Antibody-dependent Cell-mediated Cytotoxicity Activity Research Articles

Penpulimab, an Fc-Engineered IgG1 Anti-PD-1 Antibody, With Improved Efficacy and Low Incidence of Immune-Related Adverse Events.

BackgroundIgG4 anbibodies are deficient in stability and may contribute to tumor-associated escape from immune surveillance. We developed an IgG1 backbone anti-programmed cell death protein-1 (PD-1) antibody, penpulimab, which is designed to remove crystallizable fragment (Fc) gamma receptor (FcγR) binding that mediates antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and proinflammatory cytokine release.MethodsAggregation of different anti-PD-1 antibodies was tested by size exclusion chromatography, and melting temperature midpoint (Tm) and aggregation temperature onset (Tagg) were also determined. The affinity constants of penpulimab for PD-1 and human FcγRs were measured by surface plasmon resonance and biolayer interferometry. ADCC and ADCP were determined in cellular assays and antibody-dependent cytokine release (ADCR) from human macrophages was detected by ELISA. Binding kinetics of penpulimab to human PD-1 was determined by Biacore, and epitope/paratope mapping of PD-1/penpulimab was investigated using x-ray crystallography. Additionally, patients from six ongoing trials were included for analysis of immune-related adverse events (irAEs).ResultsPenpulimab demonstrated better stability and a lower level of host-cell protein residue compared with IgG4 backbone anti-PD-1 antibodies. As expected, penpulimab exhibited no apparent binding to FcγRIa, FcγRIIa_H131, FcγRIIIa_V158 and FcγRIIIa_F158, elicited no apparent ADCC and ADCP activities, and induced no remarkable IL-6 and IL-8 release by activated macrophages in vitro. Penpulimab was shown in the co-crystal study to bind to human PD-1 N-glycosylation site at N58 and had a slower off-rate from PD-1 versus nivolumab or pembrolizumab. Four hundred sixty-five patients were analyzed for irAEs. Fifteen (3.2%) patients had grade 3 or above irAEs. No death from irAEs was reported.ConclusionsIgG1 backbone anti-PD1 antibody penpulimab has a good stability and reduced host cell protein residue, as well as potent binding to the antigen. Fc engineering has eliminated Fc-mediated effector functions of penpulimab including ADCC, ADCP and reduced ADCR, which may contribute to its more favorable safety profile.Clinical Trial Registration www.ClinicalTrials.gov, identifier: AK105-101: NCT03352531, AK105-201: NCT03722147, AK105-301: NCT03866980, AK105-202:NCT03866967, AK105-203: NCT04172571, AK105-204: NCT04172506.

Abstract LB534: Development of GB2101, a novel anti-CCR8 antibody for cancer treatment

Abstract Background: Regulatory T cells (Tregs) play a crucial role in suppressing immune response and maintaining hemostasis and immune tolerance. Tregs are enriched in the tumor microenvironment (TME) and suppress anti-tumor immunity. Therefore, specific depletion of Tregs in the TME is expected to reduce anti-tumor immune suppression without affecting hemostasis in the periphery. The C-C motif chemokine receptor 8 (CCR8), highly expressed on Tregs from TME but significantly lower expressed on Tregs from peripheral blood, provides an ideal target for preferentially deletion of Tregs within tumors. GB2101 is a novel anti-CCR8 monoclonal antibody generated from hybridoma. The data from in vitro and in vivo studies indicated that GB2101 is a good PCC molecule with great potential for clinical development. Methods: GB2101 was generated by immunization of CCR8 gene plus CCR8 over-expressing cells in mice followed by hybridoma technology. The binding of the antibody to CCR8-expressing cells was detected by flow cytometry. CCL1 ligand blocking was assessed by flow cytometry and calcium flux assay. Antibody-dependent cell-mediated cytotoxicity (ADCC) function was evaluated by Jurkat-CD16A-NFAT reporter system. In vivo anti-tumor efficacy was assessed in syngeneic model using human CCR8 knock-in mice. Results: GB2101 displayed potent binding to cells expressing CCR8. GB2101 efficiently blocked CCL1 binding and CCL1-induced signals in CCR8 expressing cells. ADCC activity against target cells was observed and afucosylated version of GB2101 improved ADCC response. GB2101 significantly inhibited tumor growth in syngeneic model. Conclusions: GB2101 is a novel monoclonal antibody specifically binding to CCR8 with high affinity, and efficiently blocked CCL1-CCR8 pathway and suppressed tumor growth in vivo. Therefore, it is a very promising preclinical candidate that may be used in the treatment of multiple tumor types. Citation Format: Qinglin Du, Yiqi Cao, Xueyan Yang, Jing Lu, Fei Peng, Wenming Zhou, Liwen Liang, James Li, Shuhua Han. Development of GB2101, a novel anti-CCR8 antibody for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB534.

