients with high iNKT cells (above the median values). ADCC has a strong predictive value of cetuximab response in HNSCC. High tumoral EGFR expression and high ADCC confer a particularly long overall survival and suggest to combine this two biomarkers in order to predict clinical outcome in cetuximab-treated HNSCC patients.

L02 - Individual ADCC capability predict treatment outcome under cetuximab-based therapy in head and neck cancer?

Introduction: Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for the treatment of local advanced-head and neck squamous cell carcinoma (LA-HNSCC). The working mechanism of cetuximab include the ability to develop antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell, mainly on Natural Killer (NK) cells. Invariant NKT (iNKT) cells play an important role in the activation of immune cell subset, including NK cells, and their deficiency is related to poor clinical outcome in LA-HNSCC. The aim of this work was to investigate the predictive role of ADCC and iNKT in LA-HNSCC patients treated with cetuximab-based therapy. Patients and methods: Twenty-eight LA-HNSCC patients treated with curative intent with cetuximab-based radio-chemotherapy were analysed. Peripheral blood samples were collected at start of therapy. ADCC was evaluated as ex vivo NK-dependent activity measuring LDH release as previously standardized in our laboratory (Monteverde et al., 2015). EGFR tumoral expression was analyzed by immunohistochemistry. Results: ADCC activity above the median value (high) correlated with a better OS as compared to ADCC below the median (low) in LA-HNSCC (p = 0.033). We observed a correlation also between high ADCC and response rate (p = 0.038). By univariate analysis on OS there were ADCC activity, tumor size and HPV status as significant prognostic indicators. According to multivariate log-rank analysis only ADCC remained a significant predictor for OS (p= 0.03). If we stratified the EGFR 3+ patients into high and low ADCC performers, we found that patients characterized by both high EGFR expression and high ADCC activity showed 100% OS at 44.2+ months compared to a median OS of 23.9 months (range 2.1-53.7) for patients with high EGFR but low ADCC (p = 0.024). iNKT cells number didn't significantly correlate with OS: nonetheless, we observed a trend to a longer survival after 10 months in patients with high iNKT cells (above the median values). Conclusion: ADCC has a strong predictive value in cetuximab-treated LA-HNSCC patients. High tumoral EGFR expression and high ADCC confer a particularly long overall survival and suggest to combine this two biomarkers in order to predict clinical outcome in cetuximab-treated LA-HNSCC patients.

Abstract 2357: Therapeutic potential of the natural human IgG1k antibody pritumumab

Abstract Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. A recombinant version of the mAb was made using the GPEx® system in CHO cells. In a series of comparable studies the CHO mAb was compared with the original hybridoma. Binding specificity with both flow cytometry and immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activity confirms the comparability of the two versions. A concentration-dependent study of pritumumab-mediated ADCC suggests an EC50 of 39.6ng/ml. Further studies include establishing a xenograft model with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest pritumumab is suitable for development as an anti-tumor therapeutic. Citation Format: Ivan Babic, Rajesh Mukthavaram, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Therapeutic potential of the natural human IgG1k antibody pritumumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2357.

Abstract CT050: A Phase I/II Study of MCLA-128, a full length IgG1 Bispecific Antibody Targeting HER2 and HER3, in Patients with Solid Tumors

