ADCC Activity Research Articles

Abstract 621: ZL-1211 exhibits robust anti-tumor activity by enhancing ADCC and activating innate and adaptive immunity in CLDN18.2-high and -low expressing gastric cancer models

Abstract CLDN18.2 (Claudin18.2)-targeting therapeutic antibodies have shown promising clinical efficacy in ~30% of gastric cancers expressing high levels of CLDN18.2 and less pronounced activity in the majority of low expressing malignancies. ZL-1211 is a monoclonal antibody targeting CLDN18.2 engineered to promote enhanced ADCC with the goal of achieving more potent activity in a wider spectrum of high- and low-CLDN18.2 expressing tumors than the leading clinical benchmark. To further assess the potency of ZL-1211, we utilized a panel of gastric tumor cell lines that endogenously express CLDN18.2 and more accurately reflect target levels in human gastric tumors than engineered overexpressing models. ZL-1211 demonstrated more robust in vitro ADCC activity than clinical benchmark not only in CLDN18.2-high but also -low expressing gastric cell lines. Greater anti-tumor efficacy was also observed in mouse xenograft models with CLDN18.2-high, -medium, and low-expressing gastric cell lines in response to treatment with ZL-1211 compared to the clinical benchmark. NK cell depletion abrogated ZL-1211-mediated ADCC activity in vitro. ZL-1211 efficacy in vivo was also dependent on the presence of an NK compartment, as CLDN18.2-expressing gastric tumors grown in Balb/c nude with an intact NK function were sensitive to ZL-1211 treatment, while NOD-SCID with partially competent NK activity exhibited minimal response, and NCG without NK cells did not display any evidence of in vivo efficacy. Strikingly, NK cells strongly induced an inflammatory response in response to ZL-1211 treatment, including increased IFNγ, TNFα, and IL-6 production. Interestingly, ZL-1211 treatment led to NK cell and monocyte stimulation and enhanced CD8+ T cell activation, suggesting a mechanism of action that not only involves innate but also adaptive immunity. Taken together, our data suggest that ZL-1211 more effectively targets CLDN18.2-high gastric cancers as well as -low expressing malignancies that are not eligible for treatment with the leading clinical benchmark by inducing a robust ADCC response and activating innate and adaptive immunity to enhance anti-tumor efficacy. Clinical activity of ZL-1211 is currently under evaluation in a Phase I clinical trial (NCT05065710). Citation Format: Hiroyasu Konno, Tracey Lin, Bee-Chun Sun, Renyi Wu, Christopher Szeto, David Bellovin, Karl Hsu, Omar Kabbarah. ZL-1211 exhibits robust anti-tumor activity by enhancing ADCC and activating innate and adaptive immunity in CLDN18.2-high and -low expressing gastric cancer models [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 621.

Abstract 2902: Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies

Abstract CD20 and CD37 are co-expressed on most B-NHL and CLL tumors. Systemic depletion of normal and malignant B-cells by anti-CD20 antibodies (e.g. rituximab and obinutuzumab) and anti-CD37 antibodies (e.g. otlertuzumab and BI 836826) has been well tolerated in clinical studies. Depletion of B-cells is reversible, with the ability to repopulate B-cells from hematopoietic stem cells. Dual targeting CD20 and CD37 may improve the clinical efficacy and decrease drug resistance. PSB202 is a novel biological entity composed of two engineered monoclonal antibodies which are produced from a single stable CHO cell line - a humanized anti-CD20 antibody (PSB102) and a humanized anti-CD37 antibody (PSB107) at a 1:1 molecular ratio. In vitro binding studies demonstrated that PSB202 specifically binds to human CD20 and CD37. The antibodies’ binding to FcγRIIIa was enhanced by reducing the fucose level on each antibody component. PSB202 directly induced potent autonomous killing via target engagement. PSB202 mediated depletion of human and monkey blood B-cells and malignant human B-cell lines through ADCC activity at EC50 of double digit pM and CDC activity at EC50 of low single digit nM. Importantly, PSB202 effectively depleted B-cells in ex vivo cultures of blood cells from CLL and NHL patients who were refractive to rituximab treatment. The antitumor activity of PSB202 was demonstrated in human xenograft models in mice. PSB202 was able to reduce different human lymphoma tumors by up to ~97.9% in a dose-dependent manner, suggesting that PSB202 might be potentially efficacious against multiple types of B-cell malignancies. Synergy was demonstrated for PSB202 in combination with lenalidomide. Toxicokinetic assessment in cynomolgus monkeys showed a dose proportional exposure for both antibody components, with mean terminal elimination half-life (T1/2) of ~60 and ~68 hours for the anti-CD20 and anti-CD37 component, respectively. PBS202 was well tolerated, with observed toxicities mostly related to its activity as a B-cell depletion agent. As expected, PSB202 when administered at ≥ 10 mg/kg resulted in nearly complete depletion of B lymphocytes 24 hours following the 1st dose. B-cells returned to near baseline within 2 months after the last dose at 10 mg/kg. PSB202 can deplete normal and malignant B-cells by targeting two clinically validated targets through multiple distinct mechanisms including direct B-cell killing, enhanced ADCC activity, and CDC activity. PSB202 offers the potential to improve clinical efficacy, decrease drug resistance, provide administration convenience especially when combining with additional therapeutics, lower the cost for treatment, and reduce the unwanted side effects from chemotherapy. PSB202 is currently being evaluated in patients with previously treated, relapsed, indolent B-cell malignancies (NCT05003141). Citation Format: Paul Algate, Zhi Liu, Saying Yuan, Hua Liu, Cristina Domeier, Haiming Jiang, Yuanzhi Lao, Yanling Xu, William C. Fanslow, Ron Schoner, Wei Yan. Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies [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 2902.