Abstract 1389: In vitro killing assays for evaluation of immuno-oncology drug candidates

Abstract The need to better understand the tumor microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop novel cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Cytotoxic cells are key players in the anti-tumor immune response. CD8+ T cells, also known as cytotoxic T lymphocytes, recognize and kill cells presenting antigens bound to MHC class I molecules, such as neoantigens present on the tumor cell surface. Activation of CD8+ T cells requires at least two signals from antigen presenting cells: binding of their T cell receptor (TCR) to the appropriate peptide presented on MHC I and co-stimulation by B7 binding to CD28. However, the TME possesses several mechanisms to reduce immune cell activity, such as the expression of checkpoint inhibitors dampening the immune system. Natural killer (NK) cells represent another type of cytotoxic cells, with their innate immune activity dictating an important role in anti-tumor immunity. NK cells induce apoptosis via the release of perforin and granzymes from granules, eliminating cells lacking expression MHC I, including cancer cells that have lost expression of MHC I. However, also the activity of NK cells can be hampered in the TME, notably by the PD1/PDL1 interaction.Increasing the activity of CD8+ T and NK cells as a therapeutic anti-tumor strategy has become an important point of attention. Such strategies can have several shapes, with Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of IgG1 isotypes being one of the mechanisms that can be exploited by immunoglobulin-based therapeutics. Another way to increase the killing of tumor cells is to re-direct T cells to tumor cells by employing bi-specific antibodies targeting specific tumor associated antigens. Using this strategy, T cell activation can be concentrated in the TME, thereby also avoiding off-tumor activity. Real-time in vitro assessment of the potential enhanced killing capacity of CD8+ T and NK cells elicited by test molecules could further provide pivotal information on the functional dynamics of test molecules. Using high quality primary human immune cells, in vitro bioassays were developed with improved robustness and reproducibility to screen for the potential to enhance tumor cell killing by novel therapeutics. The ability to evaluate the compounds capacity to increase or induce a cytotoxic activity profile and thereby facilitate the anti-tumor immune response is essential in the early drug development process. Citation Format: Johan Arnold, Thibaut J. Janss, Simon Lefevre, Martijn Vlaming, Ellen Boelen, Sofie Pattijn. In vitro killing assays for evaluation of immuno-oncology drug candidates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1389.

Novel Affinity Chromatography Ligand Utilizing the Biological Interaction Between the N‐linked glycans on Monoclonal Antibodies and the FcγIIIA Receptor

Glycosylation is one of the major post‐translational modifications (PTMs) in proteins, with significant effects on protein folding, conformation, stability, and activity. Glycosylation is particularly important for therapeutic protein drugs, such as monoclonal antibodies (mAbs), as it can profoundly impact their pharmacological properties, stability, and safety. Therefore, understanding the inherent heterogeneity of mAb glycoforms is an integral part of their manufacturing processes and quality control.Most marketed mAbs belong to the immunoglobulin G1 (IgG1), IgG2, or IgG4 subclass, carrying N‐glycosylation at the conserved asparagine position 297 (Asn‐297) in the CH2 domain of the Fc region. N‐glycosylation is critical for the Fc receptor binding and consequently affects key antibody effector functions such as antibody‐dependent cell‐mediated cytotoxicity (ADCC). However, assessing ADCC activity using a cell‐based assay remains a complicated, time‐consuming, and expensive task for laboratories.Here, we present a novel workflow leveraging FcγR‐IIIA biology to identify N‐glycan compositions and how to chromatographically determine changes in glycosylation in a time and cost‐effective manner. The TSKgel® FcR‐IIIA analytical and semi‐preparative columns separate mAbs into multiple subsets based on their affinity to the FcγR‐IIIA ligand. Utilizing an increasingly acidic pH gradient, mAb glycoforms are eluted from the column in an order that correlates to their ADCC activity. The analytical TSKgel FcR‐IIIA‐NPR column provides rapid assessment of a mAb's glycoform distribution, whereas the semipreparative TSKgel FcR‐IIIA‐5PW column enables collection of the peaks for more complex orthogonal released N‐glycan analyses and N‐glycosylation profiling using peptide mapping technique.With several mAbs containing various glycosylation patterns, we directly correlated the N‐glycan nature, structure, content, and elution profile on the FcR‐IIIA columns. Furthermore, we present a direct correlation between higher ADCC activity and binding affinity on the FcR‐IIIA columns, indicating that the complexity of the mAb glycosylation pattern is reflected in the observed FcR‐IIIA elution profile. Finally, employing both qualitative and quantitative assessments, we quantified the impact of individual N‐glycan structure and composition on mAb binding to the FcR ligand, providing an additional dimension to our understanding of the FcR‐IIIA columns.This in‐depth correlation analysis provides improved tools to predict, understand, and analyze changes in FcR‐IIIA elution profiles and how changes in the N‐glycan composition influence them. Altogether, this study describes a new framework for utilizing the TSKgel FcR‐IIIA columns for glycosylation profiling, providing a powerful way to simultaneously capture, profile, and characterize the glycosylation state of a given mAb of interest in a comprehensive manner.

