Engagement Of Immune Cells Research Articles

Beyond ADCs: Harnessing Bispecific Antibodies to Directly Induce Apoptosis for Targeted Tumor Eradication

Abstract Bispecific Apoptosis Triggers (BATs) are innovative bispecific antibodies designed to simultaneously target both a tumor-associated antigen and a cancer cell’s death receptor thereby directly activating the extrinsic apoptotic pathway to induce death of cancer cells. This unique mechanism distinguishes BATs from antibody-drug conjugates (ADCs), which rely on cytotoxic drugs, and bispecific immune cell engagers such as bispecific T-cell engagers (BiTEs) and bispecific natural killer cell engagers (NKCEs), which recruit immune cells to eliminate target cancer cells. BATs offer significant potential advantages in clinical efficacy and safety over ADCs and BiTEs. Although the field is still emerging, recent advancements are highly promising, and analysis of preclinical and clinical data of DR5-targeting antibodies have been pivotal in outlining the criteria for the next generation of effective and safe medicines. Antibodies found inactive in preclinical testing were also found to be clinically ineffective, whereas antibodies with minimal preclinical results demonstrated moderate clinical activity. All clinical DR5 targeting antibodies were well tolerated by patients even at high doses (with the exception of TAS266 due to its unique design). These findings underscore the predictive value of robust preclinical models on clinical outcomes. Notably, first-in-class BAT, Cancerlysin™ IMV-M, demonstrated potent efficacy in diverse xenograft cancer models and safety in non-human primates, marking a significant advancement in developing safe and effective anti-cancer drugs.

Membrane Antigen Targeting in Acute Myeloid Leukemia Using Antibodies or CAR-T Cells

This review explores the emerging area of the therapeutic use of antibodies and chimeric antigen receptor (CAR)-T cells for the treatment of acute myeloid leukemia (AML). Through a detailed analysis of the existing literature, this paper highlights the different categories of AML antigens for immunotherapeutic targeting, the most recent applications on antibodies, including bispecific immune cell engagers and CAR-T cells, to the therapy of patients with refractory/relapsing AML The studies performed in AML patients using BisAbs and CAR-T cells have shown that only a limited number of AML patients show sustained responses to these therapies, thus underlying AML heterogeneity as a major challenge. Several studies have addressed the potential mechanisms underlying the resistance of AMLs to antibody-directed immunotherapies. A better understanding of the barriers hampering the successful development of AML immunotherapy is required. However, in spite of the limitations, the studies recently carried out have shown the peculiar sensitivity of some AML subtypes to immunotherapy and have provided the basis for future studies, such as multiplex antigen targeting, which hold the promise of successful development.

Engineered Outer Membrane Vesicles as Nanosized Immune Cell Engagers for Enhanced Solid Tumor Immunotherapy.

Although tumor immunotherapy has achieved significant success in recent years, tackling solid tumors remains a formidable challenge. Here, we present an approach that utilizes outer membrane vesicles (OMVs) from bacterial cells as scaffolds to engage immune cells in solid tumor immunotherapy. Two types of nanobodies targeting CD47/SIRPα and PD-1/PD-L1 pathways were simultaneously conjugated onto the surfaces of the OMVs in divalent and trivalent forms using orthogonal SpyCatcher-SpyTag and SnoopCatcher-SnoopTag chemistry. This resulted in the generation of an OMV-based nanosized immune cell engager (OMV-NICE) with dual-targeting abilities. In vitro assays confirmed the retention of the function of the two nanobodies on the OMV-NICE, as evidenced by the synergistically enhanced macrophage phagocytosis and T cell cytotoxicity against tumor cells. In vivo studies using a B16-F10 melanoma mouse model also revealed the superior antitumor activity of OMV-NICE compared to those of unconjugated nanobodies and OMVs alone. Subsequent mechanistic investigations further supported the enhanced recruitment of macrophages and T cells to the tumor region by OMV-NICE. Overall, this work expands the current repertoire of immune cell engagers, and the developed OMV-NICE platform holds great promise for broad applications, particularly in solid tumor immunotherapy.

