High-affinity Antibodies Research Articles

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Abstract CT136: Phase Ib study of siltuximab and spartalizumab in advanced pancreatic cancer

Abstract Background: Pancreatic adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Most patients present with advanced stages. Heightened levels of serum interleukin-6 (IL-6) are associated with carcinogenesis, immune suppression, and poor prognosis in PDAC. Preclinical data of dual inhibition of IL-6 and programmed death ligand-1 (PD-L1) facilitates CD8+ T-cell migration into pancreatic tumors and was effective in controlling tumor growth in syngeneic and genetically engineered PDAC mouse models. Siltuximab (Sil) is a chimeric monoclonal antibody which targets the IL-6 molecule specifically. Spartalizumab (Sparta) is a high-affinity ligand-blocking humanized IgG4 antibody against the PD-1 receptor. We developed a phase Ib/II trial to evaluate the safety, immunogenicity and preliminary antitumor activity of Sil and Sparta in metastatic PDAC (mPDAC). Methods: Patients (pts) with mPDAC who have progressed on at least one line of systemic chemotherapy were eligible for the trial. Other selected inclusion criteria were ≥18 years of age, ECOG PS 0-1 and measurable disease per RECIST v1.1. The phase Ib trial design is standard 3+3, with a primary endpoint of recommended phase II dose (RP2D). An additional 24 patients were planned for an expansion cohort. Sil was administered at escalating dose levels of 6 mg/kg (DL1) and 11 mg/kg (DL2), in combination with Sparta at 300 mg IV on day 1 of each 21-day cycle. Key secondary endpoints included overall response rate (ORR = CR + PR), progression-free survival (PFS) and overall survival (OS). Results: We enrolled 4 patients on DL1 and escalated to DL2/RP2D in the absence of a dose limiting toxicity (DLT). A total of 31 patients were subsequently treated at the RP2D (escalation + expansion). The median age was 68 years, 42% were male. Majority were White (63%) and 20% were Black. About 43% had head of pancreas primary tumors and 86% had liver involvement. The median number of lines of prior therapy was 3 (range 1-4). There was no objective response among the 4 patients on DL1; 1 had stable disease (SD) for 11 weeks as best response. 17 of 31 patients on RP2D had only a baseline assessment and came off study for progression of disease, prior to follow-up imaging assessment for response evaluation. Of the 14 RP2D patients with a follow-up assessment, ORR was 0% (3 were SD and 11 had progression of disease (PD) as best response), mPFS - 2.2 mos (95% CI: 1.9, 2.6), mOS - 5.5mos (95% CI: 2.8, 6.5). Treatment related adverse events (TRAEs) of any grade included fatigue (23%), anemia (18%), and increased liver enzymes (9.5%). There were no grade 4-5 TRAEs. Conclusions: The combination Sil/Sparta had an acceptable safety profile but did not elicit responses in advanced PDAC pts. Correlative studies using paired biopsies and blood are ongoing to determine how dual IL-6/PD-1 blockade impacts stromal and immune biomarkers in relationship to the clinical outcome measures. Funding and medications for the study were provided by Novartis and Recordati Rare Diseases Inc (NCT04191421) and NIH/NCI (R21CA266088). Citation Format: Olatunji B. Alese, Olumide Gbolahan, Kathleen M. Coleman, Yichun Cao, Sujata Kane, Sarah Craven, Emily Greene, Natalie Horvat, Andrew McDonald, Walid L. Shaib, Christina S. Wu, Mehmet Akce, Maria Diab, Jeffrey M. Switchenko, Gregory B. Lesinski, Bassel F. El-Rayes. Phase Ib study of siltuximab and spartalizumab in advanced pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT136.

Sustained Improvements in Clinical and Patient-Reported Outcomes and Quality of Life Through 5Years Among Ixekizumab-Treated Patients with Complete Clearance of Scalp Psoriasis by Week 60.