Abstract BACKGROUND: MCLA-128 is a novel humanized full length IgG1 bispecific antibody with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity that targets the transmembrane tyrosine kinase receptors, HER2 and HER3 present on a variety of epithelial tumor cells. In preclinical studies, MCLA-128 demonstrated potent anti-tumor activity. The primary objectives of this First-in-Human (FIH) study are to establish the MTD and Phase 2 recommended dose (RP2D) of MCLA-128 and to assess its safety, tolerability, PK, PD, and anti-tumor activity in selected patient (pt) groups. METHOD: The trial consists of 2 parts. In Part 1 (dose-escalation) patients (pts) with advanced epithelial tumors with no standard management options were enrolled with an initial accelerated 1 pt per cohort design shifting to a 3 + 3 design. The DLT evaluation period was 21 days ( = 1 cycle). MCLA-128 was administered every 3 weeks (q3w) as an intravenous infusion over 60-120 minutes. Toxicities were assessed throughout dose escalation (40 mg up to 900 mg flat dose). Part 2 consists of several expansion cohorts of pts at the RP2D (expansion phase). Here we present interim results for Part 1 of the study. RESULTS: Twenty-eight pts received MCLA-128 in 9 dose-escalation cohorts (40-160 mg in cohorts of 1 evaluable pt, 240-900 mg in cohorts of 3 to 6 evaluable pts). The most frequent drug related adverse events (AEs) observed in Part 1 were mild to moderate (G1-G2) with prompt recovery in general. AEs included infusion-related reactions (IRR), diarrhea, nausea, vomiting, fatigue, maculopapular rash, oral mucositis and G2 neutropenia in 1 pt. IRR occurred in 50% of pts with a trend for increased incidence with dose; modest and transient dose dependent cytokine release was also observed. Increasing the infusion duration to 120 min and premedication with corticosteroids was successfully implemented to mitigate IRRs and cytokine levels were no longer correlative with MCLA-128 dose levels. One drug related serious AE (SAE) has been reported: IRR G3, due to prolonged hospitalization (SUSAR). No DLTs were observed. In serum samples from 12 pts, 1 pt was positive for MCLA-128 anti-drug antibodies 36 days after starting treatment at a dose of 160 mg. A MTD was not reached in the study; PK analysis was used to support a RP2D of 750 mg q3w. Exposure to MCLA-128 increased in proportion to dose. The RP2D was calculated to maintain trough concentrations at the end of each treatment cycle significantly above the Kd of MCLA-128 as determined on HER2 amplified cell lines in vitro and above the Km in a population PK model. Preliminary signs of anti-tumor activity assessed by RECIST are encouraging. Three pts remain on study to date including 1 non-small-cell lung cancer pt who is experiencing an ongoing partial response for more than 10 months, 1 gastroesophageal junction pt and 1 colorectal cancer pt with stable disease for 5 months. In addition, 1 metastatic breast cancer pt received 5 months of MCLA-128 treatment until disease progression. CONCLUSION: MCLA-128 was well-tolerated with a favorable safety profile in pts with advanced tumors treated at doses up to 900 mg q3w. PK results and preliminary signs of anti-tumor activity support the further clinical evaluation of MCLA-128 at the RP2D of 750 mg q3w. The expansion phase of the study is ongoing at this dose level. Citation Format: Emiliano Calvo, Maria Alsina, Jan H.M. Schellens, Alwin DR Huitema, Josep Tabernero, Aurelia de Vries-Schultink, Valentina Boni, Bernard Doger, Cecile Geuijen, Robert Doornbos, Kees Bol, Martine Westendorp, Mark Throsby, Lex Bakker, Setareh Shamsili. A Phase I/II Study of MCLA-128, a full length IgG1 Bispecific Antibody Targeting HER2 and HER3, in Patients with Solid Tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT050.

Abstract 596: Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC)