Abstract 336: Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer

Abstract Introduction: MCLA-129 is an ADCC-enhanced IgG1 bispecific antibody that targets epidermal growth factor receptor (EGFR) and c-MET. The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors (TKIs). This study investigated the MCLA-129 mechanism of action in vitro and effectiveness in vivo in an EGFR exon20 insertion (ex20ins) non-small cell lung cancer (NSCLC) model. Methods: To study the effect of MCLA-129 on c-MET and EGFR dimerization, β-galactosidase enzyme complementation assays were performed. Ligand-dependent phosphorylation of EGFR and c-MET was assessed in vitro on the NCI-H1650 NSCLC cell line by immunoblotting. Shotgun mutagenesis epitope mapping was used to identify the binding epitope of MCLA-129 to EGFR. ADCC activity of MCLA-129 was determined in NSCLC cells expressing different levels of EGFR and c-MET using an ADCC reporter assay with high affinity FcγRIII 158V-variant and low affinity 158F-variant effector cells. ADCP was measured with monocyte-derived macrophages incubated with pHrodo-labeled HCC827 NSCLC target cells with readout of tumor cell phagocytosis on the Incucyte® system. For the evaluation of efficacy in vivo in the EGFR exon20ins PDX model, MCLA-129 was administered at 2.5, 8 or 25 mg/kg i.p. weekly. Results: MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and prevents receptor dimerization. Consequently, MCLA-129 inhibits phosphorylation of EGFR and c-MET as demonstrated in NCI-H1650 cells. Mutagenesis analysis identified critical binding residues of domain III of EGFR for MCLA-129 binding that are distinct from cetuximab binding. MCLA-129 displayed potent ADCC activity on NSCLC cell lines using effector cells expressing either high or low affinity FcγRIII variant. MCLA-129 demonstrated potent dose-dependent ADCP of HCC827 NSCLC cells. Finally, MCLA-129 led to significant dose-dependent regression of a patient-derived EGFR exon20ins NSCLC tumor in a mouse PDX model. Conclusion: MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple mechanisms of action including potent inhibition of c-MET and EGFR ligand binding, ligand-induced receptor dimerization and phosphorylation, ADCC and ADCP. MCLA-129 demonstrates significant growth inhibition of a patient-derived EGFR exon20ins tumor in a preclinical xenograft model. 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, including NSCLC with EGFR exon20ins, and other solid tumors. Citation Format: David J. de Gorter, Marie O'Connor, Alexandre Deshiere, Martijn van Rosmalen, Szabolcs Fatrai, Jeroen Lammerts van Bueren, Cecile A. Geuijen. Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer [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 336.

Abstract 2068: An ADCC enhanced anti-CCR8 antibody, which preferentially depletes tumor regulatory T cells and inhibits tumor growth

Abstract Tumor associated FOXP3+ regulatory T cells (Tregs) suppress antitumor immunity, thereby hindering protective immunosurveillance of tumors and hampering effective antitumor immune responses in tumor-bearing hosts. Eliminating the suppressive effects of tumor associated Tregs could, therefore, enhance antitumor activity of existing immunotherapies. CCR8 is a seven transmembrane G-coupled protein whose expression is highly upregulated on tumor associated Tregs in several types of tumors, but not on peripheral Tregs. The specific expression on tumor associated Tregs makes CCR8 an ideal target for treating cancers. The chemokine CCL1 interacts with the N terminus and second extracellular domain of CCR8. CCL1 produced by tumor associated macrophages can induce Tregs migration to the tumors. PSB114, a humanized anti-hCCR8 IgG1 antibody, was developed to block CCL1 mediated Tregs chemotaxis and deplete tumor associated CCR8-expressing Tregs. PSB114 was affinity matured by yeast display and glyco-modified to enhance its ADCC activity. PSB114 has high binding affinity for human CCR8 without significant binding to other membrane proteins, resulting in a favorable PK in hFcRn transgenic mice (T1/2 > 13 days). PSB114 is highly specific for the activated Tregs with low picomolar EC50 of depletion activity. When tested in bioassays, PSB114 enhances T cell activation and TNFα production from activated PBMCs. In a humanized mouse model, PSB114 preferentially depletes tumor infiltrating Tregs and increases NK and CD8 T cell infiltration within the tumor. Moreover, PBS114 significantly inhibited MC38 tumor growth in hCCR8 knock-in mice. These results demonstrate the potential of PSB114 for treating cancers in human. Citation Format: Jamil A. Haque, Cristina Domeier, Paul A. Algate, Zhi Liu, Wei Yan, YuFeng Peng. An ADCC enhanced anti-CCR8 antibody, which preferentially depletes tumor regulatory T cells and inhibits tumor growth [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 2068.

Abstract 313: Novel bioengineered monoclonal antibodies targeting oxidized macrophage migration inhibitory factor as anti-cancer therapeutics and companion diagnostics