Mechanism of Action and Pharmacokinetics of Approved Bispecific Antibodies.

Bispecific antibodies represent a significant advancement in therapeutic antibody engineering, offering the ability to simultaneously target two distinct antigens. This dual-targeting capability enhances therapeutic efficacy, especially in complex diseases, such as cancer and autoimmune disorders, where drug resistance and incomplete target coverage are prevalent challenges. Bispecific antibodies facilitate immune cell engagement and disrupt multiple signaling pathways, providing a more comprehensive treatment approach than traditional monoclonal antibodies. However, the intricate structure of bispecific antibodies introduces unique pharmacokinetic challenges, including issues related to their absorption, distribution, metabolism, and excretion, which can significantly affect their efficacy and safety. This review provides an in-depth analysis of the structural design, mechanisms of action, and pharmacokinetics of the currently approved bispecific antibodies. It also highlights the engineering innovations that have been implemented to overcome these challenges, such as Fc modifications and advanced dimerization techniques, which enhance the stability and half-life of bispecific antibodies. Significant progress has been made in bispecific antibody technology; however, further research is necessary to broaden their clinical applications, enhance their safety profiles, and optimize their incorporation into combination therapies. Continuous advancements in this field are expected to enable bispecific antibodies to provide more precise and effective therapeutic strategies for a range of complex diseases, ultimately improving patient outcomes and advancing precision medicine.

In silico and pharmacological evaluation of GPR65 as a cancer immunotherapy target regulating T-cell functions.

The success of cancer immunotherapies such as immune checkpoint inhibitors, CAR T-cells and immune cell engagers have provided clinicians with tools to bypass some of the limitations of cancer immunity. However, numerous tumour factors curtail the immune response against cancer and limit the efficiency of immuno-oncology (IO) therapies. Acidification of the extra-cellular tumour environment consecutive to aberrant cancer cell metabolism is a well-known promoter of oncogenic processes that also acts as an immune regulator. Yet, the suppressive mechanisms of low extra-cellular pH on anti-cancer immunity remain poorly understood. Recent reports have suggested that GPR65, a Gαs-coupled proton-sensing GPCR broadly expressed in the immune system, may act as an immune suppressant detrimental to anti-tumour immunity. So far, the immuno-regulatory properties of GPR65 in acidic milieux have mostly been documented in macrophages and myeloid cells. Our computational evaluation of GPR65's transcriptomic expression profile and potential as an IO target using public datasets prompted us to further investigate its functions in human T-cells. To this end, we identified and validated GPR65 small molecule inhibitors active in in vitro cellular assays and we showed that GPR65 inhibition promoted the killing capacity of antigen-specific human T-cells. Our results broaden the scope of GPR65 as an IO target by suggesting that its inhibition may enhance T-cell anti-tumour activity and provide useful pharmacological tools to further investigate the therapeutic potential of GPR65 inhibition.

A Novel Tetravalent Bispecific Immune Cell Engager Activates Natural Killer Cells to Kill Cancer Cells without Mediating Fratricide.

We previously reported the structure, affinity, and anticancer activity of a bivalent bispecific natural killer cell engager (BiKE) composed of one anti-CD16a VHH and one anti-HER2 VHH fused via a linker. In this study, we explored the engineering of a tetravalent BiKE by fusing two anti-CD16a and two anti-HER2 VHHs in tandem, using bivalent BiKE as a template. The tetravalent BiKE was genetically engineered, and its tertiary structure was predicted using in silico modeling. The antigen binding and affinity of the tetravalent BiKE were assessed using ELISA, flow cytometry, and biolayer interferometry. The ability of the BiKEs to kill cancer cells was evaluated through classical and residual antibody-dependent cellular cytotoxicity (ADCC) assays. Additionally, we investigated the potential for NK cell fratricide via CD16a-CD16a crosslinking. Our results revealed that the tetravalent BiKE exhibited at least 100-fold higher affinity toward its target antigens compared to its bivalent counterpart. The residual ADCC assay indicated that the tetravalent BiKE was more effective in killing cancer cells than the bivalent BiKE, attributable to its lower Koff value, which prolonged its binding to NK cell surfaces. Fratricide assays demonstrated that neither the bivalent nor the tetravalent BiKE mediated fratricide. Notably, our findings showed that daratumumab-induced NK fratricide was restricted to CD38-CD38 crosslinking and was not related to ADCC via CD16a-CD38 crosslinking. This study is the first in the literature to show the successful engineering of a tetravalent immune cell engager composed of tandem VHH units, which achieves high affinity and anticancer activity without mediating fratricide.