Identification, Efficacy, and Stability Evaluation of Succinimide Modification With a High Abundance in the Framework Region of Golimumab.

Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0–7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.

Functional antibody-dependent cell mediated cytotoxicity (ADCC) responses to vaccine and circulating influenza strains following vaccination.

Novel cell-based assays were developed to assess antibody-dependence cellular cytotoxicity (ADCC) antibodies against both vaccine and a representative circulation strain HA and NA proteins for the 2014-15 influenza season. The four assays using target cells stably expressing one of the four proteins worked well. In pre- and post-vaccine sera from 70 participants in a pre-season vaccine trial, we found ADCC antibodies and a rise in ADCC antibody titer against target cells expressing the 4 proteins but a much higher titer for the vaccine than the circulating HA in both pre-and post-vaccine sera. These differences in HA ADCC antibodies were not reflected in differences in HA binding antibodies. Our observations suggested that relatively minor changes on the subtype HA can result in large differences in ADCC activity.

Perspectives on the Efficacy of Benralizumab for Treatment of Eosinophilic Granulomatosis With Polyangiitis.

Two types of interleukin (IL)-5 antibody biologics, anti-IL-5 antibodies (mepolizumab) and anti-IL-5α receptor antibodies (benralizumab), are indicated for severe asthma. While high-dose mepolizumab is also indicated for EGPA, benralizumab is indicated only for severe asthma. Benralizumab is characterized by antibody-dependent cell-mediated cytotoxicity activity, giving them specific and rapid anti-IL-5α receptor binding abilities and the ability to target a high number of eosinophils in tissues as well as peripheral blood. Recently, reports on the efficacy of benralizumab as a treatment for EGPA have been published, along with reports on some cases that are difficult to treat with existing oral corticosteroids and mepolizumab. Therefore, we focus on the perspective of the efficacy and safety of benralizumab as a treatment for EGPA patients with steroid dependence in this review. A total of 41 patients with EGPA were treated with benralizumab. After the introduction of benralizumab, oral corticosteroids could be reduced to 10 mg/day or less in all cases and to less than 5 mg/day in 80% or more of the cases. Discontinuation of oral corticosteroids was achieved in more than 40% of patients with EGPA. Benralizumab was effective in patients with mepolizumab-refractory EGPA and intractable cardiac and neuropathy complications. Efficient elimination of eosinophils is expected to improve the remission rate of EGPA with benralizumab treatment. Although the total number of patients was small, benralizumab was safe and tolerable in a wide range of age groups, suggesting efficacy in severe cases with EGPA.

Examination of IgG Fc Receptor CD16A and CD64 Expression by Canine Leukocytes and Their ADCC Activity in Engineered NK Cells.

Human natural killer (NK) cells can target tumor cells in an antigen-specific manner by the recognition of cell bound antibodies. This process induces antibody-dependent cell-mediated cytotoxicity (ADCC) and is exclusively mediated by the low affinity IgG Fc receptor CD16A (FcγRIIIA). Exploiting ADCC by NK cells is a major area of emphasis for advancing cancer immunotherapies. CD64 (FcγRI) is the only high affinity IgG FcR and it binds to the same IgG isotypes as CD16A, but it is not expressed by human NK cells. We have generated engineered human NK cells expressing recombinant CD64 with the goal of increasing their ADCC potency. Preclinical testing of this approach is essential for establishing efficacy and safety of the engineered NK cells. The dog provides particular advantages as a model, which includes spontaneous development of cancer in the setting of an intact and outbred immune system. To advance this immunotherapy model, we cloned canine CD16A and CD64 and generated specific mAbs. We report here for the first time the expression patterns of these FcγRs on dog peripheral blood leukocytes. CD64 was expressed by neutrophils and monocytes, but not lymphocytes, while canine CD16A was expressed at high levels by a subset of monocytes and lymphocytes. These expression patterns are similar to that of human leukocytes. Based on phenotypic characteristics, the CD16A+ lymphocytes consisted of T cells (CD3+ CD8+ CD5dim α/β TCR+) and NK cells (CD3− CD5− CD94+), but not B cells. Interestingly, the majority of canine CD16A+ lymphocytes were from the T cell population. Like human CD16A, canine CD16A was downregulated by a disintegrin and metalloproteinase 17 (ADAM17) upon leukocyte activation, revealing a conserved means of regulation. We also directly demonstrate that both canine CD16A and CD64 can induce ADCC when expressed in the NK cell line NK-92. These findings pave the way to engineering canine NK cells or T cells with high affinity recombinant canine CD64 to maximize ADCC and to test their safety and efficacy to benefit both humans and dogs.