Abstract XMT-1522 is an anti-HER2 ADC that incorporates HT-19, a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery. Several parameters such as cell binding, internalization rate, cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) downstream signaling, affinity maturation, NHP cross-reactivity, normal human tissue cross-reactivity and in vivo efficacy were used to screen a wide range of antibodies to select a lead candidate optimized for use in ADC applications. In addition HT-19 was selected to be non-competitive for HER2 binding with existing therapies - trastuzumab or pertuzumab, to allow for potential combinability. In vivo efficacy as an ADC did not directly correlate with typical parameters used in antibody screening cascade such as in vitro cytotoxicity, internalization or binding affinity. The lead antibody underwent affinity maturation, and despite increases in affinity, affinity maturation significantly decreased the in vivo efficacy of the ADC in vivo xenograft models. This phenomenon was observed in all the antibody hits. HT-19 showed antibody-dependent cell-mediated cytotoxicity (ADCC) activity. When administered as a single agent in NCI-N87 gastric cancer xenograft model it had biological activity at 20 mg/kg as well as at 3 mg/kg. Consistent with the hypothesis that a non-competitive ADC is combinable with current anti-HER2 regimens, the combination of XMT-1522 with trastuzumab and/or pertuzumab showed more rapid internalization, more complete HER2 degradation, and significantly great anti-tumor activity in the NCI-N87 gastric cancer xenograft model relative to XMT-1522 alone or the combination of pertuzumab + trastuzumab. Citation Format: Natasha Bodyak, Alex Yurkovetskiy, Dmitry R. Gumerov, Dongmei Xiao, Joshua D. Thomas D. Thomas, Laura L. Poling, LiuLiang Qin, Mao Yin, Michael J. DeVit, Peter U. Park, Winnie Lee, Bianka Prinz, Donald A. Bergstrom, Timothy B. Lowinger. Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 596.

Abstract 2302: An ‘off the shelf,’ GMP-grade, IL-2-independent NK cell line expressing the high-affinity Fc-receptor to augment antibody therapeutics

Abstract A number of approved IgG1 monoclonal antibodies (mAb) used in cancer therapy, including trastuzumab, cetuximab, and rituximab, depend on natural killer (NK) cell-mediated antibody dependent cell-mediated cytotoxicity (ADCC) to induce cancer cell death, mediated through the Fcγ receptor (FcγR). Multiple studies report that survival benefit is significantly improved in cancer patients receiving IgG1 antibody therapy who have NK cells that express the high-affinity FcγRIIIa receptor (CD16-158V). Only 12% of the normal human population, however, is homozygous for expression of this high-affinity variant CD16. We hypothesized that infusion of an NK-92 cell line modified to express CD16-158V (high-affinity NK cells [haNK]) in combination with an IgG1 mAb may result in superior antitumor effects compared with IgG1 mAb alone. A GMP-grade plasmid-transfected variant of NK-92 expressing the high-affinity CD16 receptor was developed utilizing a novel transfection vector containing the erIL-2 gene, enabling the resulting haNK cells to grow independently of IL-2. Using limiting dilution, clones having identical surface molecule expression to the parent cell line were generated and shown to have long-term expression of CD16. The haNK cells displayed high ADCC in combination with rituximab, trastuzumab, and daratumumab against target cell lines that were not killed by the parental NK-92 cells. Of note, despite non-viral transfection, the plasmid is fully integrated into chromosome 5. The haNK cell line secretes about 200 pg/106 cells of IL-2, and like the parental NK-92 cell line, produces enough IFNγ to potentially stimulate an adaptive immune response. A GMP-grade master cell bank of haNK cells has been established; haNK cells can be cryopreserved with retention of ADCC activity. In vivo studies demonstrated the efficacy of haNK cells. In a multiple myeloma model of CD38+ NCI-H929 cells xenografted into NOD/SCID gamma null (NSG) mice, treatment with haNK cells plus the anti-CD38 mAb daratumumab led to prolonged survival versus mice treated with haNK cells plus an isotype control (log rank p = 0.0169), demonstrating therapeutically relevant in vivo ADCC. haNK cells have also been transfected with various CARs using mRNA, thereby enabling this platform to make a bispecific effector cell product. In summary, haNK cells are a potent and versatile platform for cellular immunotherapy, and clinical trials of these novel “off the shelf” high-affinity NK cells are scheduled for 2016. Citation Format: Laurent Boissel, Hans Klingemann, Kerry Campbell, Karen Nichols, Frances Toneguzzo, Paula Marcus, Brent Williams, Armand Keating, Patrick Soon-Shiong. An ‘off the shelf,’ GMP-grade, IL-2-independent NK cell line expressing the high-affinity Fc-receptor to augment antibody therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2302.