Abstract Background: Macrophage Migration Inhibitory Factor (MIF) has been described as a pleiotropic cytokine known to exacerbate tumor growth (Chesney and Mitchell, 2015). Due to its ubiquitous nature, MIF can be considered as an inappropriate target for therapeutic intervention. However, we discovered oxidized MIF (oxMIF) as the disease-related isoform of MIF, which is specifically present in tumor tissue (Schinagl A et al., 2016). A 1st generation anti-oxMIF monoclonal antibody (mAb) demonstrated an acceptable safety profile and efficacy signal in a phase 1 clinical trial (Mahalingam D et al., 2020). Two bioengineered 2nd generation anti-oxMIF mAbs with highly improved biophysicochemical and biological properties were generated and compared to the 1st generation anti-oxMIF mAb. Both bioengineered mAbs share identical variable domains, but distinct heavy-chain constant regions, to increase effector functions and efficacy of the therapeutic mAb ON203 and to reduce interactions with FcγRs for the radio diagnostic mAb ON102. Methods: Hydrophobicity and stability were determined by HIC and SEC. ADCC and ADCP activity were investigated by reporter and PBMC-mediated cell killing assays. Tumor penetration was assessed using IRDye 800CW or Zr89-labeled mAb in tumor-bearing Balb/c or Balb/c nude mice. PK and bioavailability were assessed in Balb/c nude mice. Efficacy was determined in PC3 xenograft models in NMRI nude mice. Results: Bioengineering significantly reduced hydrophobicity of ON203 and ON102, leading to improved stability and strongly reduced aggregation but retaining the low nM affinity for oxMIF. This further resulted in a two-fold improved bioavailability and tumor accumulation after 24-48h and a three-fold enhanced retention on day 7 in mice harboring solid tumors of the colon, when compared to the 1st generation anti-oxMIF mAb. In contrast to previously described anti-oxMIF reference, ON203 and ON102 did not mediate any unspecific release of MCP-1, IL-6, or TNF-α from PBMCs. Reporter and PBMC-mediated cell killing assays proved ADCC activity of ON203 with EC50 values of 0.1-0.8 nM and ADCP activity with EC50 values of 2.3 nM, whereas the 1st generation anti-oxMIF mAb was at least 10-fold less potent. Our findings for ON203 translated into abolishment of tumor growth in NMRI nude mice harboring human PC3 prostate cancer cells, showing superior efficacy compared to the 1st generation anti-oxMIF mAb. Conclusion: ON203 has a high potential to significantly improve efficacy compared to the 1st generation anti-oxMIF mAb, with Zr89-ON102 as companion diagnostic for patient stratification. We aim to develop these anti-oxMIF mAbs together for clinical use to create new treatment options for patients with solid tumors, with a clear rationale to combine ON203 with other immunotherapies or checkpoint inhibitors (Noe and Mitchell, 2020). Citation Format: Alexander Schinagl, Michael Thiele, Irina Mirkina, Gregor Rossmueller, Alejandro A. Puchol Tarazona, Randolf J. Kerschbaumer. Novel bioengineered monoclonal antibodies targeting oxidized macrophage migration inhibitory factor as anti-cancer therapeutics and companion diagnostics [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 313.

Abstract 5524: AK127, a novel monoclonal antibody (mAb) targeting T cell immunoreceptor (TIGIT)

Abstract Introduction: Immune checkpoint blocking antibodies achieved great clinical success, yet a large fraction of cancer patients receive minimum benefit from current immunotherapies targeted PD-1 and CTLA-4. As a new multifunctional immune checkpoint molecule, TIGIT holds promise to become an important cancer immune therapy target. AK127 is a novel humanized immunoglobulin (Ig) G1 monoclonal antibody (mAb) targeting T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT). AK127 binds to TIGIT specifically and blocks the interaction of TIGIT with its ligands, thus relieves immune suppression and promotes anti-tumor immune response. Method: The effect of AK127 on secretion of IL-2 in mixed culture of Jurkat-TIGIT and HT1080-aCD3scFv cells (ELISA) was determined in cellular assays. Binding kinetics of AK127 to C1q, FcγR were measured by Fortebio. ADCC, ADCP and CDC activities were determined in cellular assays. In preclinical in-vivo pharmacology studies, synergistic anti-tumor activity of AK127 and a bi-specific anti-PD1/CTLA4 antibody Cadonilimab (AK104) was investigated in BALB/c-hPD-1/hTIGIT transgenic mice with CT26.WT tumor model. Result: AK127 blocked the immune inhibitory signal mediated by TIGIT, resulting in enhanced IL-2 secretion. AK127 exhibited binding to FcγIa, FcγRIIIa and C1q, and eliciting ADCC, ADCP and CDC. In animal models, both AK104 (0.5 mg/kg) and AK127 (4 mg/kg) significantly inhibited tumor growth. The combination of AK127 and AK104 produced a significantly enhanced anti-tumor effect than either monotherapy, demonstrating the synergistic anti-tumor activity of AK127 combined with AK104. Conclusion: AK127 binds to human TIGIT with high affinity, competitively blocks the binding of TIGIT with its ligand CD155 and CD112, relieves immune suppression in both cellular and animal model systems, and promotes anti-tumor immune response. Citation Format: Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Chunshan Jin, Na Chen, Dennis Xia, Peng Zhang, Max Wang, Michelle Xia, Baiyong Li. AK127, a novel monoclonal antibody (mAb) targeting T cell immunoreceptor (TIGIT) [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 5524.

Abstract 324: Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers

Abstract Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein expressed during development and at lower levels in normal adult tissues. TROP2 has pleiotropic functions including formation of cell-cell junctions by mediating interactions with extracellular and intracellular proteins. In situations of TROP2 deficiency its role in normal tissues appears to be compensated by expression of EpCAM, a highly related family member. In cancer, the levels of TROP2 are increased in many epithelial tumor types. Overexpression of TROP2 confer oncogenic behavior in several in vitro and in vivo models. Both its extracellular and intracellular domains play a role in modulating interactions with ligands and/or receptors and with intracellular signaling partners to result in an oncogenic phenotype. In many solid tumors TROP2 upregulation is associated with increased tumor aggressiveness, metastasis, and decreased survival in large groups of difficult-to-treat cancers, making it an attractive target for cancer therapy. TROP2-based ADCs have been developed and one has been approved for therapeutic use in triple negative breast cancer and advanced bladder cancer. The toxicity associated with the toxin payload limits their use including in combination with chemotherapy in the earlier lines of treatment, and by the development of resistance to the payload. We isolated several anti-TROP2 heavy chain-only antibodies (HCAb) by immunization of a proprietary transgenic HCAb mouse. In this abstract, we describe the properties and activity of several of these antibodies. These antibodies are characterized by potent in vivo antitumor activity in established tumor models in NCG mice in the absence of ADCC, and by the inhibition of tumor cell viability in vitro, directly and in the absence of any effector cells. In NCG mice, which are characterized by a short antibody half-life, anti-TROP2 HCAbs displayed a T1/2 of approximately 3.5 days. These antibodies displayed very potent anti-tumor activity in established tumor xenograft models, causing tumor regression at 2 mg/kg and 80% TGI at 0.5 mg/kg in NCG mice when administered once per week. They also caused tumor regression when dosed once every three weeks in NCG mice and in tumors of very large sizes. Therefore, these antibodies directly target one or more aspects of TROP2 tumor biology that have a profound and long-lasting effect on the cancer cells. Upon glycoengineering, the afucosylated version of the antibodies displayed a strongly enhanced ADCC activity. In vitro they are characterized by different internalization rates which define several non-overlapping epitopes on TROP2. They are characterized by picomolar binding affinity to recombinant human TROP2 as determined by SPR. These antibodies bind with greater affinity to human and cyno but bind poorly to mouse, rat and rabbit. These potent naked TROP2 antibodies represent new opportunities to address TROP2 expressing cancers. Citation Format: Israel Matos, Yahya Ashraf, Hiba Zahreddine, Liying Gong, Amanda Baumholtz, Alex Zhou, Claire Bonfils, Aniel Moya-Torres, Yun Cui, Xiaowei Wang, Richard Wargachuk, Tiffany Cheng, Elijus Undzys, Shugang Yao, Jacynthe Linda Tolouse, Dominic Hou, Gordon Ngan, Luis daCruz, David Young. Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers [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 324.