Abstract P2-13-04: Final survival analysis of a phase 3 study comparing SB3 (trastuzumab biosimilar) and reference trastuzumab in HER2-positive early or locally advanced breast cancer

Abstract Background: SB3 (trastuzumab-dttb) is a biosimilar approved globally based on its similarity with reference trastuzumab (TRZ) demonstrated by thorough comparability exercises in analytical, biological, and clinical studies. In a randomized, double-blind, multicenter Phase 3 study of 875 patients with HER2-positive early or locally advanced breast cancer in the neoadjuvant setting, equivalent efficacy, similar safety, pharmacokinetics, and immunogenicity between SB3 and TRZ were shown. However, when quality attributes of TRZ were examined, downward drifts in antibody-dependent cell-mediated cytotoxicity activities (ADCC) were observed in the TRZ lots with expiry dates ranging from Aug 2018 to Dec 2019. Some of these lots of the reference product were found to be used in the Phase 3 study. After completing the Phase 3 study, patients from select countries were included in a follow-up observational study to monitor cardiac safety and survival. Here, we report the final survival results, including post-hoc subgroup analysis based on ADCC status, at a median follow-up of 68 months. Methods: During the follow-up observational study, the protocol was amended to include additional patients who originally were enrolled in the Phase 3 study but had not been followed in the observational study, in order to collect a larger sample of survival data. For these additional patients, medical records from the last assessment in the Phase 3 study through the date of enrollment in the follow-up study were collected retrospectively. As post-hoc analysis, patients in the TRZ arm were stratified into two subgroups: patients who received during neoadjuvant treatment at least one vial of TRZ with downward drift in ADCC as “Drifted TRZ”, and the others as “Non-drifted TRZ”. Event-free survival (EFS) and overall survival (OS) were assessed. Results: Of 875 patients randomized in the Phase 3 study, 538 patients (SB3, N=267; TRZ, N=271) were enrolled in the follow-up observational study: 367 patients were initially enrolled in the follow-up study, and 171 patients were additionally enrolled following the protocol amendment. The median follow-up duration was 68 months from randomization in the Phase 3 study. 54 events (20.2%) in the SB3 arm, and 67 events (24.7%) in the TRZ arm were reported (HR 0.84 [0.58, 1.20], p=0.335). 22 deaths (8.2%) and 38 deaths (14%) were reported in SB3 and TRZ arms, respectively (HR 0.61 [0.36, 1.05], p=0.073). In post-hoc analysis, of 271 patients in TRZ arm, 107 patients were grouped as “Non-drifted TRZ”, and 164 patients as “Drifted TRZ”. 19 events (17.8%) in the Non-drifted TRZ group and 48 (29.3%) events in the Drifted TRZ group occurred (HR 2.57 [1.28, 5.14], p=0.008). 9 deaths (8.4%) in the Non-drifted TRZ group and 29 deaths (17.7%) in the Drifted TRZ group were reported (HR 3.87 [1.37, 10.93], p=0.011). No difference was observed between SB3 arm and Non-drifted TRZ group in terms of EFS (HR 1.28 [0.73, 2.22], p=0.391) and OS (HR 0.99 [0.42, 2.31], p=0.975). Conclusions: Comparable long-term efficacy results in EFS and OS were shown at 68 months of follow-up, further supporting biosimilarity of SB3 to the reference product. Currently, these follow-up results represent the longest monitoring data of patients treated with a trastuzumab biosimilar for HER2-positive early or locally advanced breast cancer. Citation Format: Xavier Pivot, Mark D Pegram, Javier Cortes, Diana Lüftner, Gary H Lyman, Giuseppe Curigliano, Igor M Bondarenko, Mikhail Dvorkin, Jin Hee Ahn, Seock-Ah Im, Maria Litwiniuk, Yaroslav V Shparyk, Gwo Fuang Ho, Nikolay V Kislov, Marek Wojtukiewicz, Tomasz Sarosiek, Yee Soo Chae, Jin Seok Ahn, Hyerin Jang, Sujung Kim, Jiwon Lee, Soo Young Lee, Ye Chan Yoon. Final survival analysis of a phase 3 study comparing SB3 (trastuzumab biosimilar) and reference trastuzumab in HER2-positive early or locally advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-04.

A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells.Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs).Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3.Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. DisclosuresBattula: Tolero Pharmaceuticals: Research Funding.

Arming of iPSC-Derived NK Cells Expressing a Novel CD64 Fusion Receptor with Therapeutic Antibodies Represents a Novel Off-the-Shelf, Antigen-Targeting Strategy for Cancer