Tumor-agnostic cancer therapy using antibodies targeting oncofetal chondroitin sulfate

Molecular similarities between embryonic and malignant cells can be exploited to target tumors through specific signatures absent in healthy adult tissues. One such embryonic signature tumors express is oncofetal chondroitin sulfate (ofCS), which supports disease progression and dissemination in cancer. Here, we report the identification and characterization of phage display-derived antibody fragments recognizing two distinct ofCS epitopes. These antibody fragments show binding affinity to ofCS in the low nanomolar range across a broad selection of solid tumor types in vitro and in vivo with minimal binding to normal, inflamed, or benign tumor tissues. Anti-ofCS antibody drug conjugates and bispecific immune cell engagers based on these targeting moieties disrupt tumor progression in animal models of human and murine cancers. Thus, anti-ofCS antibody fragments hold promise for the development of broadly effective therapeutic and diagnostic applications targeting human malignancies.

CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.

Immediate myeloid depot for SARS-CoV-2 in the human lung.

In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2-dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies.

Bispecific and multispecific antibodies in oncology: opportunities and challenges.

Research into bispecific antibodies, which are designed to simultaneously bind two antigens or epitopes, has advanced enormously over the past two decades. Owing to advances in protein engineering technologies and considerable preclinical research efforts, bispecific antibodies are constantly being developed and optimized to improve their efficacy and to mitigate toxicity. To date, >200 of these agents, the majority of which are bispecific immune cell engagers, are in either preclinical or clinical evaluation. In this Review, we discuss the role of bispecific antibodies in patients with cancer, including history and development, as well as innovative targeting strategies, clinical applications, and adverse events. We also discuss novel alternative bispecific antibody constructs, such as those targeting two antigens expressed by tumour cells or cells located in the tumour microenvironment. Finally, we consider future research directions in this rapidly evolving field, including innovative antibody engineering strategies, which might enable more effective delivery, overcome resistance, and thus optimize clinical outcomes.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Abstract 6720: HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors

Abstract Introduction/Purpose: The Wagner lab has developed a protein nanoring based platform to direct human immune cells to combat solid tumors. These nanorings are bispecific, comprised of tumor-targeting and immune cell-targeting dihydrofolate reductase (DHFR2) fusion proteins. These protein nanorings direct immune cells to target and destroy cancer cells. For treating solid tumors, we aimed to target epidermal growth factor receptor (EGFR). However, targeting EGFR could lead to potential “on-target, off-tumor” effects due to expression of EGFR in healthy tissue. Matrix metalloproteinases are overexpressed in a variety of solid tumors. Therefore, to improve tumor specificity, we hypothesized that masking the immune cell targeting DHFR2 protein through a matrix metalloproteinase-2 (MMP-2) sensitive linker will prevent engagement and activation of immune cells outside the tumor microenvironment (TME). Once these protease-activatable nanorings enter the TME and bind to EGFR, cleavage by MMP-2 will lead to unmasking of the immune cell binder, thereby crosslinking and activating immune cells, causing targeted cancer cell lysis. Methods: We designed human histidine triad nucleotide binding protein-1 (HINT1) masked anti-immune cell targeting nanobody-DHFR2 fusion proteins. HINT1 was hypothesized to sterically block the engagement of immune cells outside the tumor. HINT1-anti-TCR-DHFR2 (TCR = T cell receptor) and HINT1-anti-CD16-DHFR2 were cloned, expressed, purified, and characterized. Their apparent binding affinities were determined using flow cytometry. The anti-tumor efficacy of these nanorings was evaluated against EGFR overexpressing A431 (human epidermoid carcinoma) cells. Results: HINT1-anti-TCR-DHFR2 and HINT1-anti-CD16-DHFR2 rings showed reduced binding to T and NK-92-CD16 cells, respectively. In vitro MMP-2 cleavage assay showed complete removal of the HINT1 mask within 2 hours. Western blot analysis showed that both proteins were stable in human serum up to 3 days. 2D cytotoxicity assays with protein nanorings assembled using anti-EGFR-DHFR2 and anti-TCR-DHFR2 or anti-CD16-DHFR2 with T and NK-92-CD16 cells respectively, showed a potent lysis of A431 cells. The EGFR-targeted lysis was diminished in the presence of HINT1 mask, thereby providing proof-of-concept for the steric masking of the anti-immune cell targeting nanobodies. Conclusion: Protease-activatable nanorings can selectively lyse EGFR+ tumor cells in vitro. Their antitumor efficacy and safety are currently being evaluated using a previously established in vivo mouse model. Therefore, HINT1-steric masking can serve as a simple and effective strategy to address off-tumor toxicities associated with bispecific immunotherapies. Citation Format: Abhishek Kulkarni, Debasmita Paul, Declan Dahlberg, Kostana Ligori, Freddys Rodriguez, Lakmal Rozumalski, Bruce Walcheck, Carston R. Wagner. HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6720.

Abstract 4077: T cell targeting with PD-1-selective immune cell engagers based on GlycoConnect™ (GC™-ICEs) show excellent efficacy and tolerability

Abstract Engagement of T cell cells to harness a patient’s immune system is a promising approach in immuno-oncology. An immunocytokine, i.e. a tumor-targeting antibody (fragment) genetically fused to an immunostimulatory cytokine, is specifically designed for this purpose. Fusion antibody-immunocytokines, based on molecular architectures generated by a variant of DNA recombinant technologies, have recently entered the clinic. Earlier we have demonstrated how GlycoConnect™, a site-specific conjugation technology anchoring on the native antibody glycan1, can be applied for attachment of various cytokines, including RLI or IL-15, without prior antibody engineering. The immunostimulatory activity of these GlycoConnect™ immune cell engagers (GC™-ICEs) can be modulated by tailoring linker design and payload stoichiometry. In this presentation, we demonstrate the GC™-ICE with either IL-15 or RLI can be applied to selective targeting of T cells, based on PD-1-selective binding and activation, using either protease-sensitive or non-cleavable linkers. We will show how such T cell targeting can be achieved leading to significant tumor volume reductions in syngeneic mouse models. Moreover, we have found that the GC-ICE™ can be applied without inducing systemic immune activation, thereby displaying a promising therapeutic index. In vitro and in vivo studies will be presented to showcase the biological activity of these GC™-ICEs in comparison to conventional immunocytokines obtained via genetic engineering.1Wijdeven et al., Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. mAbs 2022, 14, DOI: 10.1080/19420862.2022.2078466. Citation Format: Remon van Geel, Mick Verhagen, Elias Post, Willem Vugs, Sorraya Popal, Sander van Berkel, Floris van Delft. T cell targeting with PD-1-selective immune cell engagers based on GlycoConnect™ (GC™-ICEs) show excellent efficacy and tolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4077.