Abstract 2857: DR30303, a SMART-VHHBody powered anti-CLDN18.2 VHH-Fc with enhanced ADCC activity for the treatment of gastric and pancreatic cancers

Abstract Claudin 18.2 (CLDN18.2) is aberrantly overexpressed in gastric cancer, pancreatic adenocarcinomas, and a fraction of non-small cell lung cancer. Its tumor specificity allows CLDN18.2 to be an attractive target for developing biotherapeutics for the treatment of gastric and pancreatic cancers. DR30303 is a humanized ant-CLDN18.2 heavy chain antibody Fc fusion protein (VHH-Fc) generated via Doer Biologics’ proprietary SMART-VHHBody platform. DR30303 exhibits strong and highly selective binding to CLDN18.2, but not its close splicing variant CLDN18.1. In addition, it exhibits enhanced ADCC activity as a result of Fc engineering and low immunogenicity risk validated via in-silico T-cell epitope predictor. Its smaller size (78 kDa vs 150 kDa for conventional full-length IgG) allows DR30303 to penetrate solid tumors faster and deeper, resulting in a strong competitive advantage and differentiation compared with other anti-CLDN18.2 antibodies in the market. Single-agent therapeutic potency of DR30303 was demonstrated in various gastric and pancreatic cell-derived xenograft (CDX) mouse models with a minimum effective dose of 0.3 mg/kg dose of DR30303 led to TGI≥90%, which is significantly more potent (p<0.05) than the benchmark reference Zolbetuximab analog in same molar ratio. Moreover, DR30303 showed strong synergistic anti-tumor activities with paclitaxel, gemcitabine, and EOF (epirubicin, oxaliplatin, and fluorouracil) in patient-derived xenograft (PDX) mouse models (gastric cancer) and CDX model (pancreatic cancer). DR30303 also demonstrated favorable pharmacokinetic properties (t1/2~ 120 hours) and tolerable toxicities (MTD≥ 748.5 mg) in both SD rats and cynomolgus monkeys. Doer Biologics submitted IND to the NMPA in November 2021 and plans to initiate the Phase 1 clinical trial in H1 2022 upon IND approval. Citation Format: Yongliang Fang, Gaofeng Yao, Weixiang Zhong, Wenwen Duan, Zhenxing Zhou, Xiaofang Wen, Yonglu Chen, Jingjin Fang, Yongwei Wang, Weiqin Jiang, Junfang Xu, Yanshan Huang. DR30303, a SMART-VHHBody powered anti-CLDN18.2 VHH-Fc with enhanced ADCC activity for the treatment of gastric and pancreatic cancers [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 2857.

Abstract 3590: ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells

Abstract Despite the promise of immunotherapy for cancer treatment, nearly 80% of patients fail to respond to checkpoint inhibitor (CPI) therapy. Regulatory T cells (Tregs), which inhibit immune responses in the tumor microenvironment via multiple suppressive mechanisms, have been proposed to play a key role in those patients who are not responding to CPI therapy. Therefore, targeted depletion of Tregs should promote more effective antitumor immunity. CCR8 is a chemokine receptor that is selectively expressed on highly activated human tumor-resident Tregs, and these intratumoral CCR8+ Tregs have been shown to drive immunosuppression that leads to poor prognosis for cancer patients. Here, we demonstrate that CCR8 is highly expressed on intratumoral FoxP3+ Treg cells in multiple cancers and is absent on other major immune cell populations in tumor microenvironment including effector T cells, conventional CD4 T cells, B cells, NK cells, some FoxP3+ cells, and myeloid cells. Importantly, no CCR8 protein expression was observed on any peripheral human leukocyte subset examined. These results provide strong rationale for targeting CCR8 as a cancer immunotherapy by selectively depleting the most suppressive intratumoral Treg cells. We have developed a humanized therapeutic antibody, ZL-1218, that binds to human CCR8 with high affinity and specificity and can induce potent ADCC activity enabling strong NK cell-mediated killing of CCR8-expressing Tregs. We show that in human CCR8 knock-in mouse models bearing syngeneic tumors, ZL-1218 reduces intratumoral Treg cells and thus elicits significant tumor growth inhibition in a dose-dependent manner. We have recently explored the potential for ZL-1218 in combination immunotherapy, examining the enhanced antitumor activity when ZL-1218 is combined with anti-PD-1. Using human dissociated tumor samples, we further observed that different tumor types may induce different CCR8 expression levels on intratumoral Tregs leading to multiple, distinct CCR8+ subsets in various indications. We are currently exploring the significance of these distinct populations and the impact of ZL-1218-mediated depletion of both CCR8 high- and CCR8 low-expressing subsets in multiple indications. Together, these data support the advancement of ZL-1218 into clinical evaluation as a novel therapeutic candidate to treat human solid tumors. Citation Format: Jing Zhang, Wenhua Shi, Min Chen, Xinchuan Dai, Hongshui Liu, Shou Li, Lina Wang, Bee-Chun Sun, Monica You, Vivian Morton, Qiuping Ye, Lishan Kang, Bing Wan, Peter Brams, David I. Bellovin. ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells [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 3590.