Natural killer (NK) cells are innate lymphocytes that target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and that recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Human NK cells exclusively mediate ADCC through the IgG Fc receptor, CD16A, and studies have demonstrated that increasing the binding affinity between CD16A and therapeutic monoclonal antibodies (mAbs), mediated by the high-affinity 158V polymorphism, can augment clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we sought to arm iPSC-derived NK (iNK) cells expressing a high-affinity recombinant FcγR with various mAbs as unique tumor-targeting strategy for various malignancies.As a member of the FcγR family, CD64 (FcγRI) possesses the highest affinity and can uniquely facilitate antibody preabsorption but it is normally expressed by myeloid cells. To leverage CD64 in NK cells, we developed a novel FcγR recombinant fusion comprising the extracellular region of CD64 with the transmembrane and intracellular regions of other NK cell activating receptors, including CD16A (CD64/16A) (figure 1A). The recombinant CD64/16A engineered into a clonal master induced pluripotent stem cell (iPSC) line for mass production of off-the-shelf iPSC-derived CD64/16A NK (iNK-CD64/16A) cells, can be armed with mAbs, including various combinations thereof to enable multi-antigen targeting and to address tumor heterogeneity (figures 1B and 2).To determine optimal binding and FcR saturation of iNK-CD64/16 cells, rituximab (anti-CD20 therapeutic mAb) was added in a two-hour preabsorbtion assay (figure 3A). Using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against Raji cells, a Burkitt Lymphoma cell line, when the iNKs were preabsorbed and armed with rituximab (figure 3B). Considering the high-affinity state of CD64, we examined the effects of free IgG on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis, ADCC was evaluated in the presence of purified human IgG. Despite the high levels of excess IgG, iNK-CD64/16A cells mediated efficient ADCC when rituximab was either added to the assay (figure 4A) or preabsorbed to the cells (figure 4B), demonstrating that saturating levels of free IgG did not prevent ADCC in either setting. To determine the ability of preabsorbed and armed iNK-CD64/16 cells to retain rituximab and perform serial killing, we performed a sequential killing assay using an IncuCyte® Live Cell Analysis where preabsorbed iNK-CD64/16A cells were thawed and co-cultured with or without Raji cells for 48 hours, followed by a second round of co-culture. As shown in figure 5, iNK-CD64/16A cells armed with rituximab retain ADCC capacity and perform serial killing for an extended time.To establish that iNK-CD64/16A cells can be armed with assorted therapeutic mAbs to target other tumor-associated antigens, we next determined the ability of iNK-CD64/16A cells preabsorbed and armed with anti-HER2 mAb, trastuzumab, to target the adenocarcinoma ovarian cancer cell line SKOV-3. Indeed iNK-CD64/16A cells armed with preabsorbed trastuzumab were able to effectively kill SKOV-3 cells via in vitro ADCC by IncuCyte® Live Cell Analysis (figure 6). We next investigated in vivo ADCC using NSG mice implanted with 3x10 5 SKOV-3 cells expressing firefly luciferase intraperitoneally (IP). 10 million iNK-CD64/16A with or without preabsorbed trastuzumab were injected IP (figure 7A), and a significant reduction in tumor volume in animals treated with iNK-CD64/16A cells armed with trastuzumab compared to unarmed iNK-CD64/16A cells (figure 7B).Collectively, our data show that iNK-CD64/16A cells can be armed with various therapeutic mAbs through a unique preabsorption strategy to mediate a potent and durable ADCC activity. The versatility of mAb-armed iNK-CD64/16A cells is being further investigated in various preclinical models to further elucidate the potential of this approach to overcome antigen escape and address tumor heterogeneity. [Display omitted] DisclosuresLee: Fate Therapeutics, Inc: Current Employment. Chu: Fate Therapeutics: Current Employment. Rogers: Fate Therapeutics: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Hosking: Fate Therapeutics: Current Employment. Shirinbak: Fate Therapeutics, Inc.: Current Employment. Miller: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees. Valamehr: Fate Therapeutics, Inc.: Current Employment. Walcheck: Fate Therapeutics: Research Funding.

Bio-O2-04 - Enhancement of antibody-dependent cellular cytotoxicity is associated with treatment response to extracorporeal photopheresis in Sézary syndrome

Sézary syndrome (SS) is a rare, leukemic type of cutaneous T-cell lymphoma (CTCL), for which extracorporeal photopheresis (ECP) is a first-line therapy. Reliable biomarkers to objectively monitor the response to ECP in patients with SS are missing. We examined the quantitative and qualitative impact of ECP on natural killer (NK) cell activity in SS patients, and especially their functional ability for antibody-dependent cell-mediated cytotoxicity (ADCC). Further, we addressed the question whether the magnitude of the effect on ADCC can be associated with the anti-cancer efficacy of ECP in SS patients. We assessed numbers of NK cells, ADCC activity, and treatment response based on blood tumor staging in a cohort of 13 SS patients (8 women, 5 men) treated with ECP as a first-line therapy. Blood samples were collected before treatment start and after an average of 9 months of uninterrupted ECP treatment. NK cell numbers were reduced in SS patients compared to healthy individuals and showed a tendency of recovery after long-term ECP treatment, independent of the clinical response to treatment. Patients with marginal increase (≤1.5 AU-fold) or lack of increase in ADCC activity failed to respond clinically to treatment, while patients with an increased ADCC activity showed a reduction in blood tumor burden. NK-mediated ADCC is selectively enhanced and might be a mechanism underlying the effect of ECP while in addition it can possibly serve as a reliable biomarker to objectively monitor response to ECP in patients with SS.