Abstract 5334: Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research

Abstract Background: The preclinical evaluation of novel immune therapies requires humanized immune system (HIS) mouse models. In previous studies we have demonstrated that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T and NK cells or CD34+ hematopoietic stem cells (HSC) can be used to establish a HIS model. With the development of next-generation NOG mice, lineage-specific differentiation of immune cell sub-populations of interest can be supported. Transplantation of cell line-derived (CDX) or patient-derived (PDX) tumor xenografts on HIS mice provides a full model for human tumors for the investigation of checkpoint inhibitors (CPI), as well as novel cell therapies and immune cell engagers. Methods: HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient mice. For humanization, NOG mice and next-generation NOG strains were used: NOG, NOG-EXL, hIL-2 NOG, hIL-6 NOG and FcResolv™ NOG mice were compared to each other for lineage-specific differentiation using single donors or a mixed HSC donor pool. Engraftment of immune cells was monitored by FACS analysis of blood every four weeks. In humanized NOG mice, CDX and PDX from different entities were s.c. transplanted and used to evaluate CPI. Results: Humanized hIL-2 NOG mice showed significantly decreased survival after HSC transplantation in comparison to the other mouse strains and had to be sacrificed within the first 6-8 weeks after HSC transplantation. In the other mouse strains, transplanted HSCs engrafted and differentiated mainly into B and T cells. NOG-EXL mice displayed the highest engraftment, with up to 80% of human cells in the blood, including a higher portion of myeloid cells. In humanized hIL-6 NOG mice a higher portion of monocytes could be determined. FcResolv™ NOG mice lack murine Fc gamma receptors in order to reduce confounding interactions between residual murine immune cells and antibody-based therapies, and they engraft and differentiate human HSCs similarly to the parent NOG strain. In humanized NOG mice, selected CDX and PDX tumors successfully engrafted without significant differences in tumor growth compared to non-humanized mice. CPI treatments induced tumor growth delay in selected models. Conclusions: Next-generation NOG mouse strains are characterized by a lineage-specific differentiation of immune cells depending on integrated human cytokines. Furthermore, we established a human tumor-immune-cell model for NOG mice using different entities of CDX or PDX in combination with CPI. Our human tumor-immune cell models allow preclinical translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Louise Baskin, Jens Hoffmann. Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5334.

Abstract 1340: Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™

Abstract Natural Killer (NK) cells are highly responsive cytotoxic immune cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision guided immunotherapy in oncology. Bicycle® peptides are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle® platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA™). The NK-TICA™ consists of chemically coupled Bicycle® peptides that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell function. We demonstrate potent, selective binding of our Bicycle® peptides to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle® NK-TICA™ as a multifunctional immune cell engager will promote the modulation and anti-tumor activity of peripheral and intra-tumoral NK cells to solid tumors. Citation Format: Fay Dufort, Tucker Ezell, Alexandra Rezvaya, Kathleen Ho, Christopher Leitheiser, Philip Brandish, Kevin McDonnell, Nicholas Keen. Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1340.

Abstract 2460: Receptor antibody conjugated engager (RACE): A novel bispecific 4-1BB engager platform for enhanced immuno-oncology therapy

Abstract Background: Traditional immuno-oncology strategies, such as checkpoint blockade therapy, are evolving to immune cell engagers as a smarter strategy. The early successes of Blincyto, a CD3 engager, demonstrated its capacity to activate host pan-T cells irrespective of their TCR-pMHC restriction status. However, this broad activation has been linked to off-target immunotoxicities, thus limiting its therapeutic applicability. In response, targeting immune co-stimulatory receptors like CD28 or 4-1BB has gained attention by their expression being induced on activated immune effector cells, presenting a more targeted approach to cancer therapy. Methods: We developed RACE, a novel bispecific 4-1BB engager. This platform fuses a 4-1BB ligand (4-1BBL) trimer with antibody F(ab’)2 fragments, excluding Fc domains. A Key and lock design on the 4-1BBL trimer interface facilitates asymmetric assembly of 4-1BBL dimer and monomer in the endoplasmic reticulum, achieving over 95% assembly efficiency in mammalian systems. Results: RACE differs from conventional 4-1BB agonistic antibodies in its ability to form natural hexagonal hyper-clusters of 4-1BB receptors, enhancing NF-κB signal transduction. Unlike first-generation 4-1BB antibodies, which incurred off-tumor hepatotoxicity, RACE selectively activates 4-1BB receptors only in the presence of tumor antigens. Enhanced cytotoxicity requires successful TCR signaling complex formation between T cells and cancer cells. In CT26 and MC38 tumor models, RACE not only induced strong anti-tumor effects without hepatic or systemic toxicities but showed synergistic efficacy with anti-PD-1 antibodies as well. Conclusion: The novel RACE platform represents a potent as well as safe immuno-oncology strategy, demonstrating significant potential for clinical application. Citation Format: DaeHee Lee, Jihye Yoon, Jonggwan Jeong, Jeonghyeon Bak, KkaBi Son, Heechan Kim, Seil Jang, Minseok S. Kim. Receptor antibody conjugated engager (RACE): A novel bispecific 4-1BB engager platform for enhanced immuno-oncology therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2460.