Antibody dependent cell cytotoxicity is maintained by the unmutated common ancestor of 6F5, a Gp41 conformational epitope targeting antibody that utilizes heavy chain VH1-2

In studies on monoclonal IgG antibodies (mAbs) from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated Abs against a complex conformational epitope with contributions from both gp41 the N terminal and C terminal heptad repeat helices. Despite using the VH1-2 gene segment, known to contribute to some of the broadest neutralizing Abs against HIV, members of these Abs, termed group 76C Abs, did not exhibit broad neutralization.Because of the high number of mutations and use of VH1-2, our goal was to characterize the non-neutralizing functions of Abs of group 76C, to assess if targeting of the epitope correlates with LTNP, and to assess the maturation of these Abs by comparison to their predicted common ancestor. Serum competition assays showed group 76C Abs were enriched in LTNPs, in comparison to VRC-01. Specific group 76C clones 6F5 and 6F11, expressed as recombinant Abs, both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. Competition with group 76C recombinant Ab 6F5 confirms 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc demonstrates comparable ADCC to 6F5 and 6F11 when using gp41 constructs of both clade B and clade C. The functional capability of Abs utilizing germline VH1-2 has implications for disease control and vaccine development.

Abstract OT2-15-01: Updated analysis of MCLA-128 (zenocutuzumab), trastuzumab, and vinorelbine in patients (pts) with HER2 positive/amplified (HER2+) metastatic breast cancer (MBC) who progressed on previous anti-HER2 ADCs

Abstract Background Zenocutuzumab is a humanized bispecific full-length IgG1 antibody targeting both HER2 and HER3 with enhanced ADCC activity. The unique Dock & Block mechanism inhibits HER3 from interacting with its ligands by targeting HER2 at a different epitope than trastuzumab, that optimally positions it to block HER2/HER3 dimerization and downstream PI3K/AKT/mTOR signaling. In MBC, HER3 overexpression and/or HER3 ligand upregulation are important drivers of carcinogenesis leading to trastuzumab resistance, indicating a potential role for zenocutuzumab. Preclinical activity was seen in HER2+ breast cancer models when zenocutuzumab was combined with trastuzumab. Single-agent zenocutuzumab showed consistent antitumor activity in heavily pretreated HER2+ MBC pts. This phase 2, open-label study explored zenocutuzumab/trastuzumab/vinorelbine in MBC. Methods This open-label study planned to enroll up to 40 evaluable pts with HER2+ MBC progressing after up to 5 anti-HER2 lines of therapy including trastuzumab, pertuzumab, and an anti-HER2 ADC. This sample size with a clinical benefit rate (CBR) of 45% would provide adequate precision to exclude 30% (lower limit of 90% CI > 30%). The threshold for the CBR rate at 24 w was based on the assumption that progression-free survival (PFS) follows an exponential distribution with a median of 5 months (clinically relevant) and 3.5 months (not clinically relevant).Pts received zenocutuzumab (750 mg, 2h IV), trastuzumab (8 mg/kg loading, then 6 mg/kg), and vinorelbine (25 mg/m², D1 and 8), q3w. A safety run-in of zenocutuzumab + trastuzumab ± vinorelbine was performed. The primary endpoint of the study was CBR at 24 w (tumor assessment [TA] by RECIST 1.1, per investigator), secondary endpoints include CBR at 24 w (TA by RECIST 1.1, per central review), overall response rate (ORR), safety, biomarkers, and pharmacokinetics. Cutoff date for the efficacy endpoints was 31Mar2021. This is an updated analysis of the 2020 ASCO abstract after all patients had completed at least 6 months of treatment or discontinued. Result total of 39 pts with a median of 3 lines (range 2-5) of prior anti-HER2 therapy including TDM1 received a median of 6 (range 1-23) cycles of zenocutuzumab. In the 37 pts evaluable for efficacy and with locally confirmed HER2 overexpression (3+ IHC or 2+ IHC confirmed by FISH), CBR at 24 w per investigator was 49% (90% CI: 34-63%); ORR was 27% (95% CI: 15-42%). The CBR at 24 w was consistent across different methods of HER2 overexpression/amplification detection (local vs central laboratory) and response assessment (investigator and central independent radiological review; see table below). As of a 12Jan2021 safety data update, the most common related AEs (all grades; G3-4) were neutropenia/neutrophil count decrease (61%; 46%), diarrhea (61%; 4%), asthenia/fatigue (46%; 0), and nausea (29%; 0). The PK half-life was 117h. The correlation of HER2-HER3 pathway activation at baseline with best ORR, duration of response, PFS, and overall survival was analyzed and will be presented in the poster. Conclusion Updated analyses confirm that the efficacy of zenocutuzumab combinations with trastuzumab/vinorelbine in heavily pretreated, HER2+ MBC, with progression after TDM-1, met prespecified protocol criteria for success. The regimen is safe and well tolerated with AEs mostly related to chemotherapy. CBR with zenocutuzumab, trastuzumab, and vinorelbinePopulationNCBR at 24 wks, %. (90% CI)HER2+ by local test/TA by RECIST1.1 per investigator3749 (34-63)HER2+ by central test/TA by RECIST1.1 per investigator2955.1 (38-71)HER2+ by local test/TA by RECIST1.1 by central independent radiologist review3644 (30-59)HER2+ by central test/TA by RECIST1.1 by central independent radiologist review2850.0 (33-67) Citation Format: Erika Hamilton, Thierry Petit, Barbara Pistilli, Anthony Goncalves, Ana Alexandra Ferreira, Florence Dalenc, Fatima Cardoso, Monica M Mita, Luis Manso, Syed M Karim, Francois-Clement Bidard, Philippe Aftimos, Santiago Escriváa-de-Romaníi, Noemia Afonso, Ernesto Wasserman, Kees Bol, Viktoriya Stalbovskaya, Anastasia Vliet, Anastasia Murat, Mohamed Bekradda, Thomas Bachelot. Updated analysis of MCLA-128 (zenocutuzumab), trastuzumab, and vinorelbine in patients (pts) with HER2 positive/amplified (HER2+) metastatic breast cancer (MBC) who progressed on previous anti-HER2 ADCs [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 OT2-15-01.

