Antibody-dependent Cellular Cytotoxicity Activity Research Articles

Quantifying in vitrocomplement-dependent cytotoxicity (CDC) using advanced flow cytometry

Abstract Complement dependent cytotoxicity (CDC) is a key Fc mediated function of monoclonal antibody (mAb) therapeutics. mAb binding to antigen triggers a complex molecular cascade with sequential recruitment of serum proteins, eventuating in target cell lysis. Here we demonstrate CDC quantification in a streamlined, in vitro, advanced flow cytometry assay. Anti-CD20 mAbs were incubated with target cell lines in 96- or 384-well plates. Human serum (15%) was then added to induce CDC. Cells were labeled with iQue® Cell Membrane Integrity (R/Red) Dye to enable assessment of cell death using the iQue® Advanced Flow Cytometry Platform. Induction of CDC by anti-CD20 mAbs correlated with target cell CD20 expression, with most cell death observed with high-CD20 expressing Ramos cells. Maximal Ramos cell death induced by Rituximab and Truxima® was 68% and 70%, respectively. Comparatively, cell death of mid-CD20 expressing Raji cells was lower at 34% and 24%. No CDC was induced with CD20 negative Jurkat cells. Another anti-CD20-IgG1 mAb was compared against two isotype mutants: IgG1fut (non-fucosylated) and IgG1NQ (non-glycosylated). Their activity was profiled towards three previously described effector functions: antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP) and CDC. CDC induction was comparable between the three mAbs, however their ADCC and ADCP activity differed. The IgG1NQ did not exert ADCP or ADCC activity, whilst the IgG1fut showed more ADCC and less ADCP than the native. These data exemplify the use of advanced flow cytometry to quantify Fc function of mAbs towards a range of effector mechanisms, highlighting the potential to profile libraries of novel therapeutics in minimal time.

Reconstruction of a polyclonal ADCC antibody repertoire from an HIV-1 non-transmitting mother

Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity.

Ublituximab, a Novel, Glycoengineered Anti-CD20 Monoclonal Antibody (mAb), Demonstrates Enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) Relative to Other Anti-CD20 mAbs (P7-3.011)

Ublituximab, a Novel, Glycoengineered Anti-CD20 Monoclonal Antibody (mAb), Demonstrates Enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) Relative to Other Anti-CD20 mAbs (P7-3.011)

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

ABSTRACT Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

Abstract CT177: A phase I, first in human study of CBA-1205, glycoengineered humanized anti-DLK-1 monoclonal antibody in patients with advanced solid tumors

Abstract Background: CBA-1205 is a novel humanized anti-delta-like 1 homolog (DLK-1) antibody. It is a glycoengineered antibody by GlymaxX® technology to potentiate antibody-dependent cellular cytotoxicity activity. DLK-1 is a membrane protein with 6 tandem EGF-like motifs in extracellular region. Overexpression of DLK-1 has been reported in variety of cancer types including hepatocellular carcinoma (HCC), suggesting that DLK-1 could be an ideal target for cancer therapy. Methods: This is a first in human, multicenter, open-label Phase I study. The purpose of part 1 is to evaluate the safety and tolerability in patients with advanced solid tumors who had no standard therapy or were refractory or intolerant to standard therapies and to determine the starting dose for part 2 in HCC patients. The standard 3+3 dose escalation design was utilized in seven cohorts (0.1, 0.3, 1, 3, 10, 20, 30 mg/kg). CBA-1205 was administered at 2-week intervals in a 28-day cycle. If the patient had no Dose Limiting Toxicity (DLT) during 28 days after the first two doses, CBA-1205 administration will be continued for further cycles unless any of the criteria for discontinuation. Serum DLK-1 concentration before the start of CBA-1205 administration was analyzed using the Human Pref-1/DLK1/FA1 DuoSet ELISA (R&D Systems, Inc.). Immunohistochemical analysis of DLK-1 expression in the archival tumor specimen was determined in FFPE tissue sections using mouse anti-hDLK-1 antibody (clone DI-2-20, Chiome Bioscience). Results: 22 patients with advanced solid tumors were enrolled. CBA-1205 was well tolerated in these patients and no DLT was observed even at the highest dose tested. No ≥ Grade 3 treatment related adverse events was observed throughout the study periods. The serum concentration of CBA-1205 increased dose-proportionally. The exposure to CBA-1205 exceeded the effective concentrations observed in a mouse xenograft model. No serum anti-CBA-1205 antibody was detected. The disease control was seen in 8 patients (38.1%, all with SD). Especially, long SD was observed in 3 patients with invasive thymoma, malignant melanoma, and pancreatic carcinoma, where progression free survival (PFS) was 39, 72(ongoing, at data cutoff), and 41 weeks, respectively. The serum concentration of soluble DLK-1 was detected in 6 out of 22 patients. Patients with SD by the best overall response had a higher rate of detectable serum DLK-1 concentration and a higher mean concentration than patients with PD. No DLK-1 expression in tumor was detected by IHC method, in 14 patients who consented to use the archival tumor specimens. Conclusions: CBA-1205 was well tolerated without any severe toxicity in patients with advanced solid tumors.Part 2 of this Phase I study with two cohorts (20, 30 mg/kg) in patients with unresectable advanced/recurrent HCC refractory or intolerable to the standard therapy has been initiated. Citation Format: Masafumi Ikeda, Nobuaki Matsubara, Chihiro Kondoh, shose taoka, Yuki Katsuya, Takafumi Koyama, Noboru Yamamoto. A phase I, first in human study of CBA-1205, glycoengineered humanized anti-DLK-1 monoclonal antibody in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT177.