Abstract 6182: Artificial intelligence (AI)-powered quantification of glypican-3 (GPC3) expression facilitates patient selection for GPC3-targeted therapy in solid tumors

Abstract Introduction: GPC3 is a glycosyl-phosphatidylinositol-anchored cell surface glycoprotein that functions to control cell growth and differentiation in early development. Membrane GPC3 is highly expressed in hepatocellular carcinoma (HCC) and moderately in squamous non-small cell lung cancer (SQ-NSCLC) with limited expression in normal tissues which makes it an attractive target for immune cell engagers. This study aims to develop an AI-powered algorithm to evaluate GPC3 expression and compare it with manual immunohistochemistry (IHC) scoring in HCC, adeno-NSCLC and SQ-NSCLC. Methods: 147 NSCLC (94 SQ-NSCLC and 53 adeno-NSCLC) and 113 HCC FFPE tumor blocks were obtained from commercial sources. GPC3 protein level was quantified by a qualified IHC assay for tumor cell membrane and cytoplasm individually. PD-L1 expression is reported as tumor cell proportion score (TPS), combined positive score (CPS) and immune cell proportion score (IPS/IC) TPS, regardless of staining pattern and intensity. FFPE slides were stained for GPC3+ (GC33, Ventana), PD-L1+ (22C3, Dako) cells and then scanned at 20x using the Aperio Versa8 scanner. Machine learning (ML) models were developed using the digitized whole slide images to identify GPC3+ tumor area. Data-driven cutoffs were applied to model-generated human-interpretable features of GPC3+ percentage of tumor area to classify samples as positive and negative. As an orthogonal approach to the GPC3+ percentage cutoff method, tissue and cell annotations were used to train a convolutional neural network (CNN) to classify GPC3 positivity. Results: The epidemiology profiling results revealed that GPC3 protein is highly expressed in HCC (57.5% cases with > 1% positive cells), followed by SQ-NSCLC (52.1%) and adeno-NSCLC (5.7%). GPC3 expression was only detected in tumor cells with two staining patterns observed: membrane + cytoplasm and cytoplasm-only. There was no significant correlation between GPC3 and PD-L1 expression across tumor types, although a trend of lower level of PD-L1 was observed in the samples with GPC3 ≥50%. ML model quantification of GPC3+ and GPC3- showed high concordance with the consensus score of 2 independent pathologists on a test set of held-out samples not used in training. This concordance was comparable to that of individual annotators to consensus. Instead, GPC3 positivity results using both cutoff and CNN methods showed moderate concordance with 2-way pathologist consensus. Conclusions: Here we profiled GPC3 expression across tumor types and showed high level of expression in HCC followed by SQ-NSCLC. We have established an AI-powered digital pathology platform that can provide a standardized, scalable, and reproducible method of characterizing GPC3 positivity to support further patient selection in clinical study. Citation Format: Ellen Meng, Robin Meng, Ying Tong, Yuchen Li, Hong Wang, Paola Fiorentini, Elham Attieh, Qi Tang, Asma Kefsi, Serena Masciari, Giovanni Abbadessa, Cecile Combeau, Lei Tang, Benoit Pasquier, Rui Wang. Artificial intelligence (AI)-powered quantification of glypican-3 (GPC3) expression facilitates patient selection for GPC3-targeted therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6182.

Abstract 6709: P329G-Engager: A novel universal antibody-based adaptor platform for cancer immunotherapy

Abstract Bispecific immune cell engagers are showing promise in cancer immunotherapy. To achieve optimal efficacy, there is a growing need for patient personalization, tackling intra-tumor heterogeneity, and simplified combination therapies. Here, we present a novel modular anti-P329G adaptor platform aiming to address these issues, which is based on the recognition of the P329G mutation in the Fc portion of a targeting adaptor antibody. This P329G mutation can serve to recruit different anti-P329G effector cell engagers, including innate cell engagers, T cell engagers, costimulators or immunocytokines. Specifically, upon identifying patient-specific tumor surface targets, the primary antigen-binding IgG1 antibodies bearing Fc-silencing P329G LALA mutations are applied. Subsequently, based on the tumor’s immune profile, the best-suited anti-tumoral effector cell type is chosen for recruitment and activation. Based on this information, the secondary antibody recognizing the P329G mutation is chosen from an array of effector cell engagers with different modes of action: P329G-T cell bispecifics (P329G-TCB), ADCC-competent P329G innate cell engagers (P329G-ICE), P329G costimulatory molecules (P329G-CD28/4-1BBL) or P329G immunocytokines (P329G-IL2v). In vitro assays showed all P329G modalities inducing anti-tumoral and/or immunomodulatory cell activity. Anti-tumoral efficacy of secondary antibodies was observed only in presence of tumor-targeted P329G adaptors, while no effect was observed in their absence. The readouts consisted of tumor killing quantification, CD4+ and CD8+ T cell activation, cytokine release and cytokine receptor signaling. As a proof of concept study in vivo, an experiment in MKN-45 (CEACAM5+)-bearing humanized mice was performed, using an anti-CEACAM5 P329G-IgG adaptor and a P329G-TCB. Both tumor volume shrinkage and T cell infiltration into the tumor confirmed anti-tumoral efficacy of the platform, as compared to a conventional CEACAM5-targeted TCB. Neither the individual P329G IgG nor the individual P329G-TCB induced anti-tumoral efficacy, validating the requirement for primary and secondary antibody binding for T cell engaging activity. These results provide in vitro and in vivo evidence that the universal P329G engager platform can be used as an efficacious cancer treatment. Ultimately, this modular approach may enable off-the-shelf personalization via combination of patient-specific antibodies and universal effector cell engagers based on the patient’s tumor target and immune profile. Citation Format: Marlena Surowka, Idil Hutter-Karakoc, Diana Darowski, Christina Claus, Claudia Ferrara Koller, Anne Freimoser-Grundschober, Thomas Hofer, Andrzej Sobieniecki, Denis Assisi, Stephane Leclair, Ekkehard Moessner, Pablo Umaña, Maria Amann, Christian Klein. P329G-Engager: A novel universal antibody-based adaptor platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6709.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1341.

Cytokines in Allergic Conjunctivitis: Unraveling Their Pathophysiological Roles.

Allergic conjunctivitis is one of the common immune hypersensitivity disorders that affect the ocular system. The clinical manifestations of this condition exhibit variability contingent upon environmental factors, seasonal dynamics, and genetic predisposition. While our comprehension of the pathophysiological engagement of immune and nonimmune cells in the conjunctiva has progressed, the same cannot be asserted for the cytokines mediating this inflammatory cascade. In this review, we proffer a comprehensive description of interleukins 4 (IL-4), IL-5, IL-6, IL-9, IL-13, IL-25, IL-31, and IL-33, as well as thymic stromal lymphopoietin (TSLP), elucidating their pathophysiological roles in mediating the allergic immune responses on the ocular surface. Delving into the nuanced functions of these cytokines holds promise for the exploration of innovative therapeutic modalities aimed at managing allergic conjunctivitis.