Abstract 1407: FPA144, a therapeutic monoclonal antibody targeting the FGFR2b receptor, promotes antibody dependent cell-mediated cytotoxicity and stimulates sensitivity to PD-1 in the 4T1 syngeneic tumor model

Abstract Five Prime Therapeutics, Inc. has developed an FGFR2b-specific humanized monoclonal antibody, FPA144, to treat patients with cancer bearing overexpression of the FGFR2b receptor, and is currently in clinical trials as a single agent for gastric cancer (NCT02318329). FGFR2 gene amplification and FGFR2b overexpression occur in approximately 5% of gastric cancers and are associated with a poor prognosis in gastric cancer patients. In addition to blocking ligand binding and inducing FGFR2b internalization, FPA144 is glycoengineered for enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We have shown previously that FPA144 can produce complete and durable tumor growth inhibition in FGFR2b-over-expressing and FGFR2-amplified gastric cancer xenografts in immune-compromised mice (Gemo et al., Poster 5446, AACR 2014). In order to understand the contribution of the immune system to the mechanisms of action of FPA144, we evaluated the anti-tumor effects and immune cell recruitment of FPA144 in the 4T1 model of cancer in immune-competent mice. Although this model expresses FGFR2b, it is not FGFR2 amplified. Therapeutic treatment with FPA144 in the orthotopic 4T1 model resulted in a reduction in tumor burden (33%, P&amp;lt;0.001) and concomitant recruitment of NK cells to the site of tumor implantation, while a modified antibody lacking Fc effector function neither inhibited tumor growth nor lead to the recruitment of NK cells. Together these data support a potential role for ADCC as a mechanism of FPA144 tumor growth inhibition. In addition, treatment with FPA144 increased PD-L1 expressing cells within the tumor microenvironment, providing a strong rationale that FPA144 may combine effectively with PD-1 blockade for additional tumor growth inhibition. PD-1 blockade by the RPM1-14 antibody did not inhibit tumor growth as a single agent in the 4T1 model. Treatment with RPM1-14 in combination with FPA144, however, inhibited tumor growth by 49% (P&amp;lt;0.001), demonstrating an additive benefit of combination therapy. Overall, these data suggest that the enhanced ADCC activity of FPA144 may be critical for anti-tumor efficacy in tumors that have modest expression of FGFR2b. In addition, FPA144 may reprogram the tumor micro-environment in a way that primes the tumor for additional anti-tumor activity when combined with PD-1 blockade. Citation Format: Janine Powers, Servando Palencia, Susan Foy, Barbara Sennino, Toni Rose Hidalgo, Abigael Gemo, Thomas Brennan, Kisten Pierce. FPA144, a therapeutic monoclonal antibody targeting the FGFR2b receptor, promotes antibody dependent cell-mediated cytotoxicity and stimulates sensitivity to PD-1 in the 4T1 syngeneic tumor model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1407.

Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model.

Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo, and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

Immunoglobulin G fragment C receptor polymorphisms and efficacy of preoperative chemotherapy plus trastuzumab and lapatinib in HER2-positive breast cancer.

Lapatinib enhances antibody-dependent cell-mediated cytotoxicity (ADCC) activity of trastuzumab. FcγR polymorphisms have been associated with both ADCC and clinical activity of trastuzumab in HER2+ breast cancer (BC) patients (pts). We analyzed FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms in the CHER-LOB trial population of HER2+ BCs treated with preoperative chemotherapy plus trastuzumab (arm A), lapatinib (arm B) or both (arm C). Genotyping was successfully performed in 73/121 (60%) pts. A significant improvement in pathological complete response (pCR) rate was observed for the combination arm C, but only in FcγRIIIa V allele carriers (C vs A, 67 vs 27%, P=0.043; C vs B, 67 vs 22%, P=0.012). An independent interaction between arm C and FcγRIIIa V allele was found for pCR (odds ratio=9.4; 95% confidence interval, 2.3-39.6; P=0.003). No significant associations were observed between pCR and FcγRIIa polymorphism, and between pre-treatment tumor-infiltrating lymphocytes and FcγR polymorphisms. Our study provides evidence for a FcγRIIIa V allele-restricted pCR benefit from neoadjuvant trastuzumab plus lapatinib in HER2+ BC.