Safety and early efficacy results of a phase Ib study of nivolumab plus trastuzumab with S-1/capecitabine plus oxaliplatin for HER2-positive advanced gastric cancer (Ni-HIGH study).

276 Background: Addition of an anti-PD-1 antibody to trastuzumab reportedly enhances ADCC activity of trastuzumab, leading to an additive antitumor effect. In the KEYNOTE-811 study, pembrolizumab in combination with trastuzumab plus chemotherapy resulted in a durable response. We investigated the safety and tolerability of nivolumab plus trastuzumab combined with S-1 or capecitabine (Cape) and oxaliplatin (Ox) for patients (pts) with HER2-positive advanced gastric cancer (AGC). The safety parts of this study have already shown that there are no safety concerns (Takahari, et al. ASCO-GI 2021). Methods: This study is an open-label, non-randomized phase 1b study conducted at four centers in Japan to confirm the tolerability of addition of nivolumab to chemotherapies with trastuzumab in safety part (n = 12) followed by assessing their efficacy in expansion part (n = 30). Chemotherapy-naïve adult pts with histologically confirmed HER2-positive AGC who had measurable lesions were eligible. Pts received nivolumab (360 mg/body, day 1) and trastuzumab (cycle 1: 8 mg/kg; cycle 2–: 6 mg/kg, day 1), Ox (130 mg/m2, day 1) in combination with either S-1 (40 mg/m2 bid, days 1–14; cohort 1) or Cape (1000 mg/m2 bid, days 1–14; cohort 2) every 3 weeks until disease progression or unacceptable toxicity. Initial eligible 12 pts for safety part were enrolled alternatively to cohort 1 or 2, and patients for expansion part were allocated to either cohort by investigators’ choice up to 21 patients in each cohort. Safety and short-term efficacy data during 8 cycles (6 months) were assessed for this presentation. Results: Between November 2018 and January 2021, a total of 42 pts with HER2-positive AGC were enrolled (21 pts in each cohort). As of July 31, 2021, 29 pts discontinued protocol treatment and the rest 13 pts are still on treatment. Of the 29 pts, only 3 pts (10.3%)discontinued the treatment due to adverse event (AE). AE (≥ G3) occurred in 25 pts (59.5%) including neutropenia in 8 pts (19.0%) and diarrhea in 4 pts (9.5%). Immune-related AEs (≥ G3) occurred in 7 pts (16.6%) including diarrhea/colitis in 4 pts (9.5%). Median relative dose intensity was 100%, 78%, 73%, 75%, and 75% for nivolumab, trastuzumab, Ox, S-1, and Cape, and the percentage of dose reduction or discontinuation within initial 8 cycles were 33%, 33%, 79%, 81%, and 81%, respectively. The ORR was 76.2% (95% CI 60.6-86.9, CR 4.8%, PR 71.4%), and the DCR was 97.6%. Conversion surgery was performed in 2 pts (4.8%). 6-month PFS rate was 68.7% (95% CI 51.7-81.3). Subset results by the cohorts will be presented at the conference. Conclusions: Nivolumab, trastuzumab and either S-1 or Cape plus Ox was tolerable and showed acceptable safety and promising early efficacy results in pts with HER2-positive AGC. Clinical trial information: UMIN000034222.

Bone marrow stromal antigen 2 (BST-2) genetic variants influence expression levels and disease outcome in HIV-1 chronically infected patients

BackgroundBone marrow stromal antigen 2 (BST-2) also known as Tetherin (CD317/HM1.24), is a host restriction factor that blocks the release of HIV-1 virions from infected cells. Previous studies reported that BST-2 genetic variants or single nucleotide polymorphims (SNPs) have a preventative role during HIV-1 infection. However, the influence of BST-2 SNPs on expression levels remains unknown. In this study, we investigated the influence of BST-2 SNPs on expression levels and disease outcome in HIV-1 subtype C chronically infected antiretroviral therapy naïve individuals.ResultsWe quantified BST-2 mRNA levels in peripheral blood mononuclear cells (PBMCs), determined BST-2 protein expression on the surface of CD4+ T cells using flow cytometry and genotyped two intronic single nucleotide polymorphisms (SNPs) rs919267 and rs919266 together with one SNP rs9576 located in the 3’ untranslated region (UTR) of bst-2 gene using TaqMan assays from HIV-1 uninfected and infected participants. Subsequently, we determined the ability of plasma antibody levels to mediate antibody-dependent cellular phagocytosis (ADCP) using gp120 consensus C and p24 subtype B/C protein. Fc receptor-mediated NK cell degranulation was evaluated as a surrogate for ADCC activity using plasma from HIV-1 positive participants. BST-2 mRNA expression levels in PBMCs and protein levels on CD4+ T cells were lower in HIV-1 infected compared to uninfected participants (p = 0.075 and p < 0.001, respectively). rs919267CT (p = 0.042) and rs919267TT (p = 0.045) were associated with lower BST-2 mRNA expression levels compared to rs919267CC in HIV-1 uninfected participants. In HIV-1 infected participants, rs919267CT associated with lower CD4 counts, (p = 0.003), gp120-IgG1 (p = 0.040), gp120-IgG3 (p = 0.016) levels but higher viral loads (p = 0.001) while rs919267TT was associated with lower BST-2 mRNA levels (p = 0.046), CD4 counts (p = 0.001), gp120-IgG1 levels (p = 0.033) but higher plasma viral loads (p = 0.007). Conversely, rs9576CA was associated with higher BST-2 mRNA expression levels (p = 0.027), CD4 counts (p = 0.079), gp120-IgG1 (p = 0.009), gp120-IgG3 (p = 0.039) levels but with lower viral loads (p = 0.037).ConclusionOur findings show that bst-2 SNPs mediate BST-2 expression and disease outcome, correlate with gp120-IgG1, gp120-IgG3 levels but not p24-IgG levels, ADCC and ADCP activity.Graphical

CD40- and 41BB-specific antibody fusion proteins with PDL1 blockade-restricted agonism.