268 Enhancement of antibody-dependent cellular cytotoxicity is associated with treatment response to extracorporeal photopheresis in Sézary syndrome

Importance: Sézary syndrome (SS) is a rare, leukemic type of cutaneous T-cell lymphoma (CTCL), which can be successfully controlled with extracorporeal photopheresis (ECP). Reliable biomarkers to objectively monitor the response to ECP in patients with SS are missing. Objective: We examined the quantitative and qualitative impact of ECP on natural killer (NK) cell activity in SS patients, and especially their functional ability for antibody-dependent cell-mediated cytotoxicity (ADCC). Further, we address the question if the magnitude of the effect can be associated with the treatment response to ECP in SS patients. Materials and Methods: We collected blood samples before starting therapy and after an average of 9 months of uninterrupted ECP treatment from a case-series of 13 SS patients (8 women, 5 men). This is a single-centre study. All patients were diagnosed according to EORTC-WHO criteria and gave written informed consent to the use of their material and data for research purposes. Blood from healthy volunteers was obtained anonymously from a blood bank. Number of NK cells by flow cytometry, ADCC activity by LDH release assay and flow cytometry, treatment response based on blood tumour staging. Results: NK cell numbers were reduced in SS patients compared to healthy individuals and showed a tendency of recovery after long-term ECP treatment, independent of the response to treatment. Patients with marginal increase (≤1.5 AU-fold) or lack of increase in ADCC activity failed to respond to treatment, while an increase in ADCC efficacy paralleled a positive response to ECP. Conclusions and Relevance: NK-mediated ADCC is selectively enhanced and can serve as a reliable biomarker to objectively monitor response to ECP in patients with SS.

Abstract 231: Evaluation of pharmacologic mechanisms to overcome IgG1 antibody (Ab) resistance via quantitative systems pharmacology (QSP) modeling of antibody-dependent cell mediated cytotoxicity (ADCC)

Abstract Novel therapies that enhance ADCC have the potential to overcome Rituximab (RTX) resistance in lymphoma, which can occur due to e.g. CD20 loss, immunogenicity-induced reduction in RTX exposure, or SNPs resulting in lower CD16:Fc affinity. Nonlinear interactions between effector cells (ECs), drug, and target cell (TC) factors in ADCC, however, complicate the prediction of the net effect of potential therapeutic strategies that modulate one or more factors such as CD16 expression ([CD16]) on ECs, CD16:Fc binding affinity, or intrinsic killing propensity (g).We have developed a QSP model of the molecular-level binding reactions and cell-cell interactions governing ADCC using the anti-CD20 IgG1 antibody RTX in lymphoma as a case study. This model takes as inputs [CD16] on ECs, g, Ab Fc:CD16 binding affinity, [RTX], RTX:CD20 affinity, and [CD20] on TCs and predicts the degree of ADCC killing of tumor cells after RTX exposure. Simulations can then be used to estimate how much increase in an input parameter a potential RTX combination partner must provide to compensate for a given mechanism of RTX resistance (MoRR). The model has been successfully calibrated to ex vivo [RTX]-ADCC assays in SUDHL4 and Z138 cell lines (Herter, Mol Cancer Ther, 2013). The model and estimated parameter set well described the observed [RTX]-dependent %ADCC in both cell lines with ECs (PBMCs) from two donors with V158 and F158 CD16 SNP variants. The table below provides the model predictions of the fold increase in model parameters required to offset various MoRRs. For example, a 10-fold loss of [CD20] on TCs is predicted to cause a 100*(1 - 0.16) = 84% drop in ADCC activity, which can be overcome by a 7.8-fold increase in [CD16] on ECs. We have developed a mechanism-based simulation tool which can be used to predict the ability of novel ADCC-enhancing agents to overcome different mechanisms of IgG1 Ab resistance. Mechanism of RTX resistance (MoRR)ADCC fold change due to MoR(90% CI)Fold change in parameter required to restore ADCC (90% CI)CD16 expression on effector cells [CD16]CD16:Fc binding affinity kon16NK killing propensity g10x loss of CD20 on tumor0.16 (0.13,0.20)7.8 (6.3,9.2)10.6 (10.4,10.9)7.4 (6.1,8.7)10x loss of RTX exposure0.17(0.14,0.21)7.0(5.7,8.1)9.36(9.35,9.37)6.9(5.7,8.1)10x decrease in CD16:Fc affinity0.16(0.13,0.20)7.4(6.1,8.7)10(10,10)7.4(6.1,8.6) Citation Format: Kaitlyn E. Johnson, Maria Veronica Ciocanel, Josua Aponte, Nicolas Bajeux, Fanwang Meng, Dean Bottino. Evaluation of pharmacologic mechanisms to overcome IgG1 antibody (Ab) resistance via quantitative systems pharmacology (QSP) modeling of antibody-dependent cell mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 231.