Characterizing the effect of multiple Fc glycan attributes on the effector functions and FcγRIIIa receptor binding activity of an IgG1 antibody.

N-linked Fc glycosylation of IgG1 monoclonal antibody therapeutics can directly influence their mechanism of action by impacting IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, identification and detailed characterization of Fc glycan critical quality attributes (CQAs) provides important information for process design and control. A two-step approach was used to identify and characterize the Fc glycan CQAs for an IgG1 Mab with effector function. First, single factor experiments were performed to identify glycan critical quality attributes that influence ADCC and CDC activities. Next, a full-factorial design of experiment (DOE) to characterize the possible interactions and relative effect of these three glycan species on ADCC, CDC, and FcγRIIIa binding was employed. Additionally, the DOE data were used to develop models to predict ADCC, CDC, and FcγRIIIa binding of a given configuration of the three glycan species for this IgG1 molecule. The results demonstrate that for ADCC, afuco mono/bi has the largest effect, followed by HM and β-gal, while FcγRIIIa binding is affected by afuco mono/bi and β-gal. CDC, in contrast, is affected by β-gal only. This type of glycan characterization and modeling can provide valuable information for development, manufacturing support and process improvements for IgG products that require effector function for efficacy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1181-1192, 2016.

Hydroxychloroquine protects melanocytes from autoantibody-induced injury by reducing the binding of antigen-antibody complexes.

Vitiligo is a polygenic autoimmune disorder characterized by loss of pigmentation due to melanocyte destruction. Hydroxychloroquine (HCQ) is an effective immunosuppressant widely used in the treatment of autoimmune disorders. As generalized vitiligo (GV) is commonly considered to be a Tcell and autoantibody-induced immune disorder, the present study aimed to determine whether HCQ protects melanocytes from autoantibody‑induced disruption. Anti‑melanocyte antibodies were obtained from the serum of patients with progressive GV and the effects of HCQ on prevent the autoantibody‑induced disruption of melanocytes was observed. Cell‑based ELISA, indirect immunofluorescence and western blotting were used to analyze the autoantibody content of sera samples obtained from 32patients with progressive GV. The cytotoxicity of HCQ was detected by MTT assay, and 1µg/ml HCQ was applied to human primary melanocytes (HMCs) to examine whether it could exert protective effects against autoantibody‑induced immune injury. Flow cytometry was used to measure autoantibody binding to the surface of HMCs. Complement‑dependent cytotoxicity (CDC) and antibody‑dependent cell‑mediated cytotoxicity (ADCC) were monitored by MTT and lactate dehydrogenase‑releasing assays. The concentration of autoantibodies in sera samples taken from GV patients was significantly higher than in controls, particularly in patients who had >10% of their body surface affected by vitiligo. The majority of the autoantibodies presented in the HMCs and human keratinocytes (HKCs) and were predominantly localized to the cell surface and cytoplasm. The molecular weights of the autoantigens were identified as 30, 37‑39, 42, 53, 60‑75, 90, 100, 110, and 126kDa; the 30kDa protein was observed only in HMCs. The addition of HCQ at a concentration of 1µg/ml produced no significant cytotoxicity in HMCs and was demonstrated to reduce the binding of GV immunoglobulinG (IgG) to the surface of HMCs. HCQ also significantly decreased the effects of ADCC and CDC that were mediated by GV IgG. The present study provides evidence that HCQ dissociates autoantibody-antigen complexes on the surface of HMCs and reverses ADCC and CDC activity invitro. Thus, in addition to its effectiveness as an antimalarial therapeutic agent, HCQ may also be a promising potential treatment for patients with vitiligo.