Background: A strategy to broaden the applicability of checkpoint inhibitors is the combined use with antibodies targeting the immune stimulatory receptors CD40 and 41BB. However, the use of anti-CD40 and anti-41BB antibodies as agonists is problematic in two ways. First, anti-CD40 and anti-41BB antibodies need plasma membrane-associated presentation by FcγR binding to exert robust agonism but this obviously limits their immune stimulatory efficacy by triggering ADCC, CDC or anti-inflammatory FcγRIIb activities. Second, off tumor activation of CD40 and 41BB may cause dose limiting systemic inflammation.Methods: To overcome the FcγR-dependency of anti-41BB and anti-CD40 antibodies, we genetically fused such antibodies with a PDL1-specific blocking scFv as anchoring domain to enable FcγR-independent plasma membrane-associated presentation of anti-CD40- and anti-41BB antibodies. By help of GpL-tagged variants of the resulting bispecific antibodies, binding to their molecular targets was evaluated by help of cellular binding studies. Membrane PDL1-restricted engagement of CD40 and 41BB but also inhibition of PDL1-induced PD1 activation were evaluated in coculture assays with PDL1-expressing tumor cell lines and 41BB, CD40 and PD1 responsible cell lines or T-cells.Results: The binding properties of the bispecific antibody fusion proteins remained largely unchanged compared to their parental molecules. Upon anchoring to membrane PDL1, the bispecific antibody fusion proteins activated CD40/41BB signaling as efficient as the parental anti-CD40/anti-41BB antibodies when bound to FcγRs or cells expressing membrane-bound CD40L/41BBL. PD1 inhibition remained intact and the anti-41BB fusion protein thus showed PDL1-restricted costimulation of T-cells activated in vitro with anti-CD3 or a BiTe.Conclusions: Targeting of anti-CD40 and anti-41BB fusion proteins to membrane PDL1 with a blocking PDL1 scFv links PD1-PDL1 checkpoint blockade intrinsically with engagement of CD40 or 41BB.

A highly-specific fully-human antibody and CAR-T cells targeting CD66e/CEACAM5 are cytotoxic for CD66e-expressing cancer cells in vitro and in vivo.

Neuro-endocrine prostate cancer (NEPC) accounts for about 20% of lethal metastatic castration-resistant prostate cancer (CRPC). NEPC has the most aggressive biologic behavior of all prostate cancers and is associated with poor patient outcome. Effective treatment for NEPC is not available because NEPC exhibit distinct cell-surface expression profiles compared to other types of prostate cancer. Recently, the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) (known as CEA or CD66e) was suggested to be a specific surface protein marker for NEPC. Therefore, we identified a new, fully-human anti-CEACAM5 monoclonal antibody, 1G9, which bound to the most proximal membrane domains, A3 and B3, of CEACAM5 with high affinity and specificity. It shows no off-target binding to other CEACAM family members, membrane distal domains of CEACAM5, or 5800 human membrane proteins. IgG1 1G9 exhibited CEACAM5-specific ADCC activity toward CEACAM5-positive prostate cancer cells in vitro and in vivo. Chimeric antigen receptor T cells (CAR-T) based on scFv 1G9 induced specific and strong antitumor activity in a mouse model of prostate cancer. Our results suggest that IgG1 and CAR-T cells based on 1G9 are promising candidate therapeutics for CEACAM5-positive NEPC and other cancers.

Chimerized Anti-ICOS 314.8 Monoclonal Antibodies Inhibit Tumor Cells and Regulatory T Cells in Patients with Sézary Syndrome

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy.Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®).Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry.Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS.Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered.ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1).Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031).Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p<0.01; CI95%:-53–27). The efficacy was better than with alemtuzumab, but there was no significant difference with moga. The ADCP effect induced by anti-ICOS did not differ with moga. Interestingly, anti-ICOS were effective in 6 moga-resistant patient, as they induced 38.9±5.9% of apoptosis, compared to 12.4±4.7% with moga (p<0.001).Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab.Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. DisclosuresLopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

291 Dual-specific antibodies blocking both PD-L1 and PD-L2 engagement of PD-1 restore anti-tumor immunity

BackgroundInhibition of T cell activation and effector function via engagement of the co-inhibitory receptor PD-1 is a critical mechanism enabling tumors to evade host immunity. The two ligands for PD-1, PD-L1 and PD-L2, can be expressed by a variety of immunosuppressive stromal cells, particularly of the myeloid lineage, endothelial cells, and by tumors themselves. In addition to PD-1, PD-L1 engages B7-1 in an additional co-inhibitory interaction. Blocking only PD-1 or only PD-L1 thus does not relieve all inhibitory components of this pathway. We hypothesized that bispecific antibodies blocking both PD-L1 and PD-L2 could more fully restore tumor-specific T cell activation and potentiate anti-cancer immunotherapy. Furthermore, we speculated that enhancing the cytotoxic effector function of these antibodies might further enhance their efficacy through the depletion of tumor cells and supportive stroma.MethodsWe investigated the capacity of monoclonal antibodies capable of bivalent binding to both PD-L1 and PD-L2 to restore the function of PD-1-suppressed T cells in vitro. To assess the in vivo therapeutic efficiency of bispecific PD-Ligand antibodies with ADCC capacities, mouse IgG2a and modified human IgG1 versions were generated. We assessed their ADCC activity in vitro using a bioluminescent reporter assay, and their therapeutic efficiency in vivo in syngeneic or human-cell derived tumors.ResultsThe bispecific antibodies we generated restore the function of PD-1-suppressed T cells in vitro with equivalent efficiency to the FDA approved PD-1 antibody Pembrolizumab. Moreover, our modified human bispecific antibodies lead to significantly higher FcγRIIa activation than FDA-approved clinical human IgG1 PD-L1 antibodies in vitro. In vivo, ADCC-capable PD-Ligand bispecific antibodies suppress the growth of U2940 lymphoma in immunodeficient mice more efficiently than Rituximab, and in a syngeneic model of PD-L1/PD-L2 double positive colon carcinoma, these antibodies demonstrate superiority to PD-1 blocking antibodies to limit tumor growth and increase survival. Furthermore, treatment with our bispecific antibodies increases T cell proliferation and cytotoxicity and reduces density of immunosuppressive myeloid stroma in vivo.ConclusionsADCC-capable PD-Ligand bispecific antibodies display higher therapeutic potential than existing anti-PD-1 antibodies and represent a new class of PD-1 pathway therapeutics with significant potential for the treatment of a variety of human cancers.AcknowledgementsI would like to thank Anupallavi Srinivasamani, PhD student in Michael Curran’s lab, who performed a considerable amount of the work on this project before I joined.