Abstract 3183: Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors

Abstract Background: Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense against opsonized target cells wherein the Fc-fragment of an antibody binds to the CD16 Fc receptor (FcγRIIIa) on NK cells, triggering NK cell activation and targeted killing. Cetuximab is a human IgG1 antibody targeting epidermal growth factor receptor (EGFR) that is approved for the treatment of colorectal and head and neck cancers. Binding of cetuximab to EGRF+ cell lines has been shown to trigger ADCC mediated by NK cells. NKX101 is an investigational allogeneic NK cell therapy in development for the treatment of cancer. NKX101 is engineered to express an NKG2D-based chimeric antigen receptor (CAR) and membrane-bound IL-15. To investigate the combination of NKX101 and cetuximab, we developed in vitro assays to (i) demonstrate enhanced activity against cancer cells via CD16-mediated ADCC activity, (ii) compare ADCC activity of NKX101 cells generated from high-affinity CD16 and low-affinity CD16 donors, and (iii) evaluate the synergistic interaction between NKX101 and cetuximab. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. NucRed-labeled EGFR+ NKG2D-L+ human cancer cell lines were pre-incubated with cetuximab or isotype control and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte instrument. For synergy assessments, a dose response matrix of NKX101 cells and cetuximab was used to generate cytotoxicity values. Synergy scores for combinations were determined using the Synergy Finder Plus web application. To determine donor CD16 V/F 158 genotypes, genomic DNA was isolated from each donor and assayed using a TaqMan CD16 genotyping kit. Results: We demonstrate that cetuximab increases the anti-tumor effect of NKX101 in a dose-dependent manner. Assessment of the interaction between NKX101 and cetuximab in vitro revealed that the two agents can combine to kill cancer cells in a synergistic manner. Blocking CD16 on NKX101 cells with a neutralizing antibody significantly decreased potency of the combination, suggesting that the improvement in potency observed is a direct result of CD16-mediated ADCC activity. To determine if common CD16 polymorphisms influence NKX101 ADCC activity, we compared NKX101 cells generated from CD16 158 V/V, V/F, and F/F donors. No significant differences in potency were observed with NKX101 cells expressing the high affinity CD16 158 V/V genotype compared with those derived from CD16 lower affinity 158 V/F or 158 F/F donors. In summary, we show that the combination of an engineered allogeneic NK cell therapy, NKX101, with cetuximab leads to an increase in the specific killing of cancer cells in vitro, supporting further investigation into this combination for the treatment of solid tumors. Citation Format: Cynthia Cho, Kyle Hansen, Max Zhang, Carmel Chan, James Trager. Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3183.