Ixekizumab, a high-affinity monoclonal antibody that selectively targets interleukin-17A, is approved for the treatment of moderate-to-severe plaque psoriasis. Since scalp psoriasis can be burdensome and challenging to treat with non-systemic therapies, this post hoc analysis focused on scalp psoriasis in patients with moderate-to-severe plaque psoriasis and baseline scalp involvement. The analysis considered a holistic concept of clearance through 5years of ixekizumab treatment. Ixekizumab-treated patients with baseline scalp involvement were pooled from three multicenter, randomized, double-blind, placebo-controlled, phase 3 trials (integrated UNCOVER-1/2 and UNCOVER-3). Analyses were performed on a subpopulation of patients who achieved complete resolution of scalp psoriasis at Week 60 (i.e., Week 60 Psoriasis Scalp Severity Index [PSSI-0] responders) and on the overall patient population (i.e., Week 60 PSSI-0 responders and non-responders), which was used as a reference. Clinical outcomes (PSSI), patient-reported outcomes (Itch Numeric Rating Scale [NRS] score, Skin Pain Visual Analogue Scale [VAS]), quality of life (Dermatology Life Quality Index [DLQI]), and concurrent outcomes were assessed from baseline through 5years. Descriptive statistics of observed data were reported. After 60weeks of ixekizumab treatment, 88.4% (UNCOVER-1/2) and 75.9% (UNCOVER-3) of patients with baseline scalp involvement achieved complete clearance (PSSI-0) of scalp psoriasis. Substantial improvements in the clinical outcomes (PSSI), patient-reported outcomes (Itch NRS, Skin Pain VAS), and quality of life (DLQI) were achieved by Week 60 and sustained through Week 264 in the Week 60 PSSI-0 responders and in the overall patient population. Additionally, a significant proportion of Week 60 PSSI-0 responders achieved concurrent complete scalp and skin clearance and quality of life improvement through 5years. Continued treatment with ixekizumab provided long-term sustained scalp clearance over 5years to patients with moderate-to-severe plaque psoriasis and baseline scalp involvement, and holistic improvements occurred across clinical outcomes, patient-reported outcomes, and quality of life. NCT01474512 (UNCOVER-1), NCT01597245 (UNCOVER-2), and NCT01646177 (UNCOVER-3).

Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 μg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 μg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

A VHH single-domain platform enabling discovery and development of monospecific antibodies and modular neutralizing bispecifics against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.

Stochastic modeling of a gene regulatory network driving B cell development in germinal centers.

Germinal centers (GCs) are the key histological structures of the adaptive immune system, responsible for the development and selection of B cells producing high-affinity antibodies against antigens. Due to their level of complexity, unexpected malfunctioning may lead to a range of pathologies, including various malignant formations. One promising way to improve the understanding of malignant transformation is to study the underlying gene regulatory networks (GRNs) associated with cell development and differentiation. Evaluation and inference of the GRN structure from gene expression data is a challenging task in systems biology: recent achievements in single-cell (SC) transcriptomics allow the generation of SC gene expression data, which can be used to sharpen the knowledge on GRN structure. In order to understand whether a particular network of three key gene regulators (BCL6, IRF4, BLIMP1), influenced by two external stimuli signals (surface receptors BCR and CD40), is able to describe GC B cell differentiation, we used a stochastic model to fit SC transcriptomic data from a human lymphoid organ dataset. The model is defined mathematically as a piecewise-deterministic Markov process. We showed that after parameter tuning, the model qualitatively recapitulates mRNA distributions corresponding to GC and plasmablast stages of B cell differentiation. Thus, the model can assist in validating the GRN structure and, in the future, could lead to better understanding of the different types of dysfunction of the regulatory mechanisms.

A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single Ascending Dose Study to Evaluate the Pharmacokinetics, Immunogenicity, Safety, and Tolerability After Subcutaneous Administration of Tozorakimab in Healthy Chinese Participants.