Human Vγ9Vδ2 T cells exert anti-tumor activity independently of PD-L1 expression in tumor cells

Human γδ T cells expressing Vγ9Vδ2 T cell receptors play a crucial role in the innate immune system and have an attracted interest as effector cells in adoptive cellular immunotherapy. However, the efficacy of adoptive cellular immunotherapy for the treatment of tumors requires overcoming the immunosuppressive microenvironment. αβ T cell inhibition in the tumor microenvironment is associated with programmed death-ligand 1 (PD-L1) expression level. Vγ9Vδ2 T cells (abbreviated as γδ T cells here) exert potent cytotoxic effects in various cancers; however, γδ T cell activity in relation to the level of PD-L1 expression in cancer cells remains unclear, and the association between the PD-1/PD-L1 axis and γδ T cell cytotoxicity needs to be investigated. In this study, PD-1 blockade did not increase the cytotoxicity of γδ T cells against PD-L1high cancer cells. However, the anti-PD-L1 monoclonal antibody (mAb) enhanced the cytotoxicity of γδ T cells against a subset of cancer cells, whereas PD-L1 knockdown did not increase the cytotoxicity of γδ T cells. We also found that the expression levels of PD-L1 were positively correlated with the changes of γδ T cells cytotoxicity induced by anti-PD-L1 mAb. These observations suggest that anti-PD-L1 mAb treatment adds ADCC activity to the cytotoxicity of γδ T cells itself against PD-L1high cancer cells. The present results suggest that ex vivo expanded γδ T cells have antitumor activity independently of PD-L1 expression and may be promising effector cells for γδ T cell immunotherapy.

CD33/CD3-Bispecific T-Cell Engaging (BiTE®) Antibody Constructs Efficiently Target Monocytic CD14+ hla-DRlow IDO+ aml-MDSCs

Acute Myeloid Leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 25%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. In this study we sought to investigate whether the CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with documented pre-clinical activity against AML blasts can also target CD33+ AML-MDSCs by redirecting and activating T-cells and thereby enhance AMG 330 mediated anti-leukemic activity.First, we assessed the presence of circulating CD14+ cells that co-express CD33 but lack HLA-DR expression (HLA-DRlo) in patients with newly diagnosed AML. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14+HLA-DRlo cells was significantly increased in untreated AML patients as compared to healthy controls (28,98±4,19%, n=13 versus 3,28±0,75%, n=37) but did not correlate with the number of AML blasts. Purified CD14+HLA-DRlo cells suppressed in vitro T-cell proliferation in a concentration-dependent manner allowing us their denomination as MDSCs.Interestingly, we also observed that primary AML blasts and human AML cell lines (THP, OCI-AML, and HL-60) induced HLA-DRlo cells from healthy donor-derived monocytes that were T-cell suppressive and expressed the immune regulatory indoleamine-2,3-dioxygenase (IDO). Functional and phenotypic reprogramming of monocytes was cell-contact independent.Using ex vivo co-culturing models of primary AML blasts/AML cell lines (=target cells) and T-cells (=effector cells) we observed AMG 330-triggered antibody construct-dependent cell-mediated cytotoxicity/ADCC. Short-term (three to six days) treatment of AML patient-derived peripheral blood mononuclear cells (PBMCs) with 10 or 100 pM AMG 330 led to T-cell activation based on proliferation and on increased expression/production of CD25, CD69, CD137, CD154, IL-2, and IFN-g. The initial T-cell frequency ranging from 0,16 to 14,30% had no impact on their responsiveness. Moreover, we observed a bystander activation of NK-cells. The addition of AML-MDSCs into these co-cultures did not negatively impact ADCC activity. On the contrary, T-cells formed immunological synapses with AML-MDSCs and triggered their elimination (also in presence of AML blasts) overcoming their T-cell suppressive effect. MDSC levels in AML-PBMCs had no effect on AMG 330-mediated T-cell (and bystander cell) activation, which was further corroborated in experiments where the total CD14+ cell compartment (comprising AML-MDSCs) was removed prior AMG 330 application.However, we did observe an impact of MDSCs on BiTE® mediated T-cell proliferation, which has been shown as highly relevant for clinical activity of BiTE® antibody constructs (Zugmaier et al. Blood 2015). Here we found a 1,68-fold enhanced (p=0.001) T-cell expansion in AML-PBMCs treated with 100 pM AMG 330 following CD14+ cell depletion. Given the importance of T-cell proliferation for clinical activity this finding warrants further exploration.Taken together, our results suggest that anti-CD33/CD3 bispecific BiTE® antibody constructs may achieve anti-leukemic efficacy not only through direct T cell mediated cytotoxicity against AML blasts but also through circumventing immune evasion via MDSCs targeting. Although therapeutic targeting of MDSCs in patients has not yet been successfully accomplished, bystander killing of CD33+ MDSCs via anti-CD33/CD3-bispecific BiTE® antibody constructs could represent a very promising approach to increase the anti-leukemic T-cell response in AML patients and to reverse immune evasion. DisclosuresMougiakakos:AMGEN: Research Funding. Kischel:AMGEN: Employment. Michael:AMGEN: Employment. Dos Santos:Amgen Inc.: Employment. Jitschin:AMGEN: Research Funding. Mackensen:AMGEN: Research Funding.

The anti-SLAMF7 elotuzumab enhances ADCC activity with Th1-like γδT cells towards osteoclasts and myeloma cells

The anti-SLAMF7 elotuzumab enhances ADCC activity with Th1-like γδT cells towards osteoclasts and myeloma cells