Abstract 3199: In vitro killing assays for immuno oncology candidates

Abstract The need to better understand the tumor microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop better cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Cytotoxic cells are key players in the anti-tumor immune response. CD8+ T cells, also known as cytotoxic T lymphocytes, recognize and kill cells presenting antigens bound to MHC class I molecules, such as neoantigen present on the tumor cells. Activation of CD8+ T cells requires at least two signals from antigen presenting cells: binding of their T cell receptor (TCR) to the appropriate peptide presented on MHC I and co-stimulation by B7 binding to CD28. However, tumor environment developed different mechanisms to reduce this immune cell killing such as checkpoint inhibitors expression. In that case, the immune system will be dampened. A second type of cytotoxic cells, named Natural killer cells, are an innate immune cell type that plays an important role in anti-cancer immunity. NK cells target cells lacking MHC I expression, including cancer cells that have lost expression of MHC I. NK cells induce apoptosis in cancer cells via release of perforin and granzymes from granules, like CD8+ T cells. But here too, NK cells can be broken down by the tumor cells, notably by the PD1/PDL1 interaction. Increasing their efficacy as a therapeutic strategy has made the development of new therapeutics enhancing their anti-tumor response a priority. Those new therapeutics can have several shapes. The Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the IgG1 isotypes is one of the mechanisms that can be applied by immunoglobulin-based therapeutics. Another way to increase the killing of cancer cells is to target tumor antigens expressed by those cells using for example bispecific molecules that will enhance on the other side the T cell activation. Using primary immune cells, in vitro bioassays were developed to better screen the potential effect of new therapeutics on immune cell killing activity. Their ability to increase or induce a cytotoxic activity and facilitate the anti-tumor immune response can as a result be assessed early in the drug development process. Citation Format: Thibaut J. Janss, Juliette Lamy, Johan Arnold, Ellen Boelen, Sofie Pattijn. In vitro killing assays for immuno oncology candidates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3199.

Abstract 952: The bispecific antibody MCLA-129 impairs NSCLC tumor growth by targeting EGFR and c-MET, inhibiting ligand-induced signaling and promoting ADCC and ADCP

Abstract MCLA-129 is an ADCC-enhanced common light chain bispecific human IgG1 Biclonics® antibody specifically targeting the receptor tyrosine kinases EGFR and c-MET. It inhibits activation and downstream signaling of EGFR and c-MET induced by their respective ligands EGF and HGF and promotes elimination of tumor cells via Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Antibody-Dependent Cell-Mediated Phagocytosis (ADCP). MCLA-129 was developed to overcome c-MET signaling-dependent EGFR TKI-resistance mechanisms and was identified during an unbiased screening campaign with a focus on inhibition of ligand-induced proliferation and migration of cancer cells. Selectivity of MCLA-129 for tumor cells is achieved by simultaneously targeting both EGFR and c-MET. MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and consequently inhibits their ligand-induced phosphorylation. MCLA-129 was shown to bind critical residues on EGFR and c-MET for EGF and HGF ligand binding. GlymaxX® low fucose glycoengineering technology was used to enhance the mononuclear cell ADCC activity of the bispecific antibody. MCLA-129 demonstrated potent dose-dependent ADCC and ADCP against all NSCLC cell lines tested, which positively correlated with the EGFR and c-MET expression levels on the target cells. Significant inhibition of tumor growth of NCI-H1975EGFR L858R, T790M and HCC827/ER1EGFR del (E746, A750), c-MET amplified NSCLC cell line derived tumors was observed following MCLA-129 treatment. MCLA-129 can overcome HGF-mediated EGFR-TKI resistance, as demonstrated by testing a non-ADCC enhanced version of this antibody in a panel of NSCLC cell lines in vitro and orthotopic tumors in vivo showing that it inhibits EGFR and c-MET activity. Significant tumor regression was observed following treatment of immunodeficient xenograft NSG-hHGFki mice bearing orthotopic tumors established from HCC827EGFR del (E746, A750) cells. In addition, in an acquired erlotinib EGFR TKI resistance model, treatment of mice harboring tumors as big as &amp;gt;500 mm3 led to significant reduction of tumor size which persisted after the treatment period. Taken together these data demonstrate that MCLA-129 is a potent inhibitor of tumor growth applying various mechanisms of action, including inhibition of c-MET and EGFR signaling, ADCC and ADCP. MCLA-129 holds promise as a potential treatment for patients with NSCLC and other cancers, and warrants clinical evaluation. Citation Format: David J. de Gorter, Alexandre Deshiere, Martijn van Rosmalen, Christian Wohn, Berina Eppink, Tristan Gallenne, Rinse Klooster, Li Mao, Wenxin Xu, Liang Deng, Qingyu Shu, Wei Liu, John de Kruif, Mario Di Matteo, Massimiliano Mazzone, Mark Throsby, Cecile A. Geuijen. The bispecific antibody MCLA-129 impairs NSCLC tumor growth by targeting EGFR and c-MET, inhibiting ligand-induced signaling and promoting ADCC and ADCP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 952.