Tozorakimab is a high-affinity human immunoglobulin G1 monoclonal antibody that neutralizes interleukin (IL)-33, an IL-1 family cytokine. This phase 1, single-center, randomized, double-blind, placebo-controlled, single ascending dose study (NCT05070312) evaluated tozorakimab in a healthy Chinese population. Outcomes included the characterization of the pharmacokinetic (PK) profile and immunogenicity of tozorakimab. Safety outcomes included treatment-emergent adverse events (TEAEs) and clinical laboratory, electrocardiogram, and vital sign parameters. Healthy, non-smoking, male, and female Chinese participants aged 18-45 years with a body mass index 19-24kg/m2 were enrolled. In total, 36 participants across 2 cohorts of 18 participants were randomized 2:1 to receive a single subcutaneous dose of tozorakimab (300mg [2mL] or 600mg [4mL]) or matching placebo (2 or 4mL). Tozorakimab showed dose-dependent serum PK concentrations with an approximate monophasic distribution in serum over time and a maximum observed peak concentration of 20.1 and 33.7μg/mL in the 300- and 600-mg cohorts, respectively. No treatment-emergent anti-drug antibodies for tozorakimab were observed in any of the participants. There were no clinically relevant trends in the occurrence of TEAEs across the treatment groups. There were no clinically relevant trends over time in clinical laboratory (hematology, clinical chemistry, and urinalysis), electrocardiogram, or vital sign parameters in any treatment group. Overall, tozorakimab demonstrated dose-dependent systemic exposure in healthy Chinese participants and was well tolerated, with no safety concerns identified in this study.

Abstract 3194: Discovery of T cell receptor mimic antibody against MAGE-A4 with single B-cell cloning platform

Abstract Antibody therapeutics has been proven clinically against various cancer types and have achieved great success. Majority of the therapeutic antibodies target cell surface or extracellular proteins. Most of the tumor-specific antigens that control cell growth, proliferation, and death are intracellular, and traditional antibody therapies fail to recognize intracellular antigen targets. Such tumor specific intracellular targets consist of overexpressed self antigens, oncofetal protein, cancer testis antigens, viral protein and neo-antigens of mutant proteins. Recently, an emerging approach to target these neo-epitopes has been developed called T-cell receptor-like/TCR-mimic antibodies as they recognize similar epitopes to those of T cell receptors which target specific peptide-MHC complexes(pMHC). TCR mimic antibodies open a new avenue for targeting intracellular antigens yet there are lots of challenges for generating highly selective and potent TCR mimic antibodies. MAGE-A4, melanoma-associated antigen A4, is a member of the MAGE protein family of cancer-testis antigen (CTA). In healthy adult, MAGE-A4 expression is restricted to immune-privileged sites. But MAGE-A4 is widely expressed on many cancers such as lung cancer, head and neck squamous cell cancer, synovial sarcoma(SS), ovarian cancer, urothelial cancer and melanoma. The decapeptide GVYDGREHTV(amino acids 230-239) derived from MAGE-A4, can be presented by HLA-A*02:01 molecule, elicits specific cytotoxicity T cell response. Afami-cel, a genetically modified autologous T cell therapy against this pMHC complex, showed promising potency for patients with metastatic SS. So MAGE-A4/HLA-A*02:01 may be a good target for TCR mimic antibodies and cancer immunotherapy. In the present study, we utilized a newly established single cell cloning platform LyTARS to generate TCR mimic antibodies against HLA restricted MAGE-A4 peptide. Recombinant HLA-A*02:01 MAGE-A4 protein was used to immunize mice and plasma B cells from immunized mice were sorted and loaded onto LyTARS system. Clones that showed reactivity to recombinant HLA-A*02:01 MAGE-A4 but not to irrelevant pMHC (WT-1) were exported and sequenced. Purified antibodies were extensively characterized by the specificity, affinity, and potency by ADCC. Results demonstrated that the TCR mimic antibodies identified have sub-nano molar affinity by protein based ELISA, bind to peptide pulsed T2 cells that endogenously expressing HLA with nano-Molar apparent affinity. TCR mimic antibodies also demonstrated antibody dependent cell killing with the potency of sub-nanomolar. In summary, we have generated and extensively characterized a panel of high affinity and selective TCR mimic antibodies against HLA-A*02:01 MAGE-A4 using a newly established single cell cloning platform. Citation Format: Hao Cheng, You Wu, Tianfeng Shi, Yang Yu, Min Wang, Chenjun Jia, Wenfang Xin, Hu Liu, Teddy Yang, Danmei Yao. Discovery of T cell receptor mimic antibody against MAGE-A4 with single B-cell cloning platform [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 3194.

Abstract 2361: A PD1-antibody-attenuated IL-2 fusion protein activates immune cells leading to the inhibition of tumor growth in ex vivo human 3D tumor models

Abstract IL-2 has been prescribed to effectively treat malignancies, mainly melanoma, for decades. Its use has waned in recent years due to significant toxicity and a narrow therapeutic index. To overcome the limitations of systemic administration of IL-2, we have generated a modified IL-2 (IL2Att) with specific substitutions to reduce the binding affinity with the IL-2R. The attenuated cytokine was fused to a high-affinity anti-PD1 antibody that does not block PD-1 binding with its ligands (PD-L1 and PD-L2). The resulting antibody-cytokine fusion, anti-PD1-IL2Att, allows selective delivery of IL-2 to tumor-reactive PD1+ T cells, with limited potential to activate PD1− cells. To evaluate the effect of anti-PD1-IL2Att in a human tumor microenvironment (TME), we established a unique human 3D multi-cellular cancer system. This model system includes neoplastic and somatic cells that make up the TME, further supplemented with peripheral blood mononuclear cells (PBMC). We found that the anti-PD1-IL2Att reduced tumor cell survival more effectively in several types of these 3D cancer models (melanoma, glioblastoma, ovarian and colon cancer) than standard immune checkpoint inhibitors pembrolizumab or nivolumab. Combination treatments of anti-PD1-IL2Att with either of these agents resulted in improved immune activation and tumor cell killing. Our findings suggest that tumor-targeted attenuated cytokines such as IL-2 can promote robust tumor killing while minimizing systemic toxicity. Citation Format: Ronit Satchi-Fainaro, Opal Avramoff, Anshika Katyal, Eilam Yeini, Anne Krinsky, Yulia Liubomirski, Shai Dulberg, Galia Tiram, Ayelet Kaminitz, Jason Gill, Jessica Pedersen, Dana Bar-On, Ayala Tamir, Paul Ayton, Tetsuya Taura, David Wilson, Asaf Madi. A PD1-antibody-attenuated IL-2 fusion protein activates immune cells leading to the inhibition of tumor growth in ex vivo human 3D tumor models [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 2361.

Abstract 2366: Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy

Abstract Background: CD40 agonist antibodies have been found to augment tumor antigen presentation and have shown potential as anti-tumor treatments in human clinical trials. The clinical success of CD40 agonist antibodies has been restricted due to their limited efficacy and on-target, off-tumor toxicity. We hypothesized that toxicity associated with CD40 agonist antibodies could be overcome by activating CD40 specifically through tumor-specific antigens. Additionally, limited efficacy can be improved by rational design of CD40 bispecific antibodies (BsAbs). Herein, we present FAPxCD40, a trivalent CD40 agonist BsAb targeting fibroblast activation protein (FAP) possessing an optimized therapeutic window. Method: We generated a series of FAPxCD40 bispecific antibodies with 2, 4, and 6 CD40 valences by using a high-affinity anti-FAP antibody and a crosslink-dependent CD40 agonist VHH antibody. These BsAbs were evaluated using multiple immune cell functional assays, including assays for dendritic cell (DC) maturation, B cell maturation, and CMV-reactive T cell recall. The in vivo therapeutic window of the FAPxCD40 bispecifics was compared in CD40 humanized mice with subcutaneously transplanted FAP-positive MC38 tumor xenografts. Results: We developed FAPxCD40 BsAbs with varying valences, including bivalent FAPxCD40-2, tetravalent FAPxCD40-4, and hexavalent FAPxCD40-6. FAPxCD40-2 showed strict FAP-dependent CD40 agonist activity in both DC and B cell maturation assays, while its CD40 agonist activity upon FAP-crosslinking was modest. FAPxCD40-4 exhibited efficient CD40 agonist activity upon FAP-crosslinking, but however, it also exhibited some undesired FAP-independent CD40 agonist activity. FAPxCD40-6 efficiently clustered CD40 but marginally benefited from FAP binding, making it unsuitable as a choice. In the humanized CD40 mice model, FAPxCD40-2 effectively eliminated systemic toxicity at all tested doses. Its antitumor activity, however, is modest with TGI (tumor growth inhibition) rate of 32.8 % at a dose of 6.96 mpk. Further escalation of the FAPxCD40-2 dosage to 13.92 mpk did not result in enhanced antitumor activity with TGI rate of 46.9 %, indicating that its CD40 agonist activity may have reached a plateau. In contrast, FAPxCD40-4 broke this plateau and induce a promising antitumor response at a dose of 1.3 mpk, resulting in a 92% TGI rate. Furthermore, no liver enzyme upregulation or platelet count decrease was observed under these conditions, indicating an ideal therapeutic window. In addition, during nonclinical toxicology studies conducted on cynomolgus monkeys, it was found that doses of FAPxCD40-4 up to 99 mg/kg administered weekly for a total of four doses were well-tolerated. Conclusion: Our study demonstrates that FAPxCD40-4, a tetravalent FAPxCD40 BsAb, exhibits efficient CD40 agonist activity with an optimized therapeutic window. Citation Format: Simeng Chen, Yuan Lin, Xiaoru Zhou, Xing Sun, Changyong Yang, Cheng Liao. Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer 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 2366.

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.

Abstract 3105: Computational integration with phage library to optimize clones of DEFB4A antibodies for cancer diagnostics and therapeutics

Abstract DEFB4A, a human beta-defensin, functions as a potential cancer biomarker, with elevated expression observed in various cancers, making it a candidate for diagnostic and prognostic applications. Additionally, its role in immune modulation suggests that DEFB4A may hold promise as a target for novel cancer treatments, particularly in the context of immunotherapy. This study aimed to develop a stable and high-affinity DEFB4A antibody for clinical and pre-clinical use. To achieve this, a combination of high-diversity phage libraries and computation-assisted analysis was employed to generate single-chain variable fragments (ScFv) clones specifically targeting DEFB4A. This research presents a robust approach for the generation of DEFB4A-targeting single-chain variable fragments (ScFv) from a phage library characterized by a high diversity of 109 unique clones and high titer of 1013 cfu/mL. Computational optimization was then applied to enhance the affinity, stability, and expression of these candidates through rational design and virtual mutagenesis programs. The binding activity of the optimized mutants was validated using biophysical methods such as surface plasmon resonance (SPR) and thermal stability assays. The process showed a remarkable 100-fold increase in affinity and stability. Additionally, the selected ScFv clone was further employed in immunohistochemistry (IHC) to detect DEFB4A in breast, prostate, and gastric cancers, showing higher expression levels compared to initial clones. This study highlights the synergy of high-diversity phage libraries and computational methods in crafting DEFB4A antibodies with enhanced affinity, stability, and expression. The findings advance the field of antibody engineering, emphasizing the crucial integration of experimental and computational approaches. This work lays the groundwork for the development of high-performance biologics with diagnostic and prognostic applications across diverse cancer types. Furthermore, it demonstrates the possibility of integrating computational approaches in antibody development, offering the promise of expediting the process in the future. Citation Format: Yichen Guo, Jina Yom, Andy (Xi) Han, Zhaoying Guo, Eden Zewdu, Rachel Gonzalez, Tianli Qu, Bailey Gilmore, Xuan Liu, Wei Fu, Xiaomin Hu. Computational integration with phage library to optimize clones of DEFB4A antibodies for cancer diagnostics and therapeutics [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 3105.

Abstract 4074: Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models

Abstract Systemic administration of proinflammatory cytokines is a promising approach to treat cancer. IL-18 has been shown to promote activation of both innate and adaptive anti-tumor responses in pre-clinical models. Specifically, IL-18 has been shown to impact macrophage suppressive function as well as drive CD4+ T cells towards a TH1 phenotype. However, IL-18 therapies have been hampered by a lack of efficacy due to the suppressive activity of IL-18 binding protein (IL-18BP), which binds to IL-18 and prevents its interaction with its receptor. Several groups have taken the approach of modifying IL-18 to render it resistant to IL-18BP as a potential strategy to enhance anti-tumor activity. To explore the therapeutic benefit of delivering a conditionally-activated IL-18 to the tumor microenvironment, we have developed inducible polypeptides (INDUKINE™ molecules) consisting of mouse IL-18 or IL-18BP resistant IL-18 (BPR IL-18) tethered via protease-sensitive linkers to a high affinity antibody blockade domain, and a half-life extension (HLE) domain which improves exposure in the tumor. IL-18 INDUKINE™ molecules are inactive until reaching the tumor microenvironment, where the linkers are cleaved by intra-tumoral proteases, releasing active IL-18. Intraperitoneal (i.p.) administration of an IL-18BP resistant IL-18 (BPR IL-18) INDUKINE™ molecule led to complete tumor regression in the MC38 tumor model. In contrast, equimolar doses of wild-type IL-18 INDUKINE™ protein were not as efficacious. Moreover, BPR IL-18 INDUKINE treatment led to increased activation and frequencies of NK cells and tumor specific CD8 T cells in MC38 tumors. WTX-518 is a novel INDUKINE™ molecule that is designed to selectively deliver active human BPR IL-18 to the tumor microenvironment. WTX-518 is inducible in vitro, either when tested using a reporter assay or primary immune cells. Importantly, the active BPR IL-18 payload is resistant to IL-18BP in reporter or primary immune cell assays. WTX-518 is also selectively inducible, as incubation ex vivo with various primary human tumors led to the release of active BPR IL-18, while WTX-518 was stable in human serum and after exposure to primary cells from healthy tissues. Pharmacological and efficacy data support continued preclinical development of this innovative and differentiated engineered IL-18 therapy. Citation Format: Kristin R. Morris, Heather Brodkin, Kyriakos Economides, Daniel J. Hicklin, Julie LePrevost, Celesztina Nagy-Domonkos, Christopher Nirschl, Andres Salmeron, Cynthia Seidel-Dugan, Cierra Spencer, Zoe Steuert, William M. Winston. Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models [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 4074.

Abstract 718: CCR8 antibody drug conjugates: Targeting regulatory T cells for tumor sensitization

Abstract Background: Regulatory T (Treg) cells in the tumor microenvironment have an immune suppressive function, and their presence is associated with poor prognosis. Depletion of Treg cells may be a mechanism to sensitize tumors and represents a novel strategy for cancer immunotherapy. High expression of chemokine receptor CCR8 has been found in Treg cells in microenvironments of numerous tumors but not on Treg cells in systemic lymphoid tissues. The ability to selectively kill Treg cells in tumor environments may provide an opportunity for therapy that leaves Treg function intact elsewhere in the body where they normally mediate tolerance to self-antigen. Methods: We sought to identify CCR8 antibodies for therapeutics and designed an antibody discovery strategy to address the challenges of this target. CCR8 is a member of the G protein-coupled receptor (GPCR) family. Like many GPCRs, CCR8 is a valuable therapeutic antibody target but challenging to access due to its poor expression, membrane-dependent structure, and poor immunogenicity due to sequence conservation. We developed an antibody discovery platform (MPS) that specifically addresses each of these challenges through use of advanced immunization techniques and evolutionarily divergent host species (chickens) for robust immune responses against conserved targets. We extensively engineered CCR8 antigen to enable high expression for immunization. When sufficient antibody diversity was not obtained using our standard immunization techniques, we developed mRNA immunization techniques for this program. Antibody binding was measured using flow cytometry on transfected cells and human Treg cells. The ability of antibodies to inhibit CCR8 function was measured using intracellular calcium influx assays. Antibody internalization was measured using dye uptake assays and flow cytometry. Antibodies were conjugated with cytotoxic payloads and tested for the ability to mediate killing. Results: Using these approaches, we identified a panel of high-affinity antibodies that bound in the nanomolar or subnanomolar range with high specificity, and we selected a lead molecule to progress. We will present data from our antibody discovery campaign including mRNA immunization, binding profiles, GPCR functional data, receptor-mediated internalization, and the ability of antibody-drug conjugates (ADC) to kill CCR8+ cells, including human Treg cells, in vitro. Conclusion: Treg cells are a primary driver of the immunosuppressive microenvironment in many solid tumors. Targeting immune-suppressive CCR8+ Treg cells using ADC has potential to expand the treatment of solid tumors Citation Format: Lewis J. Stafford, Hayley Roth, Sarah Campbell, Samantha Brady, Valerie Fiers, FeiFan Yu, Andrew Tsourkas, Riley Payne, Meghan Pitts, Kyle Guldner, Tim Phillips, Breanna Tyrell, Colleen Ulke, Allison Snyder, Marianne Assogba, Holden Ohl, Ileine Sanchez, Benjamin Doranz, Joseph Rucker, Ross Chambers. CCR8 antibody drug conjugates: Targeting regulatory T cells for tumor sensitization [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 718.

Quantitative immunochromatographic assay for rapid and cost-effective on-site detection of benzo[a]pyrene in oilfield chemicals

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42–300 mg/kg, and limit of detection was 0.23–1.07 mg/kg. The recovery rate was 88–106% with a coefficient of variation of 1.46–6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection.

The role of ixekizumab in the treatment of nonradiographic axial spondyloarthritis.

Nonradiographic axial spondyloarthritis (nr-axSpA) is a subtype of SpA with undeveloped definite radiographic sacroiliitis. Tumor necrosis factor inhibitors have demonstrated effectiveness in nr-axSpA patients who do not respond to first-line therapy. More recently, accumulated data from genetic, experimental, and clinical studies revealed that IL-17 is a key player in the pathogenesis of SpA, leading to development of new biologics directly inhibiting IL-17. Among them, ixekizumab is a high-affinity monoclonal antibody that selectively targets IL-17A and has exhibited significant efficacy and acceptable safety profiles in the treatment of nr-axSpA. The aim of this paper is to narratively review the recent insights of IL-17 in the pathogenesis of axSpA and discuss the effectiveness and safety of ixekizumab in treatment of nr-axSpA.

A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

Claudin 18.2 as a novel therapeutic target.

Claudin 18.2, a tight-junction molecule predominantly found in the nonmalignant gastric epithelium, becomes accessible on the tumour cell surface during malignant transformation, thereby providing an appealing target for cancer therapy. Data from two phase III trials testing the anti-claudin 18.2 antibody zolbetuximab have established claudin 18.2-positive advanced-stage gastric cancers as an independent therapeutic subset that derives benefit from the addition of this agent to chemotherapy. This development has substantially increased the percentage of patients eligible for targeted therapy. Furthermore, newer treatments, such as high-affinity monoclonal antibodies, bispecific antibodies, chimeric antigen receptor T cells and antibody-drug conjugates capable of bystander killing effects, have shown considerable promise in patients with claudin 18.2-expressing gastric cancers. This new development has resulted from drug developers moving beyond traditional targets, such as driver gene alterations or growth factors. In this Review, we highlight the biological rationale and explore the clinical activity of therapies that target claudin 18.2 in patients with advanced-stage gastric cancer and explore the potential for expansion of claudin 18.2-targeted therapies to patients with other claudin 18.2-positive solid tumours.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Isotype switching in human memory B cells sets intrinsic antigen-affinity thresholds that dictate antigen-driven fates

Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.