Abstract 5654: A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells

Abstract Background: NEO-201 is a humanized IgG1 mAb reactive against multiple human cancers but not against most normal epithelial tissues. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells, including neutrophils, various carcinomas, and some human hematological malignancies. NEO-201 can mediate antitumor activity through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 ICI pathway. A previous study using flow cytometry demonstrated that NEO-201+/CD4+ T cells were also CD25+/CD127−/Foxp3+/CD15s+ using PBMCs from healthy donors (HD). NEO-201 can kill these Treg cells through CDC in vitro. NEO-201 does not bind to the majority of CD4+ T cells and to other immune subsets. Human gMDSCs are increased in cancer patients and are a population of immature MDSCs deriving from immature neutrophils and alternative activation of mature neutrophils. gMDSC are characterized by HLA-DR−, CD11b+, CD33+, CD15+phenotype.We have shown that NEO-201 recognizes and kill human neutrophils through ADCC. This current investigation was designed to evaluate whether NEO-201 can target and mediate ADCC activity against human gMDSCs. Methods: gMDSCs were generated from human neutrophils from 5 HD isolated using EasySepTM direct human neutrophil isolation kit. Isolated neutrophils were cultured in complete RPMI1640 medium supplemented with human GM-CSF and human IL-6 for 7 days. Phenotypic analysis by flow cytometry was performed on the generated gMDSCs using NEO-201 and mAbs against human CD33, HLA-DR, CD15, CD14, CD66b. Flow cytometry based ADCC assay was performed using gMDSCs stained with both CD33 and HLA-DR as target. PBMCs from a separate HD were used as effectors at different E:T ratios. The ADCC activity of NEO-201 was evaluated comparing the percentage of CD33+/HLA-DR− viable cells in gMDSCs incubated with medium alone to the percentage of CD33+/HLA-DR− viable cells incubated with PBMCs alone and with PBMCs plus NEO-201. Results: Flow cytometry analysis revealed that gMDSCs can be generated from human neutrophils after 7 days of culture with GM-CSF and IL-6 and that they express the following phenotype: HLA-DR−/CD33+/CD15+/CD14−/CD66b+. NEO-201 bound to the majority of these gMDSCs. NEO-201 was functional in mediating ADCC to kill these gMDSCs. Conclusion: This study demonstrated that NEO-201 can be used to identify and kill suppressive gMDSCs in addition to Treg cells. Depletion of suppressive Tregs and gMDSCs in the TME could be an effective strategy to prevent hyperprogressive disease when anti-PD-1 is used in cancer immunotherapy. These data support the rationale for the ongoing phase II clinical trial using NEO-201 in combination with pembrolizumab in checkpoint refractory patients with metastatic solid tumors. Citation Format: Massimo Fantini, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5654.

Abstract 6334: VBI-002, a CD47xICAM-1 bispecific antibody for the treatment of hepatocellular carcinoma, melanoma and non-small cell lung cancers

Abstract Intercellular Cell Adhesion Molecule 1 (ICAM-1, CD54) is a type I transmembrane protein and a member of the immunoglobulin superfamily. ICAM-1 is involved in many key processes such as cell-cell interactions, signal transduction and leukocytes trans-endothelial migration. ICAM-1 is constitutively present at low levels on healthy tissues but highly expressed in some hematological cancers and solid tumors, making it an attractive tumor target. VBI-002 is a novel human IgG1 based CD47xICAM-1 bispecific antibody with excellent safety profile and potent single agent activities in xenograft models of lymphoma, multiple myeloma and some solid tumors (Cancer Res 2022;82(12_Suppl):Abstract nr 3430). The bispecific design allows VBI-002 to selectively block CD47/SIRPα binding in tumor cells with high ICAM-1 expression. In contrast to the benchmark CD47 monoclonal antibody, VBI-002 exhibits minimal red blood cell binding and does not cause hemagglutination. In non-human primate (NHP) safety study, four weekly doses of 60 mg/kg VBI-002 were well-tolerated with only mild and reversible reduction in hemoglobin and platelets. Here we further investigate the anti-tumor activities of VBI-002 in solid tumors. Gene expression analysis suggested that ICAM-1 is highly expressed in non-small cell lung cancers (NSCLC), melanoma and a subset of hepatocellular carcinoma. Flow cytometry studies confirmed the frequent co-expression of ICAM-1 and CD47 in these tumors. VBI-002 has potent ICAM-1 dependent SIRPα blocking, antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity activities against cell lines of NSCLC, hepatocellular carcinoma, and melanoma. Furthermore, in vivo efficacy studies demonstrated that VBI-002 has potent single agent activities in cell line derived xenograft (CDX) models of NSCLC, hepatocellular carcinoma, and melanoma. Weekly dose of 20 mg/kg VBI-002 is sufficient to stop tumor growth or drive tumor regression in most models. Notably, VBI-002 is highly effective against NSCLC CDX models carrying Kras mutation and it synergizes with paclitaxel to provide additional anti-tumor activity. In conclusion, VBI-002 is a versatile novel CD47xICAM-1 bispecific antibody with potent single agent activity in various solid tumors and combined well with standard of care chemotherapies for added activity. Thus, VBI-002 warrants clinical evaluations as a single agent and in combination with standard of care chemotherapies. Citation Format: Oi Kwan Wong, Xinhua Wang, Leonard Post, Xiaocheng Chen. VBI-002, a CD47xICAM-1 bispecific antibody for the treatment of hepatocellular carcinoma, melanoma and non-small cell lung cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6334.

Abstract 1870: VBI-003, a CD47xEpCAM bispecific antibody as a potential treatment for colorectal and small cell lung cancers

Abstract CD47/SIRPα axis is an important checkpoint of innate immune system and is often exploited by tumor cells to evade host immune surveillance. In recent years, therapies such as anti-CD47 monoclonal antibodies (mAbs) and SIRPα fusion proteins that target this axis have garnered much attention. Although exciting clinical activities have been observed in hematological cancers, responses in solid tumors are less impressive and often require complex combination strategies. We previously described a novel CD47xEpCAM bispecific antibody, VBI-003, built on a human IgG1 backbone to harness the power of blocking CD47/SIRPα pathway while preserving the effector functions of IgG1 Fc (J ImmunoTher Cancer 2021;9:doi: 10.1136/jitc-2021-SITC2021.274). The bispecific design aims to improve selectivity so that SIRPα blocking and stable binding occur selectively on tumor cells where both CD47 and EpCAM are present. In vitro studies show that VBI-003 exhibits minimal red blood cell binding and does not cause hemagglutination, in contrast to benchmark CD47 mAb. VBI-003 has potent single agent activity in cell line derived xenograft (CDX) models of gastric and esophageal cancers. Here, we further explored the anti-tumor activities of VBI-003 in areas with great unmet needs such as small cell lung cancers (SCLC) and colorectal cancers (CRC). Gene expression analysis from public datasets reveals frequent expression of CD47 and EpCAM in SCLC and CRC tumors. Flow cytometry studies confirmed co-expression of CD47 and EpCAM on SCLC and CRC cell lines. Furthermore, in vitro assays showed that VBI-003 has potent EpCAM dependent SIRPα blocking, antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity activities towards SCLC and CRC cell lines. Moreover, VBI-003 demonstrated potent single agent activity in multiple CDX models of SCLC with tumor regression and often outperformed benchmark CD47 mAb. Significant tumor growth inhibition was observed in colorectal cancer models post VBI-003 treatment, with additional activity by combination with standard of care chemotherapies. VBI-003 synergized with irinotecan and outperformed monotherapy groups in CDX models of CRC. In conclusion, VBI-003 has potent single agent activity in SCLC tumor models and combined synergistically with irinotecan in CRC models. Our data support further investigation of VBI-003 as a treatment for CD47 and EpCAM expressing SCLC and CRC. Citation Format: Oi Kwan Wong, Xinhua Wang, Xiaocheng Chen, Leonard Post. VBI-003, a CD47xEpCAM bispecific antibody as a potential treatment for colorectal and small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1870.

Abstract 4531: Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study

Abstract ZL-1211, a humanized monoclonal IgG1 antibody targeting CLDN18.2, induces cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We developed ex vivo ADCC and CDC assays to study the mechanism of actions of ZL-1211. Our preclinical data has demonstrated greater antitumor activities than the clinical leading benchmark via enhanced ADCC by mutation in Fcγ domain and potent CDC activities. We used those ex vivo assays to further profile the clinical specimens from patients collected at baseline (pre-infusion) and the following cycles (post-infusion) in our phase 1 study (NCT05065710). Preliminary results in clinical samples indicated that baseline ADCC and CDC capabilities were correlated with anti-tumor activity of ZL-1211 in patients with CLDN18.2-positive locally advanced or metastatic solid tumors, suggesting probable predictive biomarkers. Results also showed that changes in circulating ZL-1211 mediated ADCC and CDC activities were dose-dependent and in accordance with the pharmacokinetic (PK) profile. In addition, we performed differential gene expression analysis and real-time PCR to compare gene expression in ZL-1211 responder and non-responder PDX models and CCLE cell lines. Our findings reveal that several immune-related genes may serve to predict the responses to ZL-1211 treatment. Other potential predictive biomarkers such as Fcγ receptor CD16A polymorphism and baseline stromal CD8 expression were also evaluated in clinical samples. Taken together, analytical approaches developed and validated from preclinical studies were successfully translated to characterize clinical samples in the current Phase 1 trial. The exploratory analysis indicated the clinical relevance of multiple biomarkers and their potential as predictive biomarkers of ZL-1211. Citation Format: Zhen Luo, Xiao Wang, Xuehuo Zeng, Jiaqing Yi, Tegan Burns, Herman Liu, Xinyu Zhang, Wenyu Liu, Karl Hsu, Hua Gong. Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4531.

Abstract 1862: Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models

Abstract Delta-like 1 homolog (DLK-1), a non-canonical notch ligand, is a membrane protein with 6 tandem EGF-like motifs in extracellular region. While the expression of DLK-1 is restricted in some neuroendocrine tissues such as adrenal gland and hypophysis in normal adult tissues, its over-expression in cancers have been reported in a variety of cancer types, such as hepatocellular carcinoma (HCC), small cell lung carcinoma, ovarian carcinoma, adrenal cortical carcinoma, pancreatic carcinoma, gastrointestinal stromal tumor, etc. Interestingly, it has been suggested that DLK-1 is a marker of cancer stem cells in HCC. CBA-1205 is a humanized antibody (IgG1/κ) targeting DLK-1, which is glycoengineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. CBA-1205 showed no toxicology issues in cynomolgus monkey GLP studies and the Phase 1 clinical trial in patients with solid tumors is under way (JapicCTI-205384). Previously, we demonstrated potent anti-tumor efficacy of CBA-1205 as a monotherapy in multiple human DLK-1 positive cancer xenograft mouse models including HCC (AACR 2019 annual meeting). Lenvatinib is an oral multi-kinase inhibitor which has been used for 1st line therapy of unresectable advanced HCC globally. The purpose of present study is to evaluate the combinatorial efficacy of CBA-1205 with lenvatinib in two HCC xenograft models (Hep3B and HepG2). Hep3B model was sensitive to lenvatinib monotherapy; oral daily dosing of lenvatinib at 3 mg/kg treatment showed 50% tumor growth inhibition (TGI) and at 10 mg/kg treatment showed more than 80% TGI. In contrast HepG2 model was less sensitive to Lenvatinib; TGI at 3 mg/kg treatment group was 20 % and that for 10 mg/kg treatment group was 50 %. When CBA-1205 (1 mg/kg, twice a week, total 4 times) was intraperitoneally administered in combination with 3 mg/kg of lenvatinib (daily, total 10 times) in Hep3B xenograft mice, synergistic and long-lasting anti-tumor efficacy was observed compared either with CBA-1205 treatment alone or lenvatinib treatment alone. Tumor growth of all mice (N=8) treated with CBA-1205 and lenvatinib showed almost complete inhibition even 2 weeks after the last dosing. More importantly, 4 out of 8 mice in CBA-1205 and lenvatinib treatment group showed tumor regression. Similar results were obtained with CBA-1205 at 1 mg/kg dosage in combination with lenvatinib at 10 mg/kg dosage in HepG2 xenograft model. In conclusion, we demonstrated synergistic and long-lasting anti-tumor efficacy of CBA-1205 in combination with lenvatinib in two different HCC pre-clinical models suggesting that the combination could be a new treatment option for unresectable advanced HCC. Citation Format: Koji Nakamura, Izumi Sakaguchi, Kota Takahashi. Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1862.

Abstract 4999: Preclinical evaluation of MCLA-129, a bispecific antibody targeting EGFR and c-MET on solid tumor cells, in comparison with amivantamab

Abstract The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors. MCLA-129 is an antibody-dependent cellular cytotoxicity (ADCC) enhanced Biclonics® that targets epidermal growth factor receptor (EGFR) and c-MET in non-small cell lung cancer (NSCLC) and other solid tumors. MCLA-129 is a potent inhibitor of tumor growth applying various mechanisms of action (MOA), including inhibition of c-MET and EGFR signaling, antibody-dependent cellular phagocytosis (ADCP) and ADCC. Here we compared the MOA of MCLA-129 with the MOA of the EGFR-c-MET bispecific antibody amivantamab. The binding of MCLA-129 to tumor cells expressing different levels of EGFR and c-MET was quantified using radiolabeled antibodies. Scatchard plot analysis showed that the affinity of MCLA-129 for both c-MET and EGFR is in the low nanomolar range. In FACS, MCLA-129 and amivantamab show similar binding to tumor cells harboring EGFR or c-MET exon14-skipping mutations. Crossblock experiments with the c-MET binding antibody onartuzumab analog showed that MCLA-129 competes for the same HGF-binding region of c-MET, which is distinct from the binding site of amivantamab according to Neijssen et al, 2021 (PMC8113745). MCLA-129 and amivantamab did not downmodulate EGFR or c-MET. ADCP was also comparable for MCLA-129 and amivantamab, as measured with monocyte-derived macrophages incubated with pHrodo-labeled target cells with readout of tumor cell phagocytosis on the Incucyte® system. In contrast, MCLA-129 displayed ADCC activity similar to or more potently than amivantamab in an ADCC reporter assay with either high affinity FcγRIII 158V-variant or low affinity 158F-variant effector cells in absence or presence of soluble EGFR and soluble c-MET. MCLA-129-induced ADCC appears to be less affected than amivantimab by the presence of soluble EGFR and soluble c-MET. In conclusion, MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple MOA including inhibition of c-MET and EGFR ligand binding, ADCP and ADCC comparable or more potent than amivantamab. A phase 1/2 clinical trial of MCLA-129 in solid tumors is ongoing. These data support the further clinical development of MCLA-129 in patients with NSCLC and other solid tumors. Citation Format: David J.J. de Gorter, Alexandre Deshiere, Arjen Kramer, Martijn van Rosmalen, Franziska Mortensen, Cecile Geuijen. Preclinical evaluation of MCLA-129, a bispecific antibody targeting EGFR and c-MET on solid tumor cells, in comparison with amivantamab. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4999.

Update on theApplication of Monoclonal Antibody Therapy in Primary Membranous Nephropathy.

When first introduced, rituximab (RTX), a chimeric anti-CD20 monoclonal antibody, brought about an alternative therapeutic paradigm for primary membranous nephropathy (PMN). Rituximab was shown to be effective and safe in PMN patients with kidney dysfunction, with. patients receiving second-line rituximab therapy achievingremission as effectively as those patients who had not previously received immunotherapy. No safety issues were reported. TheB cell-driven protocol seems to be as efficient asthe 375 mg/m2 × 4 regimen or 1 g × 2 regimen in achieving B cell depletion and remission, but patients with high M-type phospholipase A2 receptor(PLA2R) antibody levels may benefit from a higher dose of rituximab. While rituximab added another therapeutic option to the treatment regimen, it does have limitations as 20 to 40% of patients do not respond. Not all patients respond to RTX therapy for lymphoproliferative disorders either, therefore further novel anti-CD20 monoclonal antibodies have been developed and these may provide alternative therapeutic options for PMN. Ofatumumab, a fully human monoclonal antibody, specifically recognizes an epitope encompassing both the small and large extracellular loops of the CD20 molecule, resulting in increased complement-dependent cytotoxic activity. Ocrelizumab binds an alternative but overlapping epitope region to rituximab and displays enhanced antibody-dependent cellular cytotoxic (ADCC) activities. Obinutuzumab is designed to have a modified elbow-hinge amino acid sequence, leading to increased direct cell death induction and ADCC activities. In PMN clinical studies, ocrelizumab and obinutuzumab showed promising results, while ofatumumab displayed mixed results. However, there is a lack of randomized controlled trials with large samples, especially direct head-to-head comparisons. Alternative molecular mechanisms have been suggested in this context to explore novel therapeutic strategies. B cell activator-targeted, plasma cell-targeted and complement-directed treatments may lead to novel therapy paradigms for PMN. Exploratory strategies for the use of drugs with different mechanisms, such as a combinationof rituximab and cyclophosphamide and a steroid, a combination of rituximab anda calcineurin inhibitor, may provide more rapid and efficient remission, but the combination of standard immunosuppression with rituximab could increase infection risk.

Identification of broad-spectrum neutralizing antibodies against influenza A virus and evaluation of their prophylactic efficacy in mice

Influenza A virus continuously infects humans and the antigenic shifts of this respiratory virus enable it to cross the species barrier, threatening public health with the risk of pandemics. Broadly neutralizing antibodies (bnAbs) that target the antigenic surface glycoprotein, hemagglutinin (HA), of influenza A virus protect against various subtypes of the virus. Here, we screened a human scFv library, through phage display and panning against recombinant HA proteins, to discover human monoclonal antibodies (mAbs) that are broadly active. Consequently, two human mAbs, named G1 and G2, were identified, which target the HA proteins of the H1N1 and H3N2 subtypes, respectively. G1 was shown to have broad binding ability to different HA subtypes of group 1. By contrast, G2 had higher binding affinity but sensed exclusively H3 subtype-derived HAs. In a cell culture-based virus-neutralizing assay, both G1 and G2 efficiently suppressed infection of the parental influenza A viruses of H1N1 and H3N2 subtypes. Mode-of-action studies showed that the G1 antibody blocked HA2-mediated membrane fusion. Meanwhile, G2 inhibited HA1-mediated viral attachment to host cells. It is noteworthy that both antibodies elicited antibody-dependent cellular cytotoxicity (ADCC) activities by recruiting FcγRIIIA-expressing effector cells. In mouse challenge models, single-shot, intraperitoneal administration of chimeric G1 and G2 antibodies with the mouse IgG constant region completely protected mice from viral infections at doses above 10 and 1 mg/kg, respectively. The newly identified bnAbs, G1 and G2, could provide insight into the development of broad-spectrum antivirals against future pandemic influenza A virus involving group 1- or H3-subtyped strains.

A Novel Fc-Engineered Anti-HER2 Bispecific Antibody With Enhanced Antitumor Activity.

Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.

IgG N-glycan Signatures as Potential Diagnostic and Prognostic Biomarkers.

IgG N-glycans are an emerging source of disease-specific biomarkers. Over the last decade, the continued development of glycomic databases and the evolution of glyco-analytic methods have resulted in increased throughput, resolution, and sensitivity. IgG N-glycans promote adaptive immune responses through antibody-dependent cellular cytotoxicity (ADCC) and complement activation to combat infection or cancer and promote autoimmunity. In addition to the functional assays, researchers are examining the ability of protein-specific glycosylation to serve as biomarkers of disease. This literature review demonstrates that IgG N-glycans can discriminate between healthy controls, autoimmune disease, infectious disease, and cancer with high sensitivity. The literature also indicates that the IgG glycosylation patterns vary across disease state, thereby supporting their role as specific biomarkers. In addition, IgG N-glycans can be collected longitudinally from patients to track treatment responses or predict disease reoccurrence. This review focuses on IgG N-glycan profiles applied as diagnostics, cohort discriminators, and prognostics. Recent successes, remaining challenges, and upcoming approaches are critically discussed.

Activated CTLA-4-independent immunosuppression of Treg cells disturbs CTLA-4 blockade-mediated antitumor immunity.

Combination therapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) monoclonal antibodies (mAbs) has dramatically improved the prognosis of patients with multiple types of cancer, including renal cell carcinoma (RCC). However, more than half of RCC patients fail to respond to this therapy. Regulatory T cells (Treg cells) are a subset of highly immunosuppressive CD4+ T cells that promote the immune escape of tumors by suppressing effector T cells in the tumor microenvironment (TME) through various mechanisms. CTLA-4 is constitutively expressed in Treg cells and is regarded as a key molecule for Treg-cell-mediated immunosuppressive functions, suppressing antigen-presenting cells by binding to CD80/CD86. Reducing Treg cells in the TME with an anti-CTLA-4 mAb with antibody-dependent cellular cytotoxicity (ADCC) activity is considered an essential mechanism to achieve tumor regression. In contrast, we demonstrated that CTLA-4 blockade without ADCC activity enhanced CD28 costimulatory signaling pathways in Treg cells and promoted Treg-cell proliferation in mouse models. CTLA-4 blockade also augmented CTLA-4-independent immunosuppressive functions, including cytokine production, leading to insufficient antitumor effects. Similar results were also observed in human peripheral blood lymphocytes and tumor-infiltrating lymphocytes from patients with RCC. Our findings highlight the importance of Treg-cell depletion to achieve tumor regression in response to CTLA-4 blockade therapies.

Prevalence of claudin 18.2 (CLDN18.2) and the association of biomarkers with clinical activity in patients with gastric or gastroesophageal adenocarcinoma (G/GEJa) treated with zolbetuximab.

466 Background: CLDN18.2, a targetable biomarker, is a tight junction protein normally confined to gastric mucosa of healthy tissue and often retained in G/GEJa. Zolbetuximab, a chimeric IgG1 monoclonal antibody, binds to CLDN18.2 and mediates cancer cell death via antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Phase 2 FAST study results showed prolonged survival with zolbetuximab+EOX (epirubicin, oxaliplatin, capecitabine) vs EOX in CLDN18.2-positive G/GEJa. In ILUSTRO Cohort 2, confirmed partial responses (PRs) and stable disease (SD) were previously reported in 63.2% and 10.5%, respectively, of patients (pts) treated with zolbetuximab+mFOLFOX6 (modified 5-FU, folinic acid, oxaliplatin). Here is an analysis of CLDN18.2 expression and exploratory biomarkers from 2 studies of zolbetuximab alone or with mFOLFOX6 in G/GEJa. Methods: Data are from phase 1 (NCT03528629) and phase 2 (NCT03505320, ILUSTRO Cohorts 1 and 2) studies. Pts with CLDN18.2-positive locally advanced/metastatic G/GEJa received zolbetuximab alone (phase 1 [ineligible for SOC], n = 18; phase 2 Cohort 1 [≥3rd-line], n = 30) or with mFOLFOX6 (phase 2 Cohort 2 [1st-line], n = 21). Archival (any time before treatment), baseline (during screening or ≤3 months before first study treatment), and on-treatment (Cycle 3) tumor samples were collected when possible. CLDN18.2 expression and immune cell populations were assessed by IHC. Blood samples collected longitudinally while on treatment (phase 2 study) were analyzed for ADCC activity (ex vivo cell-based assay), circulating tumor antigens, and circulating tumor DNA (ctDNA). Results: Of 416 screened pts in both studies, 26.2% (n = 109), 9.6% (n = 40), and 36.1% (n = 150) had tumors that were CLDN18.2-positive in 1%–49%, 50%–74%, and ≥75% of tumor cells ( = strong to moderate staining intensity), respectively; 28.1% (n = 117) were CLDN18.2-negative (0/1+). Analysis of pre- and on-treatment tissue samples (n = 8 matched pairs) with zolbetuximab alone showed an increased trend in immune cell infiltration, with CD8+ T cells and CD163+ cells showing the most consistent trend. A trend of increased on-treatment vs baseline ADCC activity was observed in PBMCs collected from pts treated with zolbetuximab alone and with mFOLFOX6. Rapid and deep decreases in circulating tumor antigens (in pts with elevated antigen levels) and ctDNA were observed with best overall responses of both PR and SD. Conclusions: CLDN18.2 is a high-prevalence biomarker in G/GEJa. Treatment with zolbetuximab is associated with tumor and peripheral biomarker changes related to its proposed mechanism of action. Clinical responses observed in pts treated with zolbetuximab and mFOLFOX6 are associated with correlative biomarkers of activity including rapid deep molecular responses (ctDNA). Clinical trial information: NCT03528629 .

A phase I/II study of ASKB589 (anti-claudin 18.2 [CLDN18.2] monoclonal antibody) in patients with solid tumors.

397 Background: ASKB589 is a humanized IgG1 monoclonal antibody against Claudin 18.2 (CLDN18.2) with high affinity and enhanced antibody-dependent cellular cytotoxicity activity. We report preliminary safety and efficacy data from an ongoing Phase I/II, first-in-human, dose-escalation and expansion study of ASKB589 in patients (pts) with advanced solid tumors (NCT04632108). Methods: A two-part study was initiated to determine the maximum tolerated dose (MTD), safety and tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of ASKB589 as monotherapy (Part A) and in combination with chemotherapy (Part B). Each part used a modified 3 + 3 dose escalation design. Responses were assessed per RECIST 1.1 every 2 cycles (6 weeks). Adverse events (AEs) were graded using CTCAE v5.0. In Part A, pts with heavily pre-treated solid tumors received ASKB589 intravenously (IV) at doses of 0.3, 1, 3, 6, 10,15 or 20mg/kg every 3 weeks (Q3W). In Part B, pts with gastric/gastro-esophageal junction (G/GEJ) cancers received ASKB589 IV at doses of 3, 6, 10 or 15 mg/kg Q3W in combination with capecitabine plus oxaliplatin. Results: As of September 9, 2022, 51 pts received ASKB589 and no drug limiting toxicity (DLT) was observed. In expansion, pts have been enrolled into 6, 10, 15 mg/kg doses. In Part A, 17 pts were enrolled in escalation and 13 in expansion. All pts had metastatic disease, and most (90%) had ≥2 lines of prior therapy. 24 (80%) pts had treatment-related adverse events (TRAEs), the most common being nausea (53%), vomiting (43%), hypoalbuminemia (40%) and loss of appetite (30%). While the majority of TRAEs were grade 1 or 2, 3 pts (10%) had grade 3 events. For the 9 evaluable pts (G/GEJ cancer who received ≥10 mg/kg ASKB589 with at least one post-baseline tumor assessment), 5 pts (45%) had decreases in tumor size (range 1-38% from baseline). 2 pts achieved a partial response (PR) for an objective response rate (ORR) of 22%. In addition, 6 pts had stable disease (SD) for a disease control rate (DCR) of 89%. In Part B, 13 pts were enrolled in escalation and 8 in expansion. These pts were with advanced disease (95% stage IV) and had no prior systemic treatment in this setting. 17 (81%) pts had TRAEs, the most common being nausea (76%), vomiting (66%), hypoalbuminemia (52%), granulocytopenia (38%) and hypoleukemia (33%). While the majority of TRAEs were grade 1 or 2, 8 pts (38%) had grade 3 events. For the 12 evaluable pts (G/GEJ cancer who received ≥6 mg/kg ASKB589 with at least one post-baseline tumor assessment), all had decreases in tumor size (range 6-66% from baseline). 9 pts achieved PR for an ORR of 75%. In addition, 3 pts had SD for a DCR of 100%. The PK profile showed dose-related increases in drug exposure across study doses. Conclusions: ASKB589 showed a manageable safety profile up to doses of 20 mg/kg and promising antitumor activity, both as monotherapy and in combination with chemotherapy. Clinical trial information: NCT04632108 .

Antitumor activities of a defucosylated anti‑EpCAM monoclonal antibody in colorectal carcinoma xenograft models.

Epithelial cell adhesion molecule (EpCAM) is a typeI transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (KD)=2.2x10‑8 M] using flow cytometry. EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderate binding‑affinity (KD=1.5x10‑8M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.