Abstract 1170: MEN1112/OBT357, a first-in-class humanized de-fucosylated monoclonal antibody targeting CD157 positive cells in acute myeloid leukemia and myelodysplastic syndrome

Abstract Bst1/CD157 is a GPI-anchored transmembrane protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, which is expressed on some blood cells of the myeloid lineage, such as monocytes and neutrophils. The antigen is found to be highly expressed, at diagnosis and relapse, in primary samples derived from Acute Myeloid Leukemia (AML) patients, showing the brightest expression in monocyte-oriented blasts. Moreover, CD157 is expressed in high and low-risk Myelodysplastic Syndrome (MDS) patients. MEN1112/OBT357, is a humanized de-fucosylated monoclonal antibody, in clinical development (clinicaltrials.gov identifier: NCT02353143), that is characterized by high affinity and specificity against CD157, inducing potent in vitro Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). The de-fucosylation improved the binding of MEN1112/OBT357 to both high and low-affinity alleles of FcγRIIIa/CD16a, allowing stronger recognition by NK cells. Since no Complement-Dependent Cytotoxicity (CDC) activity was detected, the MEN1112/OBT357 mechanism of action should mainly rely on immune effector cells. Indeed, in an in vitro reporter assay, MEN1112/OBT357 significantly triggered ADCC activity against AML target cell lines with different expression of CD157. Since the ex vivo sensitivity assay is considered a pharmacologically and clinically relevant model to evaluate the preclinical efficacy of therapeutic antibodies in AML (1), MEN1112/OBT357 activity was tested in 38 AML primary samples, demonstrating promising efficacy in an autologous setting, both in peripheral blood and bone marrow samples, in comparison to healthy donor blood samples and independently from FcγRIIIa polymorphisms. A similar activity was also observed in CD157 positive MDS primary samples. Since Myeloid-Derived Suppressor Cells (MDSCs) have been described to be increased in the bone marrow of both AML and MDS patients and their presence to be associated with resistance to chemotherapy (2, 3), we investigated CD157 expression in MDSCs, demonstrating a high expression of the antigen in the bone marrow and peripheral blood of AML patients. Importantly, we confirmed in ex vivo experiments, the depletion of these immunosuppressive cells by MEN1112/OBT357. Overall, our data demonstrate that MEN1112/OBT357 is a novel antibody with potent ex vivo activity against blast cells in AML and MDS patients, suggesting a potential contribution of MEN1112/OBT357 in altering the immunosuppressive environment in the bone marrow niche.

Functional Similarity of Proposed Biosimilar ABP 798 with Rituximab

▪BackgroundRegulatory guidelines governing the development of biosimilars require that proposed biosimilars undergo comprehensive structural and functional characterization prior to the initiation of clinical investigation. ABP 798 is being developed as a biosimilar to rituximab. The reference product is approved in the US for treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, severe rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis. Development of biosimilars is challenging as even small variations in structure have the potential to alter the function of the molecule and thus may impact efficacy, safety, and/or bioavailability. Here we compare ABP 798 and rituximab reference product with respect to binding properties (CD20, C1q, FcRn, and Fcγ receptors), antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and induction of apoptosis. The reference product was sourced from the US (rituximab US) and the EU (rituximab EU).MethodsBinding of ABP 798 and rituximab to the CD20 antigen was characterized using a cell-based CD20 binding assay utilizing the CD20 expressing WIL2-S human B-lymphoblastoid cell line. A direct binding ELISA was used to assess the binding of the Fc domain of ABP 798 to C1q. Binding of the Fc moiety of ABP 798 and rituximab to FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa (158V and 158F) were evaluated in AlphaLISAÒ competitive binding assays. Binding to FcRn was evaluated by an AlphaScreenÒ competitive binding assay. ADCC activity was evaluated in a functional cell-based assay, with WIL2-S cells used as target cells and NK92-MI cells, stably transfected with human CD16 (FcγRIIIa [158V]), used as effector cells. CDC activity was evaluated in a functional cell-based assay using the WIL2-S cell line and baby rabbit complement. Induction of apoptosis was assessed by measuring activation of caspase 3/7 in SU-DHL-4 cells, a CD20 expressing human B cell lymphoma cell line.ResultsRelative binding (%) as compared to an ABP 798 reference standard was similar between ABP 798 and rituximab (Table 1).The dose response profiles and relative activity for ADCC and CDC were also similar (ADCC relative cytotoxicity: ABP 798, 88%; rituximab US, 85%; rituximab EU, 86%; CDC relative cytotoxicity: ABP 798, 103%; rituximab US, 102%; rituximab EU, 104%). The dose-response profile for induction of caspase 3/7 was similar between ABP 798 and rituximab as well.ConclusionsThe results presented here demonstrate that ABP 798 is similar to rituximab sourced from both US and EU in terms of biological activity across the range of tested binding and functional assays. The functional assays tested the major and minor mechanisms of action related to clinical efficacy across approved rituximab indications and these results provide a foundation for further clinical development of ABP 798. [Display omitted] DisclosuresMcBride:amgen: Employment; amgen: Other: stockholder. Maher:amgen: Employment; amgen: Other: stockholder. Sweet:amgen: Employment; amgen: Other: stockholder. Foltz:amgen: Employment; amgen: Other: stockholder. Kuhns:amgen: Other: stockholder; amgen